blood and lymph3 Flashcards

Question

Identify which coagulation factors are serine proteases

Answer 1

Serine proteases in the coagulation pathways factor II, factor VII, factor IX, factor X, Factor XI, Factor XII, and prekallikrein.

Answer 2

factor III, factor V, factor VIII, and high molecular weight kininogen.

Answer 3

almost all of the enzymes in the coagulation cascade are serine proteases, related to trypsin and/or chymotrypsin, which function by cleaving their targets at arginyl residues. The inactive precursor proteins that are activated through cleavage into active enzymes are called zymogens. Zymogens that become active serine proteases in the cascade include factor XII, Prekallikrein, factor XI, factor IX, factor X, factor VII, and factor II (prothrombin).

Answer 4

there are two main pathways leading to the activation of factor X, the extrinsic pathway and the intrinsic pathway. The extrinsic pathway, so called because it requires a factor extrinsic to the plasma (tissue factor) to function, is initiated by tissue factor binding to factor VIIa, leading to activation of factor X. Xa, with its cofactor Va, activates factor II to IIa, which then converts fibrinogen to fibrin. The separate intrinsic pathway, so called because all of the necessary components are contained in plasma, begins with activation of the contact factors (factor XII, prekallikrein, HMWK), that leads to activation of factor XI, followed by activation of factor IX which, with its cofactor VIIIa, then activates factor X, which, with its cofactor Va, activates factor II, which converts fibrinogen to fibrin. Both pathways converge at the level of activation of factor X, so the events occurring following activation of factor X are sometimes referred to as the common pathway. While the modern conception of coagulation has changed somewhat from this model, it is still useful to keep in mind when interpreting the PT and aPTT coagulation screening tests, which test the function of the extrinsic and intrinsic pathways, respectively. Two-step process of coagulation: Prothrombin + Thromboplastin + Calcium = Thrombin. Fibrinogen + Thrombin = Fibrin

Answer 5

Nearly all of the proteins in the coagulation cascade are synthesized primarily in the liver. Thus, one of the major problems observed in patients with severe liver disease is a bleeding diathesis (bleeding tendency or predisposition). Two exceptions are tissue factor, expressed on the surface of many cell types, and VWF, produced in endothelial cells and megakaryocytes. Also, factor VIII, while produced by the liver, can also be produced in other organs such as the spleen, lung, and kidney, explaining why factor VIII levels can be normal or even increased in patients with liver failure and coagulopathy. Biliary obstruction can lead to vitamin K deficiencies due to malabsorption (vit K is fat soluble)

Answer 6

factor VII has the shortest plasma t1/2 of 4-6 hours, making it one of the first factors to be depleted in some disease states. Once activated, the factors generally have a much shorter t1/2 and are rapidly inactivated, sometimes within a couple of minutes, as part of a tightly regulated control mechanism.

Answer 7

A special subset of this group of proteases are the vitamin-K dependent factors, including factors II, VII, IX, and X, along with the anticoagulant protein C. Protein S is also vitamin K dependent, though it functions in coagulation as a cofactor for protein C and not as a serine protease. These proteins share significant homology and are likely derived evolutionarily from a common ancestor. A special property that they all share is that they contain a “Gla” domain. This domain contains several (9 to 13) glutamic acid residues that undergo post-translational modification to γ- carboxy glutamic acid residues (“Gla” residues). These Gla residues are able to bind  calcium, which leads to shape change of the protein, allowing binding to an anionic phospholipid surface, which is necessary for normal protein function. This γ-carboxylation is carried out by the enzyme γ- glutamyl carboxylase in the liver. Vitamin K is required for generation of the precursor for the reaction, so vitamin K deficiency leads to inability to make the Gla residues, resulting in non-functional protein and making vitamin K deficiency an important cause of bleeding to consider.

Answer 8

due to vitamin K deficiency. Some reasons for this are decreased stores, low levels in breast milk, and developing gut flora.

Answer 9

An enzyme in the coagulation cascade that is not a serine protease. One other enzyme involved in the coagulation cascade is factor XIII, which covalently links fibrin molecules together to form a stable clot. Factor XIII is a transglutaminase that can form cross-linked amide bonds between specific lysine and glutamine residues.

Answer 10

In addition to the enzymes in the cascade, there are several cofactors that are essential for initiating or accelerating enzymatic reactions but lack intrinsic enzyme activity themselves. The cofactors are thought to work by bringing the components together and orienting them properly to make the reaction more efficient. Cofactors in the coagulation cascade include tissue factor, factor VIII, factor V, and high molecular weight kininogen (HMWK).

Answer 11

is not ordinarily expressed on cells in direct contact with the blood (endothelial cells, leukocytes). It is, however, expressed on the fibroblasts and smooth muscle cells surrounding blood vessels. With blood vessel injury, factor VIIa from the plasma will become exposed to tissue factor, leading to activation of the extrinsic pathway. Tissue factor can also become expressed on endothelial cells and monocytic blood cells under conditions of stress or injury, or when stimulated by LPS or other proinflammatory agents.

Answer 12

are both non-enzymatic procofactors with significant sequence homology to each other. Both are cleaved by thrombin to their active form, allowing them to participate in the tenase and prothrombinase complexes, respectively.

Answer 13

participates as a cofactor with the contact factors, factor XIIa, kallikrein, and factor XI.

Answer 14

is the soluble plasma protein that is cleaved by thrombin into its insoluble form, fibrin, which then participates in forming the actual blood clot. Fibrinogen is made up of 3 pairs of polypeptide chains (2 Aα-chains, 2 Bβ-chains, and 2 γ-chains) arranged into identical half molecules in an elongated polypeptide composed of three globules: a central globule containing the N-terminal domain of all of the polypeptides, and globules on each end of the molecule. With activation, thrombin cleaves off two small peptides, fibrinopeptide A and fibrinopeptide B. The release of fibrinopeptide A leads to exposure of a site on the middle domain that aligns non-covalently with a complementary site in the side domain of another fibrin molecule to form overlapping fibrils. The subsequent cleavage and release of fibrinopeptide B allows increasing aggregation of the growing fibrils. Factor XIIIa then covalently cross-links adjacent side domains, stabilizing and strengthening the developing clot.

Answer 15

plays a critical role in platelet adhesion and aggregation and also plays an important role in the coagulation cascade by serving as the carrier protein for factor VIII in the plasma. VWF is a large, multimeric protein that is produced and stored in what are known as Weibel-Palade bodies in endothelial cells and in α-granules of platelets. In the circulating plasma, it binds to and protects factor VIII, significantly prolonging its half-life (12 hrs vs 2 hrs). Patients with severe deficiency of VWF have low levels of circulating factor VIII, leading to a bleeding disorder similar to classical hemophilia A (factor VIII deficiency). They have a coexistent platelet function defect, leading to a very severe bleeding problem.

Answer 16

A common theme in the coagulation cascade is a situation where an activated enzyme will combine with a cofactor, on a negatively charged phospholipid surface and in the presence of calcium, forming an enzyme complex that accelerates the speed of the reaction several hundred- to several thousand-fold. These complexes are often referred to as “_____ase.” Examples of these complexes in the coagulation system include: extrinsic tenase, intrinsic tenase, and prothrombinase complex.

Answer 17

issue factor (a cofactor) combines with factor VIIa (a serine protease) on a phospholipid surface in the presence of calcium to bind to and activate either factor IX or factor X. This is called “extrinsic tenase” (or Xase), since it is part of the extrinsic pathway.

Answer 18

Alternatively, two members of the intrinsic pathway, Factor IXa (a serine protease) and factor VIIIa (a cofactor), can combine with phospholipid and calcium to bind and activate factor X. This complex is called intrinsic tenase.

Answer 19

Activated factor X (Xa), a serine protease, can then combine with factor Va (a cofactor), on a phospholipid surface in the presence of calcium, to bind to and activate prothrombin (factor II) to thrombin (factor IIa). This is known as the prothrombinase complex.

Answer 20

While it is useful to learn the classic coagulation cascade, our conception of how coagulation actually works has evolved. The current concept of coagulation stresses the physiologic importance of the extrinsic pathway in the initiation of coagulation. Also, instead of the intrinsic and extrinsic pathways working independently and converging at the level of factor X, there is crosstalk between them. And, the role of the contact factors is de-emphasized. A current model of coagulation divides it into three phases: an initiation phase, an amplification phase, and a propagation phase.

Answer 21

Coagulation is initiated with vascular disruption that leads to exposure of plasma to tissue factor (TF). In the plasma, a small amount (~1 %) of circulating factor VII can normally be found in its activated (VIIa) form. Free factor VIIa that is not bound to TF is inactive, which is why the small amount of circulating VIIa doesn’t initiate clotting. With exposure of tissue factor on the cell surface, it can bind factor VIIa in the presence of calcium, and the TF-VIIa (extrinsic tenase) complex can then bind factor X, leading to production of minute amounts of factor Xa. TF-VIIa also converts factor IX to factor IXa. The TF-VIIa complex can also generate more VIIa to amplify the process even more. This process, termed the initiation phase of coagulation, produces sufficient quantities of Xa to start the process of clot generation. Factor V can be slowly activated by factor Xa. Factor Xa then binds to factor Va and forms the prothrombinase complex, which generates small amounts of thrombin.

Answer 22

The amplification phase takes place on the surface of platelets which have adhered to the exposed subendothelium and been activated. In this phase, the initial procoagulant signal is amplified by small amounts of thrombin generated on TF-bearing cells. Thrombin activates Factors V and VIII to Va and VIIIa, respectively. Upon platelet activation, factors Va and VIIIa bind to the platelet surface. Xa can then form the prothrombinase complex with Va to generate small amounts of thrombin from prothrombin.

Answer 23

The propagation phase of coagulation begins with assembly of procoagulant complexes on the cell surface. The intrinsic tenase complex (factor VIIIa and factor IXa) activates more factor X on the platelet surface. The rate of activation of factor X by the intrinsic tenase complex is about 50-100 times faster than the extrinsic tenase complex, making formation of the intrinsic tenase complex critical for producing a sufficient quantity of Xa to initiate coagulation. Factor Xa then rapidly binds to factor Va (generated by thrombin in the amplification phase), forming the prothrombinase complex, which initiates conversion of prothrombin to thrombin, producing the thrombin burst necessary for fibrin-clot formation. Thrombin cleaves fibrinogen to form fibrin, leading to formation of the fibrin clot, and activates factor XIII to XIIIa, which covalently cross-links and stabilizes the forming clot. This fibrin latticework develops between and around the activated platelets, which have adhered and aggregated at the site of injury. The activated platelets provide a surface for the coagulation reactions to continue to occur, generating even more thrombin. Platelets also secrete the contents of their granules, which contain many of the components of coagulation to provide more substrate for the reactions to take place. Simultaneously, anticoagulation and fibrinolytic reactions are occurring, which will rapidly quench this process and prevent its spread beyond the site of injury.

Answer 24

At this point, it is important to emphasize the central role that thrombin plays in blood coagulation. Not only does it cleave fibrinogen to fibrin to form the actual clot. It also cleaves and activates the procofactors V and VIII to Va and VIIIa, which can then participate in the prothrombinase and intrinsic tenase complexes, respectively. It also activates factor XI to XIa, which can then go on to generate more factor IXa to participate in the intrinsic tenase complex. This all has the end result of amplifying the coagulation response and producing a burst of thrombin sufficient to form the clot. It also cleaves and activates factor XIII, which covalently links the fibrin to form a more stable clot. In addition to its role in the coagulation cascade, thrombin is the most potent known activator of platelets. One of the results of platelet activation is the translocation of the anionic phospholipid phosphatidylserine from the inner leaflet of the platelet cell membrane to the outer leaflet. Exposure provides a surface for the coagulation reactions to take place, greatly increasing their efficiency. These multiple roles of thrombin emphasize its central importance to all aspects of coagulation.

Answer 25

One could question the real role of contact factors (factor XII, prekallikrein, HMWK, and, to a lesser extent, factor XI) in this whole process. Deficiencies of factor XII, prekallikrein and HMWK exist and are not associated with a bleeding tendency. Patients with factor XI deficiency can have a mild bleeding disorder, likely related to its role in the amplification phase of coagulation through activation of factor IX. One role of factor XII may be clot stabilization, since patients with factor XII deficiency may be more prone to development of thrombotic emboli. Factor XII may also contribute to regulation of fibrinolysis and may play a role in activation of coagulation during extracorporeal membrane oxygenation (i.e., cardiac bypass), which involves exposure of the blood to foreign surfaces. HMWK is a source for bradykinin, a vasoactive peptide with multiple physiologic effects. In the end, the contact factors may play their most important role in linking coagulation and inflammation, the details of which are still being worked out. Their most important feature is that their (very rare) deficiency will lead to abnormal prolongation of the aPTT, placing them in the differential for a prolonged aPTT.

Answer 26

Fibrinolysis mechanisms to break down a clot. Both this and repair mechanisms are activated when clotting is initiated. The endothelial cells lining the blood vessels, while helping to promote clotting with damage or injury, also play a key role in minimizing risk of intravascular clotting in the resting state. And, of course, there are mechanisms to regulate and keep in check these systems, leading to a complex system of checks and balances. mechanisms to break down a clot. Both this and repair mechanisms are activated when clotting is initiated. The endothelial cells lining the blood vessels, while helping to promote clotting with damage or injury, also play a key role in minimizing risk of intravascular clotting in the resting state. And, of course, there are mechanisms to regulate and keep in check these systems, leading to a complex system of checks and balances.

Answer 27

a large and diverse group of proteins which share the feature of being able to bind to chymotrypsin-like serine proteases at their active-site serine, generally for purposes of protease inactivation. They can also perform other functions as well, such as storage or transport of the target protease. A large number of the proteins in the coagulation cascade are serine proteases and require the presence of a serine residue in their catalytic domain. Serpins have a domain called the variable reactive site loop, which is able to bind with specificity to the catalytic groove of their target serine protease. With this interaction, and sometimes the participation of a cofactor, structural changes lead to the exposure of the reactive site loop to the serine residue of the protease, which then forms a covalent bond between the two and significant structural changes in both proteins. This causes complete inactivation of both (sometimes serpins are called suicide protease inhibitors).

Answer 28

(formerly known as antithrombin-III) is a serpin that plays a critical role in anticoagulation. It targets multiple serine proteases in the coagulation cascade, probably most importantly thrombin and factor Xa, though it can also bind to and inactivate factors IXa, the VIIa-tissue factor complex, factor XIa, factor XIIa, and kallikrein. It inactivates its target by binding of an arginyl residue in its reactive site loop to the serine in the catalytic site of the protease, leading to 1:1 complex formation of serpin and protease, both of which are inactivated and cleared. Antithrombin deficiency is a well-known risk for venous thromboembolism.

Answer 29

acts as a key cofactor for antithrombin, accelerating its rate of protease inactivation by several hundred to several thousand-fold. Heparin is a polysaccharide that is a highly sulfated glycosaminoglycan. It works as a cofactor by two mechanisms: 1. A specific pentasaccharide sequence contained within the heparin polymer induces an allosteric conformational change in antithrombin that permits more efficient binding to and inhibition of the target protease. This shortened version of heparin is able to accelerate inactivation of factor Xa but not of thrombin. 2. A second mechanism in which heparin acts as a cofactor for antithrombin requires a longer form of the heparin molecule that is able to bind not only antithrombin but also its serine protease target, bringing the two molecules into close proximity to allow inactivation of the protease to occur. The longer form of heparin is required for acceleration of thrombin inactivation by antithrombin.

Answer 30

Unfractionated heparin and its derivatives (low-molecular weight heparin, fondaparinux) are important drugs used therapeutically to treat people at an increased risk of clotting. Heparin is derived from the mucosal tissues of pigs and cattle.

Answer 31

a closely related polysaccharide to heparin, is expressed on the surface of multiple cell types, including endothelial cells, and is likely the cofactor for antithrombin under normal conditions.

Answer 32

another in the serpin family. The only serine protease heparin cofactor II inhibits is thrombin. It is a cofactor for heparin and dermatan sulfate ("minor antithrombin")., which is synthesized primarily by fibroblasts and vascular smooth muscle cells. It also may play a role as an anticoagulant during pregnancy, when elevated levels of both heparin cofactor II and dermatan sulfate are seen.

Answer 33

another serpin that plays a small role in anticoagulation. It is primarily an important regulator of the classic complement pathway. Also regulates the contact factors of the intrinsic pathway (kallikrein, factor XIIa, and factor XIa).

Answer 34

a non-specific protease inhibitor, it is the most physiologically important inhibitor of activated protein C.

Answer 35

is a vitamin K-dependent serine protease. It circulates as a zymogen. It circulates as a zymogen. When thrombin is generated from prothrombin, in addition to its role as a procoagulant, it binds to thrombomodulin. Once bound to thrombomodulin, thrombin’s procoagulant activity is neutralized, but the thrombin- thrombomodulin complex on the cell surface can bind to and activate protein C.

Answer 36

a transmembrane protein constitutively expressed on endothelial cells and serves as a cofactor for thrombin. It reduces blood coagulation by converting thrombin to an anticoagulant enzyme from a procoagulant enzyme, accelerating the reaction about 1000-fold. It is analogous to the tenase and prothrombinase complexes that are part of the coagulation cascade and is sometimes referred to as the ‘protein Case” complex. Thrombomodulin expression can be down-regulated with exposure of the endothelial cells to proinflammatory agents, likely contributing to the hypercoagulability that can be seen with inflammation.

Answer 37

protein C is activated when cleaved by thrombin. Once generated, APC goes on to cleave and inactivate the cofactors Va and VIIIa, leading to decreased generation of thrombin. This reaction is enhanced by interaction of protein C with its cofactor, protein S, on an anionic phospholipid surface (i.e., the activated platelet surface). Interaction of protein S with APC alters the structure of APC alters the structure of APC and moves the APC active site closer to the membrane surface.

Answer 38

protein C’s cofactor. It is the only vitamin K-dependent factor that is not a serine protease. Only about 40% of protein S circulates in the blood in the free form, whereas the remaining 60% circulates bound to C4b-binding protein (C4bBP), a regulator of the complement pathway. This becomes important when measuring protein S levels in plasma, since protein S bound to C4bBP is inactive. Protein S probably has other roles in inhibiting coagulation outside of its role as a cofactor for protein C. Though its precise function still isn’t fully clear, it undoubtedly plays an important role as an anticoagulant as evidenced by the pro- thrombotic risk associated with deficiency.

Answer 39

protein C deficiency, which leads to increased thrombotic risk, particularly severe in the case of homozygous deficiency and can result in fatal neonatal thrombotic events.

Answer 40

it has a short half life of only 8-10 hours. Warfarin is a commonly used oral anticoagulant that functions by blocking the action of vitamin K on post-translational modification of vitamin K-dependent factors. Because of protein C’s short half-life, at onset of treatment with Coumadin it will be one of the first vitamin K-dependent coagulation factor to be depleted, leading to a transient hypercoagulable state. This becomes an important consideration when starting anticoagulation therapy with Coumadin.

Answer 41

another inherited pro-thrombotic condition related to protein C. A mutation in one of protein C’s target, factor V. It is caused by a point mutation that changes amino acid 506 of the protein from glutamine to arginine, which results in factor Va resistant to the proteolytic activation of activated protein C, so that factor Va remains activated longer than it should. Also known as APC resistance, since factor Va is resistant to inactivation by activated protein C. 3-8% of the Caucasian population is heterozygous for the factor V Leiden defect, making it one of the most common inherited risk factors for venous thromboembolism.

Answer 42

expressed constitutively by endothelial cells, is an important inhibitor of the extrinsic arm of the coagulation pathway and thus the initiation phase of thrombin generation. As factor Xa is generated by the extrinsic Xase complex (factor VIIa-tissue factor), TFPI can bind to Xa’s active site. Once surface bound, the factor Xa-TFPI complex rapidly binds and inactivates tissue factor-factor VIIa, forming a stable quaternary complex of tissue factor, factor VIIa, TFPI, and factor Xa. Factor IXa-TFPI can also bind to and inhibit factor VIIa-tissue factor, though TFPI binds to factor IXa with significantly less affinity, making this less likely to be of physiologic relevance. Ninety percent of circulating TFPI is found in association with lipoproteins, and TFPI has been implicated, in addition to its role in coagulation, to play a role in protection from atherosclerosis. Recombinant TFPI is currently being studied to see if may play a therapeutic role in the treatment of hypercoagulable states. The combination of TFPI inhibition of the initiation phase of thrombin generation and protein C pathway inhibition of the amplification phase of thrombin generation leads to a situation where, under physiologic conditions, a sufficient signal needs to be generated to reach the threshold necessary for thrombin generation to proceed to the propagation phase, serving as a mechanism to protect the body from excessive clot formation.

Answer 43

a nonspecific proteinase inhibitor that inhibits a broad range of proteinases, including thrombin, kallikrein, and plasmin, thus affecting both the coagulation and fibrinolytic pathways. It has an interesting mechanism for inactivating proteases through presentation of a “bait region” for the protease. With proteolysis of this bait region, the α2-macroglobulin molecule undergoes a rapid conformational change that traps the proteinase inside the molecule. The complex is then rapidly cleared through a receptor-mediated process.

Answer 44

Fibrinolysis is the process of clot breakdown that occurs following clot formation, allowing eventual repair of the damaged blood vessel following injury. It begins as soon as the clot begins forming. The key enzyme is plasmin,

Answer 45

a serine protease which is cleaved from its zymogen precursor, plasminogen, to its active form. Plasmin can cleave both fibrinogen (fibrinogenolysis) and fibrin (fibrinolysis) to form innumerable different types and sizes of fragments, which can be detected in the blood (collectively called fibrin degradation products or FDP). Plasmin can also break down extracellular matrix proteins, aiding in the remodeling process involved with repair of the damaged vessel. Plasminogen is synthesized in the liver and circulates in the plasma as well as being present in a wide variety of extravascular tissues and body fluids.

Answer 46

The primary activator of plasminogen in vivo. t-PA is a serine protease produced predominantly in endothelial cells. Its secretion from the endothelium is regulated by numerous important mediators of blood clotting and inflammation, such as thrombin, histamine, bradykinin, epinephrine, serotonin, and interleukins. Once released, t-PA has a very short half-life of about 2.5 minutes. It is rapidly cleared in a receptor-mediated fashion by the liver and by endothelial cells, as well as inactivated by various inhibitors, the most important being plasminogen activator inhibitor-1 (PAI-1). t-PA is a poor activator of plasmin in the absence of fibrin but efficiently activates plasminogen to plasmin upon binding to fibrin, with catalytic efficiency enhanced several hundredfold. t-PA itself can be cleaved by plasmin or other enzymes into a more active form. As a clot forms, t-PA and plasminogen bind to the fibrin being generated, localizing plasmin generation to the site of the clot. Initial plasmin degradation of fibrin increases the number of plasminogen-binding sites in the clot, further amplifying plasmin generation. Recombinant t-PA is extensively used today therapeutically for clot lysis.

Answer 47

The other main activator of plasminogen. It is synthesized by kidney cells as well as endothelial cells. It also can be secreted by tumors and is thought to contribute to metastasis by breaking down extracellular matrix to facilitate tumor invasion. It is a serine protease that is synthesized and released as prourokinase or single-chain u-PA (scu-PA). Similar to t-PA, it has a very short half-life of about 5 minutes. The prourokinase becomes bound to the clot and is then cleaved, primarily by already generated plasmin, into its active form, which can then contribute to further clot lysis. A small amount of plasmin can also be generated by cleavage of plasminogen to plasmin by the contact factors of the intrinsic pathway (factor XIIa, kallikrein, factor XIa).

Answer 48

Several mechanism exist to inhibit fibrinolysis through inhibition of plasmin itself or through inhibition of the conversion of plasminogen to plasmin. Some mechanisms include thrombin- activatable fibrinolysis inhibitor (TAFI), plasminogen activator inhibitors 1 and 2, and α2-antiplasmin.

Answer 49

is a zymogen that is synthesized in the liver and circulates in the blood in complex with plasminogen. Factor XIIIa can covalently attach it to fibrin. Like protein C, it is cleaved to its active form through binding to the thrombin-thrombomodulin complex. Once activated, it is an exopeptidase that removes basic amino acids (arginine, lysine) from the C- terminal of proteins. It targets the C-terminal of fibrin molecules and FDPs. Removal of these amino acids reduces the number of plasminogen-binding sites on fibrin, which decreases the amount of plasminogen available to t-PA or u-PA to cleave to its active form, thus down-regulating plasmin generation and slowing clot lysis.

Answer 50

The primary physiologic inhibitor of plasminogen activation, targeting t-PA and u-PA. PAI-1 is produced in multiple different cell types and has a half-life of less than 10 minutes in the blood. A major fraction of PAI-1 in blood is present in the α-granules of platelets. PAI-1 is a serpin that binds to and inactivates t-PA. Circulating levels of PAI-1 are in several-fold excess to levels of t-PA. Thus, any t-PA circulating in the blood is rapidly bound to and inactivated by PAI-1. For u-PA, PAI-1 only binds to the activated form. Deficiency of PAI-1 leads to excessive bleeding, and higher levels of PAI-1 lead to slower clot breakdown and a more thrombotic state.

Answer 51

another member of the serpin family, initially identified in human placenta. High levels are observed in the blood of pregnant women. Its role in clot lysis is not clear.

Answer 52

the primary plasmin inhibitor in blood. It is another member of the serpin family. It acts by binding to and inactivating plasmin in a 1:1 fashion. α2-Antiplasmin rapidly inhibits plasmin free in the circulation, preventing systemic fibrinogen degradation. By contrast, plasmin bound to fibrin is somewhat protected from inactivation by circulating α2-antiplasmin, leading to localization of fibrinolysis to the site of the clot. As with TAFI, factor XIIIa can covalently link α2-antiplasmin to fibrin, leading to stabilization of the fibrin scaffold in the developing clot. Individuals with deficiency show a bleeding disorder, indicating its important physiologic role in preventing fibrinolysis.

Answer 53

Under resting conditions, the endothelial cell lining employs several mechanisms to prevent thrombosis from occurring. Endothelial cells have mechanism including anticoagulation, fibrinolytic, and antiplatelet.

Answer 54

include: 1. Expression of heparan sulfate and dermatan sulfate, which act as cofactors for antithrombin and heparin cofactor II, respectively. 2. Expression of thrombomodulin, which acts as a cofactor with thrombin for activation of protein C. 3. Expression of TFPI to inhibit the extrinsic Xase complex.

Answer 55

include synthesis and release of t-PA and u-PA.

Answer 56

include: 1. Synthesis and secretion of prostacyclin (PGI2) and nitric oxide (NO), which prevent adhesion  of activated platelets and cause vasodilation. 2. Expression of an enzyme that rapidly metabolizes ADP (a potent platelet agonist) to AMP  and adenosine (a potent inhibitor of platelet function).

Answer 57

With damage or injury to the endothelial cell line through a host of mechanisms, several changes occur that promote clot formation, including: 1. Exposure and/or expression of tissue factor to initiate the extrinsic pathway. 2. Exposure of the subendothelium, leading to binding and activation of platelets, which then release substances such as ADP and thromboxane A2 which cause vasoconstriction, release procoagulant factors from their α-granules, and provide an anionic phospholipid surface for  the coagulation reactions to take place. 3. Release of von Willebrand factor from the Weibel-Palade bodies of the endothelial cells.

Answer 58

With exposure of the subendothelium, von Willebrand factor (VWF) binds to the subendothelial matrix. Circulating platelets adhere to the subendothelium through platelet integrin receptor interactions with VWF, collagen, and other subendothelial components. Binding leads to platelet shape change and activation as well as platelet aggregation. This leads to release of platelet granules containing many of the coagulation proteins as well as platelet agonists and vasoactive substances that cause vasoconstriction to help stem blood loss. In addition, phosphatidylserine, normally kept on inner leaflet of the cell membrane, is translocated to the outer leaflet, providing a surface for the coagulation reactions to take place. This part of the hemostatic process, involving platelet adhesion, aggregation, and activation, is sometimes referred to as “primary hemostasis.”

Answer 59

At the same time, tissue factor, either constitutively expressed on cells in the extravascular space or exposed on damaged endothelial cells in the area of the injury, becomes exposed to the small amount of factor VIIa circulating in the plasma, leading to initiation of the extrinsic coagulation pathway. This leads to activation of factors IX and X. A prothrombinase complex made up of factor Xa and factor Va, on the anionic phospholipid surface of the platelet and in the presence of calcium, is able to bind and activate prothrombin to thrombin. The generated thrombin is able to activate multiple factors, including factor XI, factor VIII, and factor V, leading to amplification of the coagulation process. This then leads to the propagation phase of coagulation, with generation of a burst of thrombin. Thrombin cleaves fibrinogen to fibrin to form a fibrin network around the activated platelets in the area of injury. Thrombin also cleaves and activates factor XIII, which covalently cross-links the fibrin, stabilizing the clot. This process, involving activation of the coagulation cascade and formation of the fibrin clot, is sometimes referred to as “secondary hemostasis.”

Answer 60

Thrombin simultaneously carries out several other functions. It is a potent activator of platelets, supporting development of the platelet plug and generating more phospholipid surface for coagulation to take place. It also initiates the anticoagulation process through activation of protein C, which goes on to inactivate factors V and VIII, and contributes to regulation of the fibrinolytic process through activation of TAFI. Thrombin itself, along with several of the other serine proteases that participate in coagulation, is rapidly inactivated and cleared by antithrombin, a reaction which is greatly accelerated by heparin or heparin-like molecules expressed on endothelial cells. At the same time, the extrinsic pathway is inhibited by TFPI.

Answer 61

t-PA is being released by the injured endothelial lining. t-PA (primarily), as well as u-PA, binds to the forming fibrin network and cleaves plasminogen to plasmin. Plasmin begins the process of breaking down the forming clot (fibrinolysis). Uncontrolled fibrinolysis is avoided by rapid inactivation of plasmin (particularly circulating plasmin) by α2-antiplasmin. Also, t-PA and u-PA are being inactivated by PAI-1.

Answer 62

Adhesion, activation, and aggregation of platelets to form a platelet plug constitute the first events in formation of a clot (primary hemostasis). The platelet plug is stabilized by formation of a fibrin network generated through the coagulation cascade (secondary hemostasis). Optimal numbers and function of platelets are key to cessation of bleeding from small vascular injuries. Disorders of platelet number or function can lead to bleeding from the skin, mucous membranes, brain, or other sites.

Answer 63

The circulating platelet is a small anuclear discoid cell ~2-3 microns in diameter that arises from megakaryocytes, with a maturation time of 4-5 days and a circulating life span of 9-10 days. In patients with normal spleen size, 80% of platelets are circulating and 20% are in the spleen. In some pathologic states (e.g., hypersplenism), the spleen may contain up to 90% of platelets. The bone marrow reserve of platelets is limited and can be rapidly depleted after sudden platelet loss or destruction. Newly formed platelets are larger in size and termed megathrombocytes. Platelets do not have a nucleus, but they do contain mitochondria. They have three kinds of functional granules: dense, alpha, and lysosomal granules. Dense granules contain ATP, ADP, serotonin, and calcium. α-granules contains a number of proteins essential for platelet function, including  procoagulant proteins (fibrinogen, factor V, von Willebrand factor, etc), platelet-specific  factors for platelet activation, and growth factors such as platelet-derived growth factor. Lysosomal granules contain acid hydrolases.  Platelets have an extensive system of internal membrane tunnels, called the surface-connected canalicular system, through which the contents of the platelet granules are extruded during platelet aggregation and secretion. Platelets also have a cytoplasmic framework of monomers, filaments, and tubules that constitute the cytoskeleton and allow shape change with activation.

Answer 64

Platelets play several important roles in hemostasis, including adhesion to the vascular subendothelium at sites of injury to begin the hemostatic process, activation of intracellular signaling pathways leading to cytoskeletal changes and release of intracellular granules to enhance platelet plug formation, aggregation to form the platelet plug, and support of thrombin generation by providing a phospholipid surface for the coagulation cascade to take place. These processes are a continuous and dynamic interaction of vessel, platelet, and plasma components. The endothelial cells of intact vessels prevent blood coagulation by secretion of a heparin- like molecule and through expression of thrombomodulin, which when bound to thrombin activates protein C and S. Intact endothelial cells prevent platelet aggregation by the secretion of nitric oxide and prostacyclin, inhibitors of platelet activation.

Answer 65

With vessel injury, subendothelial components are exposed. Circulating von Willebrand factor (vWF) adheres to the damaged, exposed subendothelium. Under conditions of high shear flow, circulating platelets then contact the exposed subendothelium in a rolling fashion and adhere by interaction between glycoprotein Ib (GP1b)on the platelet surface and vWF. With exposure to soluble agonists such as thrombin, ADP, epinephrine, and thromboxane A2, or to adhesive proteins in the subendothelial matrix such as collagen and vWF, the platelet integrin GPIIb-IIIa (αIIbβ3) increases its affinity for vWF, leading to tighter binding. GPVI also interacts directly with collagen in the subendothelium. Numerous ligands in the subendothelium, such as collagen, laminin, and fibronectin, also interact with β1 integrins on the platelet surface. All of these interactions lead to firm adherence of the platelet to the subendothelial surface.

Answer 66

With adherence to the injured vessel wall, platelets undergo shape change through cytoskeletal activation, becoming more spherical with extended pseudopods and spreading over the exposed subendothelium. The contents of platelet granules are released. Soluble agonists, including thrombin, thromboxane A2, epinephrine, and ADP, interact with their respective G protein coupled platelet membrane receptors, leading to intracellular signaling and calcium mobilization. Calcium activates phospholipase A2, which releases arachidonic acid from phospholipids. Cyclooxygenase (COX-1) then converts arachidonic acid to prostaglandin H2, which in turn is converted to thromboxane A2 by thromboxane synthetase. Thromboxane A2, along with other agonists, is released, acting to further amplify platelet activation. With platelet activation, membrane reorganization also occurs, with switching of the phospholipid phosphatidylserine from the inner to the outer membrane leaflet, making it available to interact with clotting factors that then lead to thrombin generation.

Answer 67

With platelet adhesion and with binding of soluble agonists to receptors to amplify platelet activation, GPIIb-IIIa is converted to a high-affinity state where it can bind fibrinogen and vWF. Binding of the membrane protein talin to GPIIb-IIIa is the last step to mediate the change from a low-affinity to a high-affinity state. GPIIb-IIIa can then bind fibrinogen, which acts as a bridge to lace platelets together into an aggregate. Thombin generated through activation of the coagulation cascade then converts fibrinogen to fibrin to stabilize the platelet plug.

Answer 68

Several tests are used to evaluate platelet function. A CBC with peripheral blood smear provides the platelet count and allows evaluation of platelet size and granularity. A bleeding time (or, more commonly used now, a platelet function analyzer or PFA-100 test) is a diagnostic test for platelet dysfunction. To do a bleeding time, a small incision in the skin is made using a standardized template and the time until cessation of bleeding is measured. A normal bleeding time is less than 9 minutes. A hemophiliac with a normal platelet count and normal platelet function will have a normal bleeding time. A platelet count below 100,000/uL will lead to a prolonged bleeding time, as will a qualitative platelet disorder. Platelet aggregation studies are done to evaluate platelet aggregation in response to a set of agonists, including thrombin, ADP, epinephrine, collagen, arachidonic acid, and ristocetin (an antibiotic which causes vWF to bind to GP1b, inducing platelet aggregation).

Answer 69

Major classes of drugs which inhibit platelet function include cyclooxygenase inhibitors such as Aspirin and nonsteroidal anti-inflammatory drugs such as Ibuprofen, ADP receptor inhibitors such as Ticlopidine (Ticlid) and Clopidogrel (Plavix), and GPIIb-IIIa receptor antagonists such as Abciximab.

Answer 70

(a low platelet count) can be due to decreased platelet production, increased platelet destruction or consumption, or sequestration of platelets in the spleen. A normal platelet count is between 150,000 and 400,000/uL. Spontaneous hemorrhage and increased risk of hemorrhage with trauma or surgery may be seen with platelet counts <50,000/uL, and with platelet counts less than 10- 20,000/uL, life-threatening spontaneous hemorrhage, such as spontaneous intracranial hemorrhage, can be seen. Thrombocytopenia due to decreased platelet production can occur with primary bone marrow disorders such as aplastic anemia, myelodysplasia, and leukemia. It can also occur with bone marrow invasion by metastatic cancer, myelofibrosis, or infections such as tuberculosis. Toxins such as chemotherapeutic drugs, chemicals, and exposure to radiation can injure the bone marrow and lead to thrombocytopenia. And, severe nutritional disorders such as B12 or folate deficiency can affect megakaryopoiesis. Finally, rare congenital disorders can lead to a decreased platelet count. The most common cause of thrombocytopenia due to increased destruction is immune thrombocytopenic purpura (ITP), formerly known as idiopathic thrombocytopenic purpura.

Answer 71

In patients with ITP, autoantibodies develop which are directed against platelet antigens, leading to their removal by macrophages of the reticuloendothelial system of the liver and spleen, a similar mechanism to that seen with autoimmune hemolytic anemia. There are two forms of ITP: acute and chronic. Acute ITP is usually seen in children or young adults, often preceded by a viral infection. Onset of the thrombocytopenia is sudden and can be severe. Patients present with petechiae and nosebleeds. Recovery is generally within 2 to 6 weeks without treatment or after treatment with steroids. Chronic ITP is more common in adults and is often associated with concurrent autoimmune disorders (e.g., SLE or rheumatoid arthritis), lymphoma, or HIV, although most cases remain idiopathic. Spontaneous remissions are infrequent, and most patients require treatment. The most commonly used treatment options include corticosteroids, intravenous immunoglobulin (IVIG), and splenectomy. Steroids work by dampening proliferation of the B cell clone making the autoantibody. An effect is usually seen within 7 to 10 days of starting treatment. IVIG acts by blocking splenic Fc receptors to prevent their binding to antibody-coated platelets, with an effect being seen within 1 to 2 days. Splenectomy works by removing the site of autoantibody-induced platelet removal and leads to lasting responses in 60 to 70% of patients.

Answer 72

occurs when a patient develops antibodies to platelet antigens not present on the patient’s own platelets. It can occur in the setting of a patient receiving platelet transfusions, or it can occur in the neonate through passive transfer of maternal IgG alloantibodies across the placenta. Drug-induced immune thrombocytopenia can occur when an antibody recognizes a neoepitope created by the binding of a drug to a platelet surface glycoprotein. Heparin-induced thrombocytopenia (HIT) can occur in patients on heparin therapy and can be associated with development of thromboemboli due to platelet activation.

Answer 73

include DIC, sepsis, thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS). Thrombocytopenia with these disorders is due to increased platelet consumption.

Answer 74

TTP is characterized by fever, renal insufficiency, microangiopathic hemolytic anemia, mental status changes, and thrombocytopenia. It occurs when endothelial damage occurs from a variety of mechanisms (e.g., infection, immune complexes, HIV, pregnancy, cancer), leading to abnormal release of unusually large vWF molecules from storage sites. These large vWF multimers mediate platelet adhesion and aggregation, forming diffuse platelet plugs in small arterioles. The large multimers are present because of the absence of a metalloprotease called ADAMTS13 that normally digests the vWF into smaller multimers. While a congenital form of the disease exists, most of the time it is acquired through development of autoantibodies to ADAMTS13. Treatment is with plasmapheresis to remove the large vWF multimers and replace the missing ADAMTS13.

Answer 75

HUS has a similar presentation but tends to more often be associated with renal failure and tends to occur more often in children. It is due to damage to the endothelial lining, usually by a bacterial toxin, leading to platelet adhesion and activation and microthrombi formation. It is often a self-limited process and is generally treated with supportive care alone

Answer 76

the most common congenital bleeding disorder. It can also be an acquired problem if antibodies develop against the vWF molecule. It can be due to an inadequate amount of vWF or it can be due to mutations in the vWF gene leading to abnormal protein function. vWF plays a key role in adhesion of platelets to injured vascular endothelium. Lack of vWF leads to abnormal platelet/endothelial interaction, leading to a primary hemostatic bleeding disorder characterized by mucosal and skin bleeding. VWF also serves as a carrier protein for factor VIII. So, with severe deficiency, defects in secondary hemostasis can be seen as well, due to a functional factor VIII deficiency. Lab tests for diagnosis of vWD include the bleeding time or PFA-100, which will be abnormally prolonged with vWD, a factor VIII level, a von Willebrand antigen test to measure the amount of vW protein, and a test of von Willebrand activity, also known as the ristocetin cofactor activity, which measures function of a patient’s von Willebrand protein using donor platelets. vWF multimer assays are occasionally obtained when evaluating for a qualitative defect in vWF function. A commonly used treatment for vWD is DDAVP (arginine vasopressin), which enhances release of vWF from endothelial stores. It is effective for treatment of type 1 vWD (partial quantitative deficiency) but not type 2 (qualitative defects) or type 3 (near-complete absence of vWF). Factor replacement can also be used is some situations. Patients should be told to avoid aspirin and other platelet inhibiting agents.

Answer 77

a rare autosomal recessive disorder where expression of GP1b on the platelet surface is reduced, leading to a defect in platelet adhesion. Platelet aggregation studies only show abnormal aggregation with ristocetin.

Answer 78

Storage pool deficiencies can occur, with a deficiency of either dense granules or α-granules. Deficiency of α-granules is known as gray platelet syndrome. Several syndromes can be associated with a lack of dense granules. These disorders can also be acquired when platelets pass across abnormal vascular surfaces (such as cardiopulmonary bypass apparatus) leading to partially degranulated platelets. Disorders can also be due to rare defects in signal transduction pathways within the platelets.

Answer 79

Rare inherited defects include afibrogenemia, which leads to both primary (platelet plug formation) and secondary (formation of cross-linked fibrin) hemostatic defects. The defect in primary hemostasis is due to lack of fibrinogen for binding to GPIIb-IIIa to allow platelet aggregation. The defect in secondary hemostasis is due to lack of fibrinogen for formation of cross-linked fibrin. Patients have platelet-type mucosal and cutaneous bleeding as well as deep muscle hematomas more characteristic of coagulation defects. Glanzmann thrombasthenia is a rare autosomal recessive bleeding disorder caused by absent or defective GPIIb-IIIa. Platelets can adhere but are unable to aggregate in response to normal agonist stimuli. Patients have petechiae and easy bruising.

Answer 80

Brisk bleeding from obvious trauma suggests a local vascular defect. Prolonged or recurrent bleeding is more likely a generalized hemostatic disorder. Sudden resumption of bleeding from an injured site raises the possibility of excessive fibrinolysis or abnormal crosslinking of fibrin. Multiple site bleeding suggests a more severe, generalized hemostatic disorder. Mucocutaneous bleeding (bruising, petechiae, epistaxis, menorrhagia, prolonged oozing after tooth extraction, increased bleeding after aspirin intake) is indicative of a defect in primary hemostasis (platelet disorder or von Willebrand disease) while soft tissue/joint/deep bleeding is more consistent with a defect in secondary hemostasis (coagulopathy). Findings on physical exam may suggest an underlying disorder, such as petechiae with thrombocytopenia, an enlarged spleen and lymph nodes with chronic infections or malignancies, signs of liver disease such as jaundice or edema with liver coagulopathy, or musculoskeletal abnormalities and joint disease with the hemophilias. Complaints or signs of easy bruising are common in children and many elderly people without an underlying bleeding disorder. It is rare, however, for children < 1 year of age to show bruising. Trauma (accidental or non-accidental) should be considered as a cause of multiple or unusual bruises at any age. Large (>2 inches in diameter) or indurated purpuric lesions should lead to an evaluation for a bleeding problem. Recurrent, brief nosebleeds are frequently seen in children. Nosebleeds that occur every 1-2 months, last longer than 10 minutes, involve both nares, and require medical attention or transfusion are suspicious of a bleeding defect.

Answer 81

Platelet count and blood smear to evaluate for thrombocytopenia or other hematologic abnormalities. Bleeding time or platelet function analyzer (PFA-100) to evaluate primary hemostasis. APTT as a screening test for the intrinsic coagulation pathway. PT/INR as a screening test for the extrinsic coagulation pathway. Thrombin clotting time (TCT) to evaluate for fibrinogen defects, the presence of fibrin split  products, or heparin effects. Fibrinogen level. Further testing is based on results of the basic screen and clinical suspicion. If the initial studies are negative in a patient with a definitive history of bleeding, further diagnostic studies should be done (under the guidance of a hematologist) to evaluate for such things as mild hemophilia, factor XIII deficiency, and fibrinolytic defects. Mild cases of von Willebrand disease may require repeated testing to establish a diagnosis if clinical suspicion remains. Occasionally, screening tests are obtained prior to a surgical procedure and abnormalities, such as an isolated prolonged APTT, are seen in an otherwise asymptomatic patient. Such a patient may have a factor XII deficiency (not associated with bleeding) or a lupus anticoagulant (associated with thrombosis, not bleeding). Or, they may have a mild form of hemophilia (factors VIII, IX, or XI deficiencies) or von Willebrand disease. Appropriate factor levels to rule out factor deficiency and a 1:1 mixing of normal plasma with patient plasma to evaluate for a lupus anticoagulant or factor inhibitor can then be done.

Answer 82

Red cells do no carry MHC antigens in humans, and the antigens they do carry are much less polymorphic in the population (many fewer alleles). The white cells that come along in transfusion are recognized and destroyed. Platelets do bear HLA (class I) and when people repeatedly need platelets, they may develop an alloimmunization problem, in which case HLA typing becomes necessary.

Answer 83

are glycolipids found on the surface of all body cells, including red cells. The lipid backbone spans the plasma membrane and the terminal sugars confer the antigenic specificity A, B, or O. people who are O are somewhat protected from pancreatic cancer and much less likely to develop venous thromboembolic disorders. Antigens are assembled by a set of glycosyl transferases that first builds the H antigen, the basic core sugar chain which almost everybody has. Then a final glycosyl transferase, of which there are three alleles, can act. The O allele is an amorph (it does not code for a working transferase and so group O only have the H antigen). People who are group A have a glycosyl transferase allele, which puts an additional sugar on the H antigen and people who are B have a different allelic form of this enzyme, which adds a different sugar. Group AB individuals have both the A and B antigens on their red cells, because they have both the A and B transferases from their parents. There are some people who lack the transferase gene that puts the final sugar on the core and thus do not express even the H antigen, so there is no substrate for the A or B glycosyltransferases to modify. This is the Bombay phenotype (Oh) and it is rare. All blood, even type O, is foreign to such people.

Answer 84

glycoproteins with the same sugar, found in the body fluids of people who have the secretor (Se) phenotype.

Answer 85

the antigens that are not the same as your own are foreign to you and you will become immunized to them. Thus a person who is group A will make antibody foreign to B but not A. a person who is O will have antibodies to both A and B.

Answer 86

An isoantibody that causes agglutination of cells of genetically different members of the same species. Measuring their titer can be use in the diagnosis of B cell immunodeficiency, since they should begin to appear in the blood between 3 and 6 months of age, as antigen exposure occurs. Isohemagglutinins are of the IgM class. There are variant A and B types (A2, A3, Ax, Bx, etc.) in which the A or B antigen is expressed rather weakly; such people may be typed incorrectly or with difficulty in the blood bank. Suppression of ABO antigens can be seen in some diseases such as leukemia. In addition, titers of isohemagglutinin can be low in the elderly and in hypogammaglobulinemia. Any of these conditions can lead to an “ABO discrepancy” (lack of correlation between ABO phenotype as determined by cell and serum typing) which must be resolved. DNA typing is possible. AB is the rarest blood group, O the most useful as blood donors, and group B people are the best looking.

Answer 87

the second most important blood group system. Rh antigens are on proteins coded for at two loci; one is for the alleles d/D and the other for c/C and e/E. The most important allele is D. D is dominant over d (the d allele, another amorph, is heavily mutated and does not make a protein). Ninety-two percent of U.S. blacks are Rh+; 85% of whites. Rh(D)- is rare in sub-Saharan Africa. There are no “naturally occurring” isohemagglutinins for Rh; it’s a protein, and not ubiquitous in nature, so you don’t make antibody to it unless you’re Rh(D)- and become immunized with Rh(D)+ red cells.

Answer 88

Individuals with the rare Bombay phenotype (hh) do not express H antigen (also called substance H), the antigen which is present in blood group O. As a result, they cannot make A antigen (also called substance A) or B antigen (substance B) on their red blood cells, whatever alleles they may have of the A and B blood-group genes, because A antigen and B antigen are made from H antigen. For this reason people who have Bombay phenotype can donate red blood cells to any member of the ABO blood group system (unless some other blood factor gene, such as Rhesus, is incompatible), but they cannot receive blood from any member of the ABO blood group system (which always contains one or more of A and B and H antigens), but only from other people who have Bombay phenotype.

Answer 89

this is the first process after donating blood. All donor units of blood are typed for ABO and Rh, and tested for syphilis, hepatitis B and C, HIV, and West Nile Virus antibody. In most blood banks the red cells are also typed for a select list of “minor” blood group antigens as well. Reverse typing is also performed, making sure that the isohemagglutinins in the plasma are appropriate for the determined red cell type. Plasma and cells are then separated, and banked.

Answer 90

this occurs before transfusing a patient. Prior to transfusion, recipients are typed for ABO and Rh and their plasma screened for expected and unecpected antibodies (using a panel of normal, phenotyped red cells). By first matching ABO and Rh (major crossmatch), next you must ask if there are other antibodies in the recipient’s plasma, which can react with antigen on this donor’s red blood cells. If there are and you give the blood anyway, the worst case scenario will be generalized complement-mediated hemolysis, and free hemoglobin deposited in the kidneys, which can lead to acute renal failure. The lab will also do a minor crossmatch in which plasma from the prospective recipient is mixed with red cells from the prospective donor. The big question is, will this recipient’s plasma destroy the incoming red cells. Donor red cells are first suspended in saline and a drop of the recipient’s plasma added. The tube is then centrifuged gently and the supernatant checked for redness, which would indicate that hemolysis had occurred (the plasma contains complement, of course). The pellet is resuspended and examined for clumping of the red cells. If either test is positive that unit cannot be used. If there is no hemolysis or agglutination, the blood and the recipient are considered “compatible.” Group O red blood cells can be given to recipients of almost any phenotype (universal donor). Type AB people can be universal recipients.

Answer 91

This is a test which uses antibody against human Ig to detect human Ig on the surface of red blood cells (direct test) or in plasma (indirect).

Answer 92

asks, is there antibody already on these cells I am interested in? Add antibody against human IgG to RBCs of patient. If they had some IgG already sticking to RBCs surface, than this antiglobulin could cross link it and the cells would agglutinate.

Answer 93

asks, is there unexpected antibody to red cell antigens in the plasma of this potential recipient? Add recipient plasma to donor RBCs and then wash off any unbound proteins. If there was antibody against the cell, it might bind but not agglutinate them. But if you now added the antiglobulin, it will cross-link, it will cross-link the bound antibodies, and that would agglutinate the cells. This is an indirect antiglobulin test and is done whenever there are questions about the major crossmatch. If the cells now agglutinate, there must have been antibody to them in the plasma, because antiglobulin alone won’t react with red cells. Memory aid: There’s always one more step in an indirect test.

Answer 94

This is also called erythroblastosis fetalis. It occurs in Rh(D)+ babies of Rh(D)- mothers. In the last trimester, and especially at the time of delivery, some red cells from the baby enter the mother’s circulation. If she is Rh(D)- and the baby is Rh(D)+, she may make anti-Rh(D). No problem for the baby, who isn’t there anymore. But in a subsequent pregnancy with another Rh(D)+ fetus, the mother’s antibodies, formed after the first pregnancy and boosted by the second, can cross the placenta and destroy the fetus’ red blood cells. In addition, each subsequent pregnancy with an Rh(D)+ fetus boosts her response. The fetus will be born jaundiced. This can be dangerous: high levels of bilirubin (a breakdown product of hemoglobin) can cross the blood-brain barrier and damage the basal ganglia, resulting in cerebral palsy or, if there is very severe damage, fetal death. The disease is preventable if, at the time that the mother delivers her first Rh(D)+ baby, she is given IgG antibody to Rh(D) (Rh-immune globulin), the most familiar brand being Ortho’s RhoGAM. These antibodies combine with the fetal red cells, opsonizing them, and they are destroyed before they get a chance to immunize her. Note: she is not made tolerant¾just not immunized. She must receive Rh immune globulin each time there is a chance of being immunized by Rh(D)+ cells: this includes all subsequent normal deliveries, abortions, fetal manipulations, amniocenteses, etc. We’re visited by an Rh(D)- woman who’s pregnant; she had two previous miscarriages. Did they immunize her against Rh(D)? We take some of her plasma and add it to ABO-compatible, Rh(D)+ cells. We see nothing, but did it bind? So we now wash the cells and add antiglobulin; they agglutinate. She is, in fact, already immunized. A later development in the prevention of HDN is the practice of giving a shot of RhoGAM to Rh negative women at 28 weeks gestation, to prevent immunization by small transplacental bleeds during the third trimester. The Rh-immune globulin would also be given at or shortly after delivery, if the child turns out to be Rh(D)+.

Answer 95

Normally, isohemagglutinins are IgM, probably because the ubiquitous antigens that stimulate them are T- independent (carbohydrates often are). So they don’t cross the placenta. Anti-Rh antibodies are usually IgG (being anti-proteins), and do cross the placenta. Occasional people do make IgG isohemagglutinins. This is especially true of group O people. So A or B fetuses of these women are at some risk of ABO hemolytic disease. There is no RhoGAM-like antibody for this.

Answer 96

These are antibodies to one antigen which bind, fortuitously, to another; a fancy name for cross-reactive antibodies. A good example is an antibody that appears in the serum of a patient with infectious mononucleosis; it is really in response to a viral antigen, but it happens to react also with horse red blood cells, giving us a quick and cheap presumptive test (the Monospot) for mono. Another example is an antibody that people with syphilis make; there is a similar phosphodiester group in the bacterium Treponema pallidum and the phospholipids that can be extracted from beef heart (cardiolipin), so a simple test can be performed that doesn’t require Treponema.

Answer 97

patient history will clue you in if the bleeding disorder is congenital or an acquired abnormality. Bleeding after tonsillectomy or other surgical procedures and/or after teeth extraction is very suggestive of a congenital coagulopathy. Determining whether there is a history of exposure to agents that cause liver disease, other serious medical illness and medications will help determine whether there are causes for an acquired coagulopathy.

Answer 98

The protime (PT) measures the procoagulant activity of the factors VII, X, V, II and fibrinogen. This is the extrinsic pathway and the lower part of the coagulation cascade. The protime normal range is generally between 9 and 12 seconds, however this value is based on the potency of the material (thromboplastin) that is used to start the reaction in the laboratory. Therefore, the results are also reported as compared to an international normalized ratio (INR). An INR of 1.0 would be a normal value. The protime can be long because of a deficiency of any of the above mentioned factors, but the most common situation results from a deficiency of the vitamin K dependent factors, VII, X, and II either because of a lack of vitamin K or inadequate liver function. The drug, Warfarin, because of its affect in inhibiting the vitamin K dependent reactions, also results in a prolonged protime. The protime is used to monitor Warfarin therapy.

Answer 99

The activated partial thromboplastin time (PTT) measures the procoagulant activity of the entire pathway. However, it is most sensitive to deficiencies of the higher numbered factors, especially XI, VIII and IX. It is not affected by deficiencies of Factor VII. The PTT can be prolonged also by anticoagulant drugs such as heparin or acquired anticoagulants such as fibrin split products. The normal range in most laboratories is usually 25-32 seconds. The PTT is used to monitor heparin therapy. Patients with hemophilia will have a prolonged PTT.

Answer 100

The thrombin time measures the procoagulant activity of fibrinogen and is also very sensitive to the anticoagulant effect of heparin or fibrin split products. The normal range is usually 12-18 seconds, if there is heparin contamination, fibrinogen deficiency or an abnormal fibrinogen it will be prolonged.

Answer 101

The bleeding time measures the platelet and vessel interaction, as well as the number and the function of platelets. It is performed by making a standardized cut with a simplate bleeding time device on the forearm. The time to clotting is then measured. This test is very operator dependent and takes meticulous attention to detail. In addition, it is affected by abnormalities in the skin. The normal bleeding time is generally between 2 and 9 minutes. A severe decrease in platelet count (less than 20,000-30,000) will cause a prolongation of the bleeding time, as will von Willebrand disease or abnormalities in platelet function. Other factor deficiencies do not prolong the bleeding time.

Answer 102

A new device, the Platelet Function Analyzer, can perform an in vitro bleeding time. It also can determine platelet response to agonists.

Answer 103

Hemophilia A (Factor VIII deficiency) is the most common cause of a severe bleeding tendency (1 in 5,000 male births, 30% new mutations). Hemophilia B (Christmas Disease or Factor IX deficiency) is ten times less common than Factor VIII deficiency. These two syndromes cause identical clinical problems and specific factor assays must be done to distinguish the two disorders. These two disorders are X-linked. This means that females are carriers and with rare exceptions, it is only the male offspring that are severely affected. The deficiency of Factor VIII or IX results in a prolonged PTT. In general, the longer the PTT, the more severe the hemophilia is. Assuming that pooled plasma from a normal population would give a value of 100%, we classify the hemophilia patients as to the residual percentage of factor activity they have. Many centers now offer genetic testing. A PCR test for an inversion on the long arm of the x chromosome can identify 40% of severe Hemophilia A patients.

Answer 104

Less than 1% factor activity. These patients suffer from spontaneous hemorrhaging into their joints, muscles, soft tissues, retroperitoneal space and unfortunately, sometimes the central nervous system. Prior to specific factor replacement therapy this illness results in early death. If repeated bleeding in the joints is uncorrected in these hemophiliacs, very severe arthritis and eventual total destruction of the joints results. Current treatment with recombinant or purified factor products is very effective in preventing or stopping bleeding.

Answer 105

2% to 5% factor activity. It usually takes some degree of trauma to cause bleeding in these patients.

Answer 106

> 10% factor activity. Mild hemophiliacs only bleed after trauma and do not develop the chronic joint disease that the more severely affected patients do. However, when they do sustain trauma and develop joint or soft tissue bleeding, they need specific factor therapy just as urgently as the more severely affected patients in order to get resolution of the bleed. The absolute minimum levels of factor required for surgery are somewhere above 50%. Therefore, these patients will have hematoma formation, wound breakdown and prolonged disability if not replaced with factor aggressively during surgery. Because mild hemophiliacs do not present with spontaneous hemorrhage, in general they are diagnosed after a bad traumatic event or after a bad result from surgery. Once one mild hemophiliac is discovered, a vigorous attempt to screen all possibly involved family members should be made, since the other affected individuals may not come to medical attention until they have developed a serious bleed.

Answer 107

30% to 100% factor activity. Because of lyonization of the x chromosome, the carriers of hemophilia A or B can be mildly affected themselves (30% factor level) or can be completely normal. Carrier females with bleeding are called symptomatic carriers and require life-long hemophilia follow-up. All potential carriers should be tested both for genetic counseling and also because those who have low factor levels will also require factor replacement after trauma and during surgery in order to get optimal results. Because factor levels cannot be solely used to determine carrier status, there are various molecular genetics methods available and families are encouraged to be evaluated.

Answer 108

Another common congenital bleeding disorder is Factor XI deficiency. The levels are usually greater than 5% of normal so that spontaneous bleeding is quite rare in these persons. This deficiency is autosomal recessive so that both men and women can be affected. It is quite common in Ashkenazi Jews in the United States and in various populations in the Middle East. The classic presentation is post-operative hemorrhage since most people do not have spontaneous bleeding. The PTT will be prolonged and a specific factor assay for Factor XI must be done to make the diagnosis.

Answer 109

Factor VII deficiency is quite rare but it can cause a severe bleeding disorder similar to Hemophilia A or B. In Factor VII deficiency only the protime (PT) is prolonged. The PTT is normal. When a patient is found with a prolonged PT, then a Factor VII assay is done and a classification of severity similar to hemophilia is done. Both men and women are affected because Factor VII deficiency is an autosomal disease. The severe patients are thought to be homozygous for mild factor VII deficiency.

Answer 110

von Willebrand disease is the most common milder congenital bleeding disorder. The von Willebrand protein is an extremely large protein that circulates in the plasma in a series of multimeric forms, that has two functions. One function is to adhere platelets to exposed collagen at the site of a wound. Therefore, von Willebrand protein is necessary for adequate platelet function and in von Willebrand disease, the bleeding time will be prolonged. The second function of von Willebrand protein is to carry Factor VIII. Without von Willebrand factor, Factor VIII has a very short half-life in the plasma. Therefore, if von Willebrand protein is decreased, the Factor VIII level will also be decreased. If the Factor VIII level is decreased severely, then the PTT will be prolonged. When we screen a patient for von Willebrand disease, we evaluate both functions of the von Willebrand protein. A standard screen would include: a bleeding time performed with the simplate bleeding device or PFA-100, a PTT, Factor VIII activity level, a level of the von Willebrand antigen (which describes how much of von Willebrand protein is present) and a test of the function of the von Willebrand protein known as the von Willebrand activity or ristocetin cofactor. In addition, we can determine the pattern of multimeric forms of the protein in the plasma. von Willebrand disease can result as either a deficiency of normal von Willebrand protein (type I) or the presence of an abnormal protein (type II). Treatment with DDAVP (arginine vasopressin) is very effective in type I and less effective in type II, which is why we are interested in classifying the patients. von Willebrand disease is an autosomal dominant disease, therefore, both men and women are affected. Because of the abnormality in platelet function, patients often bleed from mucosal membranes, and nose bleeds, GI bleeds and menorrhagia are major clinical problems. In general, they have high enough Factor VIII levels to prevent joint or muscle bleeding, although they bleed after surgery if not corrected with DDAVP or factor concentrates.

Answer 111

Bleeding timeor PFA-100–assess platelet function. vWF antigen – assess amount of factor present. vWFactivity–assess function of factor to aggregate platelets with ristocetin. Factor VIII activity–assessability of vWF to carry VIII. Multimeric analysis by electrophoresis–determine loss of large forms or  abnormal bands. RIPA–(Ristocetin-induced-platelet-aggregation)–determine hyperaggrebility to  ristocetin to diagnose Type 2b.

Answer 112

Partial quantitative deficiency (type 1), Qualitative deficiency (type 2), Abnormal clearance by platelets (type 2b), Severe or total quantitative deficiency (type 3).

Answer 113

An occasional patient will make a specific antibody against one of the coagulation factors. Though extremely rare, the most common of these is an acquired Factor VIII inhibitor. The patients often have other autoimmune illness, are postpartum or are of advanced age. They present with soft tissue and muscle bleeding, and usually marked hematomas of their skin or mucosal bleeding. The only laboratory test to be abnormal will be the PTT, which will usually be greatly prolonged. A mixing test should be performed. When normal plasma is mixed with the patient’s plasma, the PTT will not correct after two hours of incubation. This is because the antibody in the patient’s plasma will bind and inhibit the normal Factor VIII and the PTT will remain prolonged. This disease is confirmed by assaying specific factor levels. It is also possible to measure a titer of the inhibitory antibody. This disease has a 25% mortality due to bleeding, but an excellent long-term prognosis since most patients respond to immune suppression or spontaneously remit. Much less common than the Factor VIII inhibitor, are inhibitors to von Willebrand protein (acquired von Willebrand disease), Factor II (seen in lupus patients) and Factor V (seen in post-op or ICU patients).

Answer 114

Heparin in Sample, Hemophilia A and B (Factor VIII or IX Deficiency), Factor XI Deficiency, Factor XII Deficiency (patients do not bleed), Acquired Hemophilia, von Willebrand Disease, and Lupus Anticoagulant (patients do not bleed).

Answer 115

Protime relatively more prolonged than PTT, Liver disease, Vitamin K deficiency, and Warfarin or rat poison ingestion

Answer 116

Disseminated intravascular coagulation (DIC)

Answer 117

Most of the coagulation factors are synthesized by the liver. Therefore, abnormalities in hepatic function can cause deficiencies of the clotting factors. This is especially true for Factor V and for the vitamin K dependent Factors, II, VII, IX, and X. In very severe liver disease, the fibrinogen can be low also. Because the factors that are usually decreased affect the protime more than the PTT, the usual pattern of coagulation tests in a patient with liver disease, will be a prolonged protime with a relatively less prolonged PTT. The longer the protime is, the more severe the deficiency and the more likely that the PTT will also be slightly prolonged. If the fibrinogen level is decreased, the thrombin time may also be prolonged. These patients may also have consumption of platelets by their spleen due to portal hypertension, in which case they may have a decrease in platelet count as well. In patients with a prolonged protime due to liver disease, it is important to make sure that there is not a component of vitamin K deficiency exacerbating the problem thus, frequently trials of vitamin K therapy are done. Severe liver disease also causes abnormalities of the fibrinolytic system. There may be deficiencies of the endogenous anticoagulants also, such as antithrombin and protein C and S.

Answer 118

Vitamin K deficiency and warfarin administration which interfere with the vitamin K utilization, are very common causes of a prolonged protime with normal or slightly prolonged PTT. Three of the vitamin K dependent Factors II, VII and X affect the protime mostly. Vitamin K is readily obtained from the diet so that in general vitamin K deficiency is a disease of patients who have no oral intake and have their gut flora (which can make vitamin K) killed by broad spectrum antibiotics. Unless vitamin K is replaced in a patient in the intensive care unit, who is NPO, on antibiotics and critically ill, he will become deficient, with increasingly prolonged protime in approximately five days. Since warfarin (Coumadin) interferes with vitamin K utilization, vitamin K deficient hospitalized patients are much more sensitive to small doses of warfarin than a normal patient and over-anticoagulation can quickly occur. Long acting fat soluble rat poisons are used in suicide attempts. These patients present with extremely prolonged protime and PTT and require very intensive vitamin K therapy for months, until the poison is eliminated. Vitamin K deficiency is also a problem for the normal new-born infant. In developed countries, they are treated right after birth to prevent this complication.

Answer 119

Massive trauma, hemorrhagic or septic shock, amniotic fluid embolism, burns, acute leukemia or transfusion and drug reactions can all cause disseminated intravascular coagulation. In this situation the coagulation cascade is activated in the vascular system with the result that fibrin and platelet microthrombi form and plug capillaries and cause tissue infarction. At the same time, some factors and platelets are consumed so that the patient develops multiple coagulation factor deficiencies and often hemorrhage results. The most consistent laboratory findings are that the fibrinogen level has decreased markedly and the platelet count is low. Because the fibrinolytic system is activated in order to try and remove the fibrin-platelet microthrombi, fibrin cleavage products are released into the circulation, and these are known as fibrin split products, fibrin degredation products or D-dimer and can be measured in the laboratory. These fibrin split products inhibit the PTT assay, as well as the thrombin time, with the result that both of these tests will be markedly prolonged. In addition, Factor VIII and Factor V, which are the cofactors of coagulation, can be consumed. The usual pattern of laboratory abnormalities is shown in the Table below. Note that the PTT is increased relatively much more than the protime. This pattern of abnormalities is best distinguished from liver disease abnormalities in that the protime is usually the least affected (in contrast to liver disease) and the fibrinogen level is much lower in DIC than in the usual case of liver disease. When the underlying illness which caused the DIC is corrected, the fibrinogen will return quickly to normal and the platelet count will rise.

Answer 120

Prolonged PT, Greatly prolonged PTT Prolonged thrombin time, Low platelet count, Low fibrinogen level, Increased fibrin split products, and Increased D-dimer

Answer 121

the formation and propagation of clot within the vasculature; this term refers to an abnormal or pathologic process with imbalance in the hemostatic system. This generally occurs when some combination of stasis (slowed blood flow), inflammation and/or vessel wall injury is present in a person with an increased baseline propensity for thrombosis (e.g. inherited or acquired hypercoagulable state). Hypercoagulable states exist when there is chronic damage to vessel walls, excess of procoagulant factors or a deficiency of anticoagulant factors or fibrinolytic activity.

Answer 122

often associated with interaction of multiple risk factors. For the patient who has had a thrombotic episode in the absence of a defined precipitating condition (recent surgery, trauma, neoplasm, pregnancy, or prolonged immobilizing illness, etc.) or recurrent episodes of thrombosis, or thrombosis at an early age and is otherwise healthy, or a severe, life threatening thrombosis, or thrombosis at an unusual site (e.g. mesenteric or cerebral venous thrombosis), or a family history of thrombosis,  it is appropriate to search for an underlying hemostatic defect that can contribute to a “hypercoagulable state.”

Answer 123

The lupus anticoagulant is a very common acquired abnormality which results in a hypercoaguable state. The anticoagulant is an IgG antibody, which reacts against phospholipid in the platelet membrane or endothelial cell. The syndrome caused by the lupus anticoagulant is called the Antiphospholipid Antibody Syndrome or APS. The lupus anticoagulant is detected because it prolongs the PTT since the antibody binds up the phospholipid which is added to the test tube in order to start the reaction. Even though the PTT is prolonged in vitro, the patients do not have a bleeding tendency, instead they have a thrombotic syndrome which includes deep vein thrombosis, pulmonary embolism, thrombotic strokes and recurrent miscarriage due to thrombotic disease of the placental blood vessels. When normal plasma is mixed with the plasma from a patient with the lupus anticoagulant, the PTT does not correct. If, however, the patient’s plasma is first mixed with a source of phospholipid such as platelets, the antibody will be absorbed out of the plasma by the phospholipid and a repeat PTT will show partial correction. There are other methods also of detecting the lupus anticoagulant such as the dilute Russell’s Viper Venom Test (dRVVT). This is a sensitive test for the lupus anticoagulant because Russell’s Viper Venom directly activates factor X. Low amounts of venom and phospholipid are used in the assay and if antiphospholipid antibodies are present they will bind the phospholipid and prolong the clotting time. Adding extra phospholipid will overcome the antibodies and correct the time. It is extremely important to diagnose the lupus anticoagulant because these patients with thrombotic tendency must be distinguished from other patients with prolonged PTT’s who have a bleeding tendency, since treatment is entirely different. Although, it is named the lupus anticoagulant, a minority of the patients have lupus. They may have other autoimmune illness, malignancy, a recent infection or it may be drug induced by antibiotics or anti-psychotic drugs.

Answer 124

The four most common familial congenital conditions which cause a hypercoagulable syndrome are deficiencies of antithrombin, protein C and protein S or resistance to protein C (Factor V Leiden). The first three of these diseases are autosomal dominant and only modest decreases in the plasma levels of these proteins are associated with a risk of thrombosis. The homozygous deficiencies of protein C and protein S are frequently fatal at birth. Heterozygous patients generally present after puberty with recurrent venous thrombotic disease. The diagnosis can be made with specific assays of the activity or the amount of protein present. Factor V Leiden hypercoagulability results from a mutation in Factor V so that it is not inactivated by protein C. The heterozygous state is common in people of European ancestry. Often, more than one hypercoagulable condition is required to cause individuals with Factor V Leiden to thrombose.

Answer 125

B cells in blood can be measured by counting cells with surface immunoglobulin. Most labs use instead the markers CD19 or CD20 because they are more specific (a macrophage or PMN could have immune complexes on its surface, bound to its Fc receptors, and score as a false positive). A fluorescent molecule is coupled to a monoclonal antibody (mAb) to CD19 or 20, which is then mixed with a blood sample. For T cells we use mAbs to CD3 (total T cells), or to CD4 or CD8. Fluorescent cells can be counted under a microscope that has a UV light source and appropriate filters, or by flow cytometry. Double positive is when there is both CD4 and CD8 which would be present in the thymus because that is where T cell development occurs (double positive). Double negative lymphocytes would be B cells.

Answer 126

used to evaluate serum protein to test the humoral system. Different band lengths are quantified to show relative amount of immunoglobins. It is not very sensitive to small abnormalities. For example, it could not pick up selective IgA deficiency, because IgA runs pretty much together with the much larger IgG (γ globulin) band. Electrophoresis can be done on urine (to look for “Bence Jones protein,” free immunoglobulin light chains seen in patients with multiple myeloma) and on cerebrospinal fluid, where oligoclonal (a few clones) peaks in the IgG region are sometimes seen in multiple sclerosis.

Answer 127

to measure levels of individual immunoglobulin classes or subclasses. mmunodiffusion can be used to measure any other multivalent antigen (one that can form a precipitate with an appropriate antibody), for example the individual complement or clotting components, if you have a specific antiserum. Gels of this type can be purchased ready-to-go. They’re fairly cheap, but slow for a big hospital lab, which uses quicker tests that can be automated.

Answer 128

Antibodies against autoantigens in the nucleus are best observed using human cells grown on a slide. To do the test, the slide is fixed with an agent (alcohol or acetone) that makes the cells’ plasma membranes permeable so that antibodies can penetrate to the interior, and patient’s serum is dropped on the slide. After washing, fluorescein-labeled goat anti-human IgG (occasionally, anti-IgA or –IgM) is added. A further wash, and the slide is examined under the UV microscope. Experienced rheumatologists can tell much not only from the presence of fluorescence (indicating antinuclear antibodies,) but also from the pattern— speckled, diffuse, nucleolar, etc.

Answer 129

(IgM anti-IgG) is detected by its ability to agglutinate latex particles if they have been coated with IgG (“passive” agglutination, because the particle isn’t the antigen). This is a simple and cheap test, which anyone can do.

Answer 130

in serum often are insoluble in the cold. If you suspect a Type III disease, put a sample of serum in the fridge and examine it after 1 – 7 days for a precipitate; this precipitate is called a mixed cryoglobulin, to distinguish it from the pure (monoclonal) cryoglobulin that is occasionally seen in multiple myeloma.

Answer 131

can be used to evaluate Type II and Type III immunopathology. It can be used to identify antibody in a patient’s tissues (direct immunofluorescence) or in their blood (indirect).

Answer 132

is very like immunofluorescence, but uses a final antibody labeled instead with an enzyme, typically peroxidase, which produces a brown or black product. These slides can be observed with an ordinary microscope and archived for a long time (fluorescence fades with time.)

Answer 133

A simple ELISA test is the best way to evaluate for a specific antibody. As is done in the HIV antibody screen, antigen is coupled to a plate, then the test serum is added; if there is antibody to the antigen it will bind. It is then identified using an enzyme-coupled antibody to the specific class of the expected serum antibody.

Answer 134

A throat swab is smeared on a slide, and fluorescent-labelled antibodies to known bacterial antigens are poured over it. A test for antigen.

Answer 135

Antibodies to bacteria can often be detected quickly, sensitively, and specifically by immunofluorescence: A smear of the suspect bacterium, grown in culture, is prepared, the patient’s serum is layered over it, and the slide is then washed. Next, fluorescein-labeled goat anti-human Ig is added; the slide washed again, and looked at under a microscope by UV light. If the bugs fluoresce, there was antibody in the serum. Known bacteria are placed on a slide, and the patient's serum is poured over. After washing, fluorescent- labelled goat anti-human Ig is poured over. A test for antibody.

Answer 136

If an antigen is at least divalent, an excellent technique is a sandwich or capture ELISA to measure its concentration in a biological fluid or other solution. Let’s say it’s a patient’s serum and we’re measuring myocardial creatine kinase isoform MB in the serum, which would be elevated shortly after a heart attack. You start by making or buying two monoclonal antibodies to human CK-MB, each to a different epitope. Put one mAb on a plate so that it’s stuck there. That’s the capture antibody. Add the patient’s serum. Wash off anything that isn’t bound. Then add the second antibody, which will stick to the other epitope on the antigen in proportion to how much antigen’s there. The second antibody has an enzyme coupled to it¾usually peroxidase—and it completes the sandwich. Now add a colorless peroxidase substrate (S) that produces a colored product (P). Finally, measure the intensity of the product color in a plate spectrophotometer, also called an ELISA reader. Small molecules, with only one epitope, can’t be measured in a capture assay. For them we use a variety of competition assays.

Answer 137

using a kit, the throat swab is extracted in a tube of buffer, and the extract passed through a membrane to which Strep antigens stick because there’s a dot of anti-Strep antibody coupled to it. To detect this binding, the kit provides another antibody against Strep to which liposomes (little fat droplets with a water interior) have been bound; the water contains a dye. This preparation is also passed through the membrane, and it sticks if antigen has been trapped by the dot. Detergent is added to pop the liposomes, and so the spot turns color if there was Strep antigen in the extract. Something similar to this goes on in a dipstick pregnancy test (in which antibodies are used to detect chorionic gonadotrophin in the urine).

Answer 138

for screening suspected bacterial meningitis, you can obtain sets of tiny latex beads coated separately with antibodies to 4 common bacterial culprits (They are Hemophilus influenzae B, Group B streptococci, Strep. pneumoniae, and Neisseria meningitides). Add beads to a drop of the patient’s cerebrospinal fluid; if any agglutinate, it’s because they have been cross-linked by bacterial antigen that was in the CSF.

Answer 139

the best way to evaluate Th1 activity is to expose skin to common antigens to which most people will have DTH. A good set is: Candida, streptokinase/streptodornase (SK/SD,) trichophytin, mumps, tetanus, tuberculin. Read at 24-48 hours. Challenge DTH test: over 98% of normals will become “sensitized” (immunized) to dinitrofluorobenzene in about 10 days if it’s painted on their skin. This is like intentionally inducing poison ivy. One can also stimulate T cells in mononuclear leukocyte preparations (lymphocytes + monocytes) with the T cell mitogens PHA or Con A, and observe either proliferation or IL-2, IL-4, or IFNg production. Mitogens are plant (usually) proteins that bind certain sugar sequences; they are probably part of the plant’s defense system against things like fungi. Some of these sugar sequences are also found on human cells; ConA and PHA both bind sugars associated with the T cell receptor complex, fooling T cells (all of them) into thinking they are recognizing antigen. These tests are sometime useful to do because total numbers may be normal while function is impaired. In infants, a chest x-ray may be very useful to see how the thymus looks. Lymphoid biopsy may be necessary in suspected primary immunodeficiency. A biopsy of rectal mucosa is often less traumatic to the patient. Killer cell assays are done in research labs.

Answer 140

The machines are called flow cytometers because they take cells in suspension and pump them through an orifice so small that the cells emerge in single file in a very fine stream. Lasers illuminate the cells and light emitted or scattered by each cell is collected by photomultipliers connected to a fast computer system. Light scatter gives information about cell size and cytoplasmic granularity, and if the cell has bound a fluorescent- tagged antibody, the fluorescent light emitted is quantified. Monoclonal antibodies to many cell surface molecules are available, and can be bought tagged with different dyes—or even quantum dots—so that they can be used simultaneously (see the diagram). Other dyes can be used, like propidium iodide, which reacts quantitatively with DNA, becoming fluorescent, so that you can tell where a cell is in the cell cycle by its DNA content. By using multiparameter cytometry, you can ask questions like, what percentage of cells in the blood bears the CD34 marker that is seen on hematopoietic stem cells? Are they cycling or resting? The machine examines 10,000 individual cells in a second. If cells are fixed and permeabilized, flow cytometry can be used to detect internal antigens, such as cytokines (not yet secreted) or transcription factors; this is how we identify Treg, for example. Pro-B, pre-B, immature and mature B cells are distinguished by fluorescence microscopy using Abs to IgD, IgM, and H or L chains, on both fixed (permeabilized) and intact cells, so you can distinguish whether a molecule is within a cell, or on its surface.

Answer 141

Decreased blood flow (venous stasis), inflammation of or near the blood vessels (altered vessels), and intrinsic alteration in the nature of the blood (altered coagulability).

Answer 142

Symptoms of deep vein thrombosis (DVT) and pulmonary embolus (PE) can be vague and non-specific, making it difficult to make a diagnosis at times and making it important to have a high index of suspicion. It is estimated that up to 50% of DVT and PE are asymptomatic or undetected. The clinical signs and symptoms of DVT reflect the obstruction by clot of the deep veins in an extremity. On the severe end of the spectrum, complete obstruction of a proximal vein, such as a massive iliofemoral thrombosis, can produce nearly complete obstruction of venous outflow from an extremity, leading to a condition called phlegmasia cerulean dolens (an extremely swollen, blue, painful leg). Lesser degrees of obstruction can produce pain, pitting edema of the distal extremity, and a warm, dusky, reddish-blue discoloration of the skin caused by enhanced superficial venous blood flow. Sometimes these physical signs can be very subtle, requiring good light and asking the patient to stand for a few minutes to appreciate differences in size, warmth, color, or edema between normal and involved legs.

Answer 143

One consequence of extremity DVT can be postthrombotic syndrome due to chronic venous insufficiency and chronic venostasis. Affected extremities become chronically swollen and painful and show dark skin discoloration. Cutaneous ulcers can develop, usually around the ankle when a leg is affected. Recurrent bouts of leg pain and swelling can occur due to intermittent obstruction of blood flow in the absence of formation of new thrombi.

Answer 144

Another dreaded complication of DVT is PE, which occurs when part of a thrombus breaks off and travels through major veins, past the right heart, and into the pulmonary artery circulation until it becomes lodged. Lung tissue past the thrombus cannot participate in gas exchange and can infarct. If a clot involves both pulmonary arteries (saddle embolus) cardiovascular arrest and death can occur. DVT restricted to the calf veins uncommonly results in clinically important PE and is rarely associated with a fatal outcome. In contrast, inadequately treated DVT involving the popliteal or more proximal leg veins is associated with a 20 to 50% risk of clinically relevant recurrence and is strongly associated with both symptomatic and fatal PE. Classic signs and symptoms include sudden chest pain, dyspnea, anxiety, cough, syncope, and cyanosis. Hemoptysis can occur uncommonly, and patients can present with cardiac arrest and sudden death. Patients with small but recurrent PE can develop chronic dyspnea and chronic pulmonary hypertension with elevated right heart pressures. Significant mortality can occur if an unrecognized PE goes untreated.

Answer 145

Because of the often vague or non-specific signs and symptoms, diagnosis of DVT or PE based on history and physical alone can be difficult and unreliable. Once a venous thrombus is suspected, further testing is required to make a definitive diagnosis. Evidence-based algorithms using test sensitivity and specificity and pretest probability have been developed to aid in diagnosing DVT and PE.

Answer 146

A very useful screening test in these algorithms is the D- dimer assay. D-dimers can only be formed when cross- linked fibrin has been degraded by plasmin through fibrinolysis. (see diagram at left) So, in order for D-dimers to be formed, there must be formation of a clot, making the D-dimer assay an indirect measure of clot formation. D-dimer is a very sensitive but not specific test with high negative predictive value for DVT (i.e., a negative results rules out DVT – a positive result, however, doesn’t rule it in). With a positive D-dimer screening test, further studies should be performed. Venous ultrasound +/- US- Doppler (which can measure blood flow and pressure in blood vessels) has >95% sensitivity and specificity in patients with symptomatic DVT. For diagnosis of PE, spiral CT scan of the chest and ventilation/perfusion (V/Q) scans are the most commonly performed studies. An embolus in the pulmonary artery circulation can be directly visualized on CT scan. For the V/Q scan, two types of imaging are done. A gaseous radionuclide is inhaled to evaluate which parts of the lungs are being aerated with breathing. Then, another radionuclide is injected to assess how well blood circulates through the lungs. Mismatch between ventilation and perfusion (i.e., a localized area that ventilates well but is not perfused) may indicate a pulmonary embolus.

Answer 147

occur under conditions of high shear stress, a condition where con willebrand factor is critical for platelet adhesion. They are composed primarily of aggregated platelets, containing small amounts of fibrin and few red cells, making them appear white in color (white thrombi). If they become large enough to lead to complete arterial occlusion, ischemia and infarction of the downstream tissues occurs. Clinical manifestations are dependent upon the organ involved (heart attack with coronary artery occlusion, stroke with cerebral artery occlusion, gut ischemia with mesenteric artery occlusion, etc). Abnormalities of blood flow which can contribute to development of thrombi include hypertension and turbulent blood flow at arterial branch points and at sites of focal atherosclerosis. Abnormalities of the blood vessel can include intraluminal vascular endothelial cell injury, atherosclerotic plaque rupture, hyperhomocysteinemia, aneurysm formation, and vessel dissection. Altered coagulability can be due to platelet activation, hyperviscosity such as may occur with certain malignancies, and thrombocytosis.

Answer 148

typically develop under conditions of slow blood flow (low shear stress). They are primarily composed of large amounts of fibrin containing numerous red cells (“red thrombi”). Stasis can be due to numerous factors, such as right-sided heart failure, pre-existing venous thrombosis, extrinsic vascular compression by tumor, immobility, obesity, and chronic venous insufficiency. Vascular factors contributing to venous thrombosis can include direct trauma or surgery, extrinsic compression, presence of a foreign body such as an IV catheter, and vascular endothelial cell injury due to exposure to toxins or excess levels of homocysteine. Altered coagulability can be due to inherited or acquired disorders of procoagulant proteins, deficiency of anticoagulant proteins, deficient fibrinolysis, and other factors such as use of oral contraceptives, pregnancy, malignancy, hyperhomocysteinemia, hyperviscosity, and the presence of antiphospholipid antibodies. Increased age also contributes to increased risk for thrombosis, likely due to multiple factors.

Answer 149

in the acute setting, heparin (to prevent further clot formation) and a fibrinolytic agent such as tPA (to lyse the existing clot) are indicated. Time is of the essence due to the risk for tissue ischemia and infarction with delay in initiation of treatment. With arterial thrombi the pathophysiology of thrombus formation is primarily related to platelet activation and aggregation. Thus, in the more long-term setting where prevention is the main goal of therapy, antiplatelet agents are indicated in most situations. These can include aspirin (inhibits cyclooxygenase), the thienopyridines such as ticlopidine (Ticlid) and clopidogrel (Plavix)(ADP receptor antagonists), and the glycoprotein IIb/IIIa inhibitors such as abciximab (Reopro).

Answer 150

since activation of the coagulation cascade and formation of a fibrin clot is the main pathophysiologic mechanism, agents that inhibit coagulation are indicated. In the acute setting, unfractionated or low molecular weight heparin are used initially. These drugs act as cofactors to potentiate the activity of antithrombin III. In the long-term, low molecular weight heparin can be continued (requiring twice daily subcutaneous injections), or the patient can be switched to oral anticoagulation with warfarin (Coumadin). Warfarin acts by inhibiting the activity of vitamin K dependent enzymes (Factors II, VII, IX, X, Protein C, Protein S). Because it doesn’t affect the coagulation factors that have already been synthesized and released into the circulation, warfarin is not effective until enough time has passed to allow turnover of the factors. Of those factors, factor VII has the shortest half-life (5 hours) while the other factors have half-lives of 24 to 48 hours. Because factor VII will have the greatest effect on the PT/INR, the PT/INR will be prolonged before full anticoagulation has taken place. For that reason, it is important to maintain patients on heparin anticoagulation therapy at least 5 days after starting the warfarin to insure that full anticoagulation has taken place. Warfarin has a long half-life which is affected by many drugs and foods, so careful and regular monitoring of the PT/INR in patients taking warfarin is necessary to prevent under- or overdosing.

Answer 151

Duration of anticoagulation therapy is determined on an individual basis based on a number of factors. The goal of treatment is prevention of DVT recurrence or clot extension and prevention of PE. Key in the decision-making process is determining if the DVT occurred due to transient risk factors (such as surgery, temporary immobilization, trauma, or pregnancy) or if the patient has an underlying hypercoagulable disorder or ongoing risk factors that require longer treatment. The location and severity of the clot also influences clinical decision making. The table on the following page shows one recommended approach.

Answer 152

Family history of abnormal blood clotting, abnormal blood clotting at a young age, Thrombosis in unusual locations or sites, such as veins in the arms, liver (portal), intestines (mesenteric), kidney (renal) or brain (cerebral), Blood clots that occur without a clear cause (idiopathic), Blood clots that recur, A history of frequent miscarriages, and Stroke at a young age.

Answer 153

(Activated Protein C Resistance). Due to an autosomal dominant mutation of the factor V gene that leads to partial resistance to inactivation through proteolytic cleavage by protein C. Factor V Leiden is inactivated 10 times more slowly than normal factor Va. Resistance to inactivation leads to increased risk for thrombosis. Factor V Leiden is the most common inherited predisposition to hypercoagulability in Caucasian populations of northern European background (up to 15%). Heterozygotes have a 4- to 7-fold increased lifetime relative risk of developing venous thromboembolism, and homozygotes are estimated to have an 80- fold increased lifetime relative risk. Long-term anticoagulation therapy is not necessary for heterozygotes unless they experience more than one thrombotic event or life-threatening thromboembolism. Asymptomatic patients should not be treated but should be informed of increased thrombotic risk associated with other risk factors such as surgery, oral contraceptive use, or pregnancy.

Answer 154

This is the second most common inherited predisposition to hypercoagulability and has an autosomal dominant inheritance pattern. The mutation appears to lead to elevated concentrations of plasma prothrombin. Heterozygotes have a 2-6-fold increased lifetime relative risk of VTE. Indications for treatment are similar to those for factor V Leiden. Heterozygotes for both mutations (factor V Leiden and prothrombin G20210A) should have long-term anticoagulation following a first thrombotic event.

Answer 155

Protein C is a vitamin K-dependent plasma protein that, when activated, inactivates factors Va and VIIIa to inhibit coagulation. Deficiency is inherited in an autosomal dominant fashion. Most affected patients are heterozygotes with ~50% of normal protein C levels. The estimated increased lifetime relative risk of VTE is up to 31-fold. Homozygous deficiency of protein C leads to neonatal purpura fulminans, an often fatal disease associated with extensive venous or arterial thrombosis at birth and levels of protein C <5% of normal. Heterozygotes can also develop warfarin-induced skin necrosis. This problem typically occurs in patients started on large doses of warfarin in the absence of concomitant heparin therapy. Warfarin decreases the already low protein C levels more rapidly that the vitamin K-dependent procoagulant factors, leading to exacerbation of the basal hypercoagulable state. Asymptomatic patients should not receive anticoagulation but should avoid other risk factors and should receive prophylaxis for high-risk procedures such as surgery. Those with a single thrombotic event should receive 6-12 months of anticoagulation and those with more than one event, a single life-threatening event, or a strong family history of thrombosis should receive long-term anticoagulation. Protein C concentrates are available for those with homozygous deficiency.

Answer 156

Protein S is another vitamin K-dependent plasma protein that facilitates the anticoagulant activity of activated protein C. As with protein C, deficiency is inherited as an autosomal dominant trait. Patients can present with VTE or with arterial thrombosis, including stroke. Risk for thrombosis is higher when combined with other risk factors. Neonatal purpura fulminans and warfarin-induced skin necrosis can be seen. The estimated lifetime increased relative risk of thrombosis is up to 36-fold. Treatment principles are the same as those for protein C deficiency.

Answer 157

Antithrombin III regulates coagulation by inactivating thrombin as well as factors Xa, IXa, XIa and XIIa. AT-III deficiency is inherited in an autosomal dominant fashion. Most patients are heterozygous, having ~50% of normal activity levels. Homozygous deficiency is usually fatal in utero. The estimated increased lifetime relative risk of thrombosis is up to 40-fold. Deficiency of AT-III can sometimes lead to heparin resistance, since heparin acts as a cofactor for AT-III and, thus, lack of AT-III limits the therapeutic effectiveness of heparin. Asymptomatic patients should not be treated. Patients with a single thrombotic event should receive at least 3-6 months of anticoagulation. Patients with massive VTE or PE should be considered for thrombolytic therapy. Pregnant patients or patients undergoing surgery should receive prophylaxis, including AT concentrate administration.

Answer 158

can be inherited or acquired. The mechanism underlying increased risk for thrombosis may be enhanced platelet activation and adhesiveness due to endothelial cell injury.

Answer 159

(>150%) can lead to a 3-6 fold greater relative risk of VTE. The risk increases to 11-fold with activity levels >200%.

Answer 160

(plasminogen deficiency, tPA deficiency, etc) can lead to increased risk for thrombosis.

Answer 161

are additional important acquired risk factors for thrombosis as well as increased risk for obstetric complications and fetal death. Paradoxically, while these antibodies lead to increased risk for both venous and arterial thrombosis, in the laboratory they lead to prolongation of the APTT (hence the name “lupus anticoagulant”). This is a laboratory artifact due to in vitro inhibition of phospholipid-dependent coagulation assays and does not reflect the way they act on the coagulation system in vivo.

Answer 162

have been a well-recognized acquired risk factor for thrombosis since their introduction in the 1960’s. The likely mechanism is alteration in levels of coagulation factors leading to a net increased risk for thrombosis. The risk is greatly increased in combination with other risk factors. For example, factor V Leiden alone leads to a 4-7 fold increased relative risk for thrombosis while factor V Leiden in combination with oral contraceptive use leads to a 30-35 fold increased risk.

Answer 163

includes heparin and oral anticoagulants, fibrinolytic agents, and anti-platelet agents. Antithrombotic drugs disrupt hemostasis. Therefore there is a fine balance between efficacy and toxicity. Patients on anti-coagulant therapy must be carefully monitored to avoid hemorrhage.

Answer 164

Anticoagulant drugs interfere with the coagulation cascade and prevent formation of thrombin, which converts fibrinogen to fibrin. 

Answer 165

Fibrinolytic drugs promote lysis of clots by increasing formation of plasmin, a serine protease that degrades fibrin. 

Answer 166

Anti-platelet drugs inhibit formation of platelet products or block platelet adhesion thus preventing platelet aggregation and clot formation.

Answer 167

Occurs naturally in the granules of mast cells. For therapeutic use it is extracted from porcine intestine or bovine lung.

Answer 168

includes unfractionated heparin, low molecular weight heparin, and fondaparinux

Answer 169

Proteoglycan containing covalently linked sulfated polysaccharide chains of varying length. Mean molecular weight of 12,000 daltons. It has the highest negative charge density of any known macromolecule.

Answer 170

(lovenox, enoxaparin, dalteparin, nadroparin) - Produced by chemical or enzymatic depolymerization of heparin to one-third the size of heparin (~4500 Daltons).

Answer 171

Synthetic pentasaccharide corresponding to the minimal sequence in heparin for binding antithrombin.

Answer 172

Heparins bind to antithrombin III, a natural protease inhibitor present at high concentrations in plasma that inactivates coagulation factors. Binding of heparin to antithrombin III greatly increases the rate of thrombin inactivation by 1000X. Heparin also accelerates the rate of decay of IXa, Xa, and XIIa by antithrombin III. Once thrombin is bound to the antithrombin /heparin complex, heparin is released and can be reused. The inactivation of coagulation factors by heparin prevents the conversion of fibrinogen to fibrin by thrombin. Differences in the mechanism of action of unfractionated heparin, LMWH and fondaparinux is due to polysaccharide chain length. Only heparin containing at least 18 saccharide units (>5400 daltons) binds to the antithrombin/ thrombin complex. Low molecular weight heparin (<5400 dalton) and fondaparinux do not inhibit thrombin by antithrombin but selectively bind to and inactivate factor Xa by antithrombin.

Answer 173

is not absorbed from the GI tract so it cannot be given orally. It is given I.V. for an immediate effect or sub-cutaneously for a delayed effect. has poor pharmacokinetics, poor bioavailability and relatively short half-life (1-5 hr) resulting in an unpredictable dose response. requires hospital admission and careful monitoring. does not cross the placenta therefore it is the drug of choice during pregnancy.

Answer 174

are given subcutaneously and effects are more prolonged due to a longer half-life. have better bioavailability and more predictable dose response than unfractionated heparin and require less monitoring (outpatient treatment).

Answer 175

Venous thrombosis and pulmonary embolism (rapid onset of action). Used with oral  anticoagulants and fibrinolytic drugs. Fondaparinux recently approved for this use. Management of unstable angina or acute myocardial infarction. During and after coronary angioplasty or stent placement. During surgery requiring cardiopulmonary bypass. Kidney dialysis  

Answer 176

Bleeding - The anticoagulant effect of heparin disappears within hours of discontinuation of the drug. If life-threatening hemorrhage occurs, the effect of heparin (and LMWH) can be reversed with protamine sulfate, a positively charged compound that neutralizes heparin. Heparin-induced thrombocytopenia syndrome (HIT)

Answer 177

Platelet count decreases (>50%) in 3-5% of patients 5-10 days after heparin. Caused by development of antibodies to platelet factor 4/heparin complexes. The antibodies bind to and activate platelets resulting in a prothrombotic state (venous thromboembolism, arterial thrombosis, myocardial infarction, stroke). Thrombocytopenia is less common with LMWH.

Answer 178

Argatroban (Novastan) - A small molecule inhibitor. | Lepirudin (Refludan) - Recombinant form of hirudin, the anticoagulant from leeches.

Answer 179

Due to the contaminant oversulfated chondroitin sulfate (rarely if ever found in nature). Activation of the contact system (production of bradykinin and complement activation). Development of improved screening methods

Answer 180

includes warfarin (Coumadin) and new oral anticoagulants (direct thrombin or factor Xa inhibitors).

Answer 181

the most commonly used oral anti-coagulant.

Answer 182

Warfarin is a derivative of dicumarol, which was discovered as a component in spoiled sweet clover that was responsible for a hemorrhagic disease in cattle. Warfarin (Wisconsin Alumni Research Foundation) initially was made (1948) for rodent control. It is a vitamin K analogue. Warfarin inhibits enzymes that use vitamin K as a cofactor. Several coagulation proteins (II, VII, IX and X) undergo vitamin K-dependent gamma carboxylation of N-terminal glutamates. Vitamin K undergoes oxidation/reduction during the reaction. The recycling of vitamin K to the reduced form by reductases is inhibited by warfarin resulting in depletion of vitamin K. Coagulation proteins lacking gamma carboxylation cannot bind calcium and are non-functional.

Answer 183

It is rapidly absorbed (90 min), has good bioavailability and a long half-life (36-48 hr). The full antithrombotic effect of warfarin is not achieved until the existing coagulation factors in the circulation are removed (requires 2-3 days).

Answer 184

Venous thromboembolism (in combination with heparin, which acts more rapidly). Systemic embolism in patients with prosthetic heart valves or atrial fibrillation. Stroke, recurrent infarction, or death in patients with acute myocardial infarction.  

Answer 185

Hemorrhage. The drug is stopped and vitamin K is administered (takes 24-48 hr for  reversal since coagulation factors have to be resynthesized). Plasma can be  transfused to replace coagulation factors. Warfarin cannot be used during pregnancy since it crosses the placenta and is  teratogenic

Answer 186

Drugs that inhibit platelet function (aspirin). Drugs that decrease vitamin K synthesis by intestinal microbes (antibiotics). Drugs that displace warfarin from plasma proteins. This effectively increases the  concentration of active free drug (clofibrate, phenytoin). Drugs that reduce the metabolism and elimination of warfarin in the liver  (cimetidine, amiodorone, phenylbutazone).

Answer 187

Drugs that increase the metabolism by inducing metabolic enzymes in the liver (barbiturates, rifampin). Drugs that decrease warfarin absorption from the GI tract (cholestyramine).  

Answer 188

Direct thrombin or Factor Xa inhibitors

Answer 189

Rapid onset of action, Absence of food interactions. Do not require monitoring Disadvantages of direct thrombin or factor XaContraindicated with kidney disease. Greater GI bleeding than with warfarin. Short half-life. Cost (20X>warfarin). No antidote available to reverse effects

Answer 190

FDA approved 2010 in atrial fibrillation. Oral prodrug converted to dabigatran, a potent, direct inhibitor of thrombin. Lower rates of stroke and systemic embolism than with warfarin. Less intracranial hemorrhage (but increase in myocardial infarction). Should not be used in patients with prosthetic heart valves (inferior to warfarin)  

Answer 191

FDA approved 2012 for atrial fibrillation 

Answer 192

FDA approved 2011, 2012 for atrial fibrillation, VTE. Synthetic anti-coagulants that directly inhibit Factor Xa. Superior to warfarin in preventing strokes and emboli for treatment of atrial fibrillation

Answer 193

Convert plasminogen to plasmin, a protease that degrades fibrin clots. Includes t-PA, urokinase (u-PA0, and streptokinase

Answer 194

Serine protease produced by recombinant DNA technology. Newer modified forms can be given as bolus injections and have prolonged half-life (retoplase, lanoteplase, tenecteplase). The t-PA type fibrinolytic drugs bind fibrin, which increases cleavage of plasminogen to plasmin.

Answer 195

Enzyme obtained from renal cells in culture that converts plasminogen to plasmin. Does not bind fibrin.

Answer 196

Non-enzymatic protein obtained from hemolytic streptococci (less expensive than t-PA). It forms a complex with plasminogen, which becomes activate and converts to plasmin.

Answer 197

Acute myocardial infarction (AMI) - Used in combination with aspirin. Ischemic stroke. Effective if administered within 3 hr after stroke. Deep vein thrombosis. Used in conjunction with heparin/warfarin treatment. Pulmonary embolism.  

Answer 198

Hemorrhage from the lysis "physiologic thrombi" at sites of vascular injury. Induce a systemic lytic state due to the increased formation of plasmin which destroys other coagulation factors (V,VIII). Allergic reaction and formation of antibodies to streptokinase.

Answer 199

Platelets provide the initial hemostatic plug at sites of vascular injury and contribute to pathological thrombi. Antiplatelet drugs are used to treat acute coronary syndrome (angina, myocardial infarction) characterized by atherosclerotic plaque rupture and platelet-mediated thrombosis. Three classes of anti-platelet drugs inhibit formation of platelet products (aspirin), prevent activation/aggregation (ADP receptor antagonists), and block adhesion proteins (glycoprotein IIb/IIIa inhibitors).

Answer 200

Irreversibly inactivates cyclooxygenase preventing thromboxane A2 formation by  platelets. Since platelets cannot synthesize new proteins (i.e. cyclooxygenase) the effect is  permanent, lasting the life of the platelet (7-10 days). Aspirin is used in combination with thrombolytic therapy after AMI and thrombotic  stroke. Used for prevention of AMI and stroke in high-risk patients with atherosclerosis.

Answer 201

ADP receptor antagonists. Oral antiplatelet agents that bind to ADP receptor (P2Y12) on platelets. They block platelet activation by ADP, which inhibits secretion of alpha granules and blocks expression of adhesion proteins GPIIb/IIIa. They are rapidly absorbed but have slow onset of action (maximum effect in 5-7 days) because they are prodrugs that are metabolized in the liver to the active intermediate (variable patient responses). The thienopyridines bind irreversibly to platelets and their effect lasts for the platelet life span (7-10 days). Used for preventing cardiac events in patients with atherosclerosis and unstable angina in combination with aspirin. Used for patients with aspirin intolerance. Clopidogrel has fewer adverse effects. Prasugrel (approved 2009) is the most potent and shown in clinical trails to be better than clopidogrel in preventing death and MI (increased risk of bleeding and cancer).

Answer 202

(approved 2010) Oral ATP analogue that binds reversibly to the ADP receptor P2Y12. More rapid action than thienopyridines since it does not require metabolic  activation. Greater platelet inhibition than clopidogrel. PLATO results (2009) comparing ticagrelor with clopidogrel for treatment of acute  coronary syndrome showed that ticagrelor reduced the rate of death without an increase in overall major bleeding.

Answer 203

Glycoprotein IIb/IIIa is an adhesion protein (integrin) on the surface of platelets that is a receptor for fibrinogen. The GPIIb/IIIa inhibitors block the receptor and prevent platelet aggregation. Adverse effects include bleeding and thrombocytopenia. These can be reversed by platelet infusions.

Answer 204

Monoclonal antibody fragment against the glycoprotein receptor, GPIIb/IIIa. It is used following coronary angioplasty and for unstable angina. Prevents restenosis, recurrent AMI and death when used with aspirin and heparin. Also used in combination with thrombolytic drugs for AMI. Given i.v.

Answer 205

A cyclic peptide inhibitor of a binding site on GPIIb/IIIa that is used for unstable angina and during angioplasty. 

Answer 206

Small molecule inhibitor used for treatment of unstable angina and myocardial infarction.

Answer 207

diagnosis is made by detecting infection with HIV-1, the AIDS virus. People are ‘seropositive’ if they have antibody to HIV, which is the most common way in which infection is first detected; once they get symptoms of opportunistic infections or Kaposi’s sarcoma, or their Th (CD4+) cells fall below 200/μL of blood, it’s AIDS. (Normal range: 500-1000/ μL)

Answer 208

AIDS is caused by a virus called HIV-1, for Human Immunodeficiency Virus. HIV-2 has been isolated in West Africa, but has not gone global. HIV is a nontransforming retrovirus, that is, an RNA virus that carries no oncogene, and reproduces itself by copying its RNA into DNA by means of its own enzyme, reverse transcriptase. It is similar to visna virus of sheep, equine infectious anemia virus, and the feline immunodeficiency virus, all of which cause slow, ultimately fatal illnesses, and so the group are referred to as lentiviruses. It is most closely related to a Simian Immunodeficiency Virus, SIV. It is thought that HIV-1 evolved recently from SIV, perhaps as recently as the 1940s in Zaire (now the Democratic Republic of Congo). The first sera in the USA with antibody to HIV-1 are found in 1978; in Africa, some sera from 1959 are positive, and HIV-1 sequences have been cloned from a blood sample of 1959 from D.R. Congo. Thus this seems to be a relatively new virus, which has jumped from simian to human and not yet adapted to its new host (similar in that respect to Ebola and Marburg viruses). Very recent data suggest that the virus’s virulence may be declining in at least some parts of Africa.

Answer 209

the most antigenically variable pathogenic virus we have encountered. Reverse transcriptase is a highly error-prone enzyme, without proofreading capability. It makes a mistake about once in 100,000 base replications, so infected people have many variants in their body.

Answer 210

It is sexually transmitted so frequent sex is risky if it involves partners who might have the virus themselves. Any lesion on or injury to mucous membranes increases risk. Injection of blood containing virus is highly risky, although much less so than with blood containing hepatitis virus. In over 3000 reports of accidental exposures of health care workers in the USA to HIV, only nine were documented to have become antibody-positive. We do not think that use of drugs per se is risky, nor use of sexual stimulants like amyl nitrite. Heterosexual contacts now account for more than half of new cases worldwide, and more than half of those are women and girls.

Answer 211

In the years before the first HIV drug (1981-1987) 95% of all infected people died of tumors or opportunistic infections. A few were identified as “long-term survivors” (LTS). Of these, some are homozygous for a 32-base pair deletion in the gene for a chemokine receptor, CCR5 (they were CCR5D32). CCR5 is an HIV coreceptor. The mutated allele occurs at a 10% frequency in Caucasians, but is very rare in other populations. A different group of LTS were “elite controllers.” They became infected but did not progress to AIDS. Two-thirds of them have the HLA-B57 allele. They make effective CTL to HIV peptides presented in HLA- B57. There is a good correlation between HIV-specific CTL numbers and prognosis.

Answer 212

spherical enveloped virion with a central cylindrical nucleocapis. At the virion core lie 2 identical ssRNA pieces (a dimer). Associated with these are a nucleocapsid (NC) proteins bound to the RNA and the 3 essential retroviral enzymes, protease, reverse transcriptase and integrase. Surrounding the RNA dimer lies the capsid shell which has icosahedral symmetry. The proteins that constitute this chell are called capsid proteins (CA). the major capsid protein is p24 (this can be measured in the serum to detect early HIV infection). The rest of the virus has the same structure as influenza. Proteins under the envelope are called matric proteins. These proteins serve to hold the glycoprotein spikes that traverse the lipid bilayer membrane (envelope). The surface glycoproteins are gp 120 and gp41.

Answer 213

all retroviruses posses, in their RNA genome, two endng long terminal repeat (LTR) sequences, as well as the gag, pol, and env genes.

Answer 214

flank retroviral genome and serve as sticky ends and promoter/enhancer function. Sticky ends are the sequences, recognized by integrase, that are involved in insertion into the host DNA. Transposons, moble genetic elements, have similar flanking DNA pieces. Promotor/enhancer function allows proteins to bind to LTRs after they are incorporated into the host DNA to modify viral DNA transcription.

Answer 215

codes for proteins inside the HIV envelope: nucleocapsid (NC), capsid (p24), and matrix (MA) proteins that are antigenic.

Answer 216

encodes the vital protease integrase and reverse transcriptase enzymes. The only way the retroviridase maintain their current pol position in the race to cause human disease is with these unique enzymes. Protease is vital HIV enzyme that cleaves gag and pol proteins from their larger precursor molecules (post translational modification). New drugs have been developed that block the action of the HIV enzyme, protease. Therapy with these protease inhibitors reduces HIV levels and increase CD4 T-lymphocyte cell counts.

Answer 217

codes for the envelope proteins that, once glycosylated, form the glycoproteins spikes gp 120 and gp 41. Together they are called gp 160 and bind to CD4 receptors on T cells. There are hypervariable regions in this protein where point mutations occur in multiples of 3 to preserve the reading frame. (other genes have this too, including reverse transcriptase)

Answer 218

After a single exposure, infected people develop high blood virus levels (>105 copies/mL) that peak at about 6 weeks. There is a loss of CD4 cells in the gut mucosa, and an associated increase in gut permeability. HIV spreads systemically. Antibody to HIV peaks by 9 weeks, whereupon virus levels fall sharply, but not to zero. This new level is the patient’s “set point” and it seems to reflect the abilities of their immune system, rather than those of the virus. The mean incubation period (infection to AIDS) estimated from transfusion-acquired HIV infection, where it could be most precisely timed, it was about 9.5 years without treatment. When the virus enters the body, it may adhere to a lectin on dendritic cells called DC-SIGN. Taken up by this means it is not harmed, and thus uses the DC as a Trojan horse to get to the lymph nodes where the Th are.

Answer 219

HIV binds by its envelope glycoprotein, gp120, to the CD4 molecule on the surface of Th cells. This induces a conformational change in gp120, which allows it to now bind a co-receptor, one of the chemokine receptors, CCR5 or CXCR4. When a person is first infected, almost all the virus is CCR5-tropic. In turn, binding the chemokine receptor changes the conformation of the gp41 glycoprotein that is associated with gp120, exposing a very hydrophobic region that literally melts away the T cell’s membrane, so the cell and virus fuse. The virus can thus inject its core into the cell, activate its reverse transcriptase and make a double-stranded DNA copy of its RNA. The DNA moves into the nucleus. Helped by a viral integrase, it is then inserted into a random break in the host cell’s DNA as latent virus. We know little about how latency is regulated, or whether it is harmless to the cell. It may be that HIV goes latent in resting cells and replicates productively in activated ones. By using all three reading frames, the small HIV genome (9749 bases) encodes 9 genes: the gag, pol, and env genes that all retroviruses have, and 6 others that regulate latency and virulence. By alternative RNA splicing, and protease-mediated cleavage of 3 large precursor proteins (HIV makes its own protease) it can make 16 polypeptides.

Answer 220

may die rapidly; become persistent virus-producers; or enter latency. In the first case, as viruses bud en masse from the infected cell, they tear so many holes in the membrane that the cell dies. In the early, pre-AIDS stage of the disease, the clearance rate of virus and the replacement rate of CD4 cells are incredible: it has been estimated that the entire population of virus is replaced daily, and CD4 cells every 3 days. A very significant behavior of the virus is this: when the virus is replicating, gp120/gp41 is made early, and it becomes inserted into the cell’s plasma membrane. This allows fusion of the infected cell to nearby uninfected CD4 cells, and a syncytium forms. In this way the virus can spread without an extracellular phase. This could be part of the reason the antibody patients make seems to be useless. With time, CD4 cells are gradually lost; it looks like simple exhaustion of the ability to make more of them. This is commonly expressed as a falling blood CD4/CD8 ratio (the normal ratio is from ~1.5 to 3). An accelerating fall in this ratio, or an absolute CD4 count below 400/mL, are poor prognostic signs. When the immune system can no longer cope, opportunistic infections take hold. During the long seropositive period, the major site of HIV persistence is memory Tfh cells in the lymph nodes. They are able to suppress viral replication but not eliminate the virus DNA from their nuclei. If such a cell chances to get activated by its correct antigen, it will develop into a clone of virus-producing cells. This disruption of Tfh function leads to, or is accompanied by, a gradual dysregulation of B cells, which early on can be hyperactivated, and later become exhausted so that antibody production begins to decline.

Answer 221

The most common condition is to be seropositive without symptoms, as are about a million people in the United States. After the acute infection, there is a phase of clinical latency—viral infection without symptoms—that may last years. Good therapy can keep the next stages from developing. Next would be the development of a minor opportunistic infection (OI) like Candida albicans (a yeast) of the mouth, esophagus, or rectum. There may be fevers, night sweats, weight loss and fatigue. With the appearance of major opportunistic infections (including TB) or malignancy (commonly Kaposi sarcoma, less commonly Burkitt lymphoma or other lymphoma), or an absolute CD4 count below 200, the full-blown AIDS picture is present. This progression, as we said, is with treatment no longer inevitable. Because the brain also has cells that can be infected by HIV, including macrophages and microglia, there is a not-uncommon late AIDS dementia complex which is terribly distressing for patients and family. It is probably the consequence of toxic cytokine release by virus-activated phagocytes.

Answer 222

primarily of types that require T cell-mediated immunity, as might be expected given the virus’ primary target. We see viral infection, including cytomegalovirus, hepatitis and especially herpes simplex and zoster. We see fungi, especially Candida albicans and Pneumocystis jirovecii. Protozoan infections, such as Toxoplasma, Cryptosporidium (which causes a sometimes-fatal diarrhea), and Isospora are very serious. Infections with opportunistic intracellular bacteria¾usually Mycobacterium avium complex or MAC, and more and more commonly, M. tuberculosis¾are frequent. In fact, TB is the leading cause of death in people infected with HIV. High-grade, extracellular bacterial pathogens are less of a problem, possibly because the ability to make Tfh-independent antibody responses to capsular polysaccharides is preserved.

Answer 223

is a tumor of the endothelial cells lining lymphatics. It is caused by KSHV (Kaposi’s sarcoma herpesvirus,) also called HHV8 (human herpesvirus 8).

Answer 224

The patient will often have made the diagnosis. The most common test is for antibody to HIV. Antibody is measured by an ELISA which has a certain false-positive rate, so a positive ELISA must be confirmed by Western Blot analysis, in which standardized viral protein preparations are separated by electrophoresis, blotted and fixed to nitrocellulose, and then ‘stained’ with the patient’s antibodies, which must bind to the correct viral proteins (gp120, gp41) for the test to be considered a true positive. Very small amounts of the virus RNA itself are now detected by the polymerase chain reaction (PCR), and this is very useful for following therapy. In patients who can be gotten down to about 50 viral particles/mL or lower and kept that low, disease progression seems to be halted. The antibodies that patients make are obviously not protective. Though they bind to the virus, they do not block attachment to and infection of Th cells. There are neutralizing epitopes on the virus, but they are shielded by carbohydrate and not readily available to B cells. Typically, if a patient does make neutralizing antibody, the virus rapidly mutates and escapes. But broadly- neutralizing antibodies are possible.

Answer 225

Reverse transcriptase (RT) is unique to retroviruses, using their RNA template to create DNA, so it’s a target. There are two classes of RT inhibitors: nucleosides (NRTI), which are competitive inhibitors and chain-terminators; and non-nucleoside (NNRTI) inhibitors, which bind a hydrophobic pocket on the enzyme that changes the conformation, and thus the activity, of the catalytic site. Because escape from inhibition due to mutation is so common, using each of these classes of drugs together greatly lowers the odds of escape.

Answer 226

The gag, pol, and env proteins are made as a single gag-pol-env polyprotein which the virus cleaves using its own protease, which therefore has become a drug target for protease inhibitors.

Answer 227

binds to part of gp41 so that it cannot change conformation to fuse the viral membrane with the helper cell’s. It is a small peptide fusion inhibitor.

Answer 228

a small-molecule CCR5 antagonist that blocks viral entry into CD4+ cells. It binds to the transmembrane portion of CCR5, causing changes in the conformation of the external receptor so that it no longer engages gp120.

Answer 229

When the viral DNA copy reaches the nucleus, a viral integrase function, part of the RT complex, inserts it randomly into the cell’s DNA. Raltegravir, an integrase inhibitor, which received FDA advisory panel approval in 2007, blocks that step, and has been shown to be effective in patients with RT inhibitor-resistant strains of HIV.

Answer 230

combines two NRTIs and a third drug from a different class; in the typical first-line formulation that would be an NNRTI. These can all go in a single one-a-day pill, which increases compliance considerably. The cost of caring for an AIDS patient exceeds $25,000/year in the USA, which is of course greater than the health budgets of most of the world’s villages. The ethical and practical problems surrounding trials and prices of new drugs, especially in the Third World, are formidable. However, several generic pharmaceutical companies around the world have defied US and other patent laws and prepared cheap 3-drug combination ART pills that are available in sub-Saharan Africa for about $100- $150/year. Only about 9.7 million of the 35 million HIV+ people get them (but that’s up from 50,000 in 2002).

Answer 231

Safe sex, safer addictions. You don’t get AIDS from casual contacts. Male circumcision is very effective and a growing practice in parts of Africa. Condoms work if they are used, and stay intact. Spermicides don’t, but an anti-HIV drug (tenofovir) incorporated in a barrier gel had partial effectiveness in a South African trial in 2010. Prophylactic ART protects the non-infected member of a couple, and ART therapy to pregnant HIV+ mothers protects the fetus. The virus is not hardy, and common disinfectants (alcohol, Clorox) kill it readily. Health care practitioners should use hepatitis precautions. If you do flow cytometry or even centrifuge human blood, be sure you understand the production of aerosols by your machine. All HIV seropositive people should be treated with ART. Early treatment probably increases survival and definitely decreases transmission. In November 2010 a trial of a two- reverse transcriptase inhibitor pill found that highly sexually-active men had a 44% decrease in HIV infection compared to placebo (both groups were instructed in other prevention strategies.) Compliance was a problem; the most compliant subjects were protected much more effectively.

Answer 232

Although there was initial excitement, a large vaccine trial fizzled (May 2003); it produced good antibody responses but did not, overall, decrease infection rates. Because we need a vaccine that can preferentially stimulate Th1 cells and CTL; the current vaccines seem to be best at inducing antibody responses, and antibody doesn’t protect very well (otherwise, seropositive people wouldn’t get sick). In 2009, a large trial (called RV144) in Thailand reported significant protection for the first time, though the effect was disappointingly modest. The key epitope on HIV seems to be well-concealed within the gp120/gp41 complex and almost invisible to B cells. However, it has been shown many times recently that a small amount of the antibody about 20% of HIV-positive people make is broadly neutralizing (bnAb); it can block infection by almost all HIV strains and mutant forms. The most interesting are ones directed against the site on the gp120 that binds to CD4; this site can’t mutate much, because if it did, the virus would no longer be infective.

Answer 233

1. HIV exhibits tremendous global genetic diversity. 2. Its immense mutational capacity allows evasion of both T and B cell immunity. 3. HIV goes latent in the host genome, from which it cannot be eliminated by conventional antiretroviral drugs. 4. There has been no known example of spontaneous immune clearance, to use as the basis for data-driven vaccine design. 5. Although bnAbs have been found, they are rare, only found in a subgroup, take years to develop, and are extensively hypermutated; no method exists now for induction of these Abs by immunization. 6. But a lot of smart people think that they can figure out a way to make these epitopes immunogenic in everybody.

Answer 234

1. People with immunodeficiencies, particularly of T cells, have a higher incidence of tumors, e.g. AIDS patients have a higher rate of Kaposi sarcoma, Burkitt lymphoma, and some other tumors. Organ transplant recipients taking powerful immunosuppressive drugs (and therefore immunodeficient) had a 25 to 100-fold increase in tumors relative to healthy controls. People treated with chemotherapy may have a 14-fold increased risk of developing secondary leukemia. 2. Activated T cells that recognize tumor-associated antigens can easily be identified. The presence of lymphocytes in a tumor (tumor-infiltrating lymphocytes or TIL,) many of which are tumor-specific, is a good prognostic sign. 3. A small percentage of tumors, mainly melanomas and some lymphomas, spontaneously regress, presumably due to an immunologic response. There are limitations to the hypothesis, however. First: the tumors that immunodeficient and immunosuppressed people get are not a random sample of all the tumors that can happen; rather, they tend to be tumors of the lymphoid system, and of the skin, but rarely lung or breast. Second: Nude mice (mice with no thymus) should get tumors very readily, but in fact spontaneous tumors are rare in these mice. Probably because these mice have very high levels of natural killer (NK) cells, which are not part of the traditional (T and B cell) immune system but as you know can be quite tumoricidal, directly or via ADCC.

Answer 235

a dynamic process that consists of immunosurveillance and tumor progression. It is made up of three phases: elimination, equilibrium, and escape.

Answer 236

also known as immunosurveillance, includes innate and adaptive immune responses to tumour cells. For the innate immune response, several effector cells such as natural killer cells and T cells are activated by the inflammatory cytokines, which are released by the growing tumour cells, macrophages and stromal cells surrounding the tumour cells. The recruited NK cells macrophages produce interleukin 12 and interferon gamma, which kill tumour cells by cytotoxic mechanisms such as perforin, TNF-related apoptosis-inducing ligands (TRAILs), and reactive oxygen species. Tumor cells exhibit a variety of metabolic abnormalities compared to normal cells, and these can lead to the expression of DAMPs which activate innate immunity. Cytokine secretion and antigen presentation on dendritic cells activate T cells, and so macrophages and cytotoxic T cells infiltrate the tumor. If the abnormal clone is successfully eradicated, the process ends.

Answer 237

The next step in cancer immunoediting, during which tumor cells that have escaped the elimination phase and have a non-immmunogenic phenotype are selected for growth. It is the longest of the three processes in cancer immunoediting and may occur over a period of many years. During this period of Darwinian selection, new tumor cell variants emerge with various mutations that further increase overall resistance to immune attack. In most clinically relevant tumors, lymphocytes infiltrate the tumor, but do not fully destroy it. Instead the tumor and lymphocytes exist in equilibrium. This may be analogous to the situation with Epstein-Barr virus in the bone marrow, or Varicella in dorsal root ganglia; long as the immune response is strong the virus is kept in latency. But biologic equilibria are dynamic, and changing conditions—the host’s immunity drops for some reason, or further mutations accumulate in residual tumor cells—can eventually lead to reactivation.

Answer 238

During the escape phase, tumor cell variants selected in the equilibrium phase have breached the host organism's immune defenses, with various genetic and epigenetic changes conferring further resistance to immune detection. It’s been known for many years that tumors fight back when the immune system attacks them. For example, it is common to find tumor-specific CTLs surrounding tumor clusters in biopsies. But they aren’t able effectively to kill the tumor cells. In the past decade it’s been found that CTL have at least two “checkpoint inhibitor” surface receptors which, if engaged by corresponding ligands on the cell they are interacting with, signals downregulation of their cytotoxic activity. These clearly play a normal role in immune regulation. But it’s easy to imagine that when CTL first arrive, they kill most of the tumor cells, but a few cells that happen to have upregulated the inhibitory ligands escape. They are the ones that grow out, and with time, the entire tumor will be made up of CTL-resistant cells. The checkpoint inhibitors are CTLA-4 and PD-1. Tumors evolve many escape mechanisms. Some modify their tumor-associated antigens (see below) until the host does not have T cells against them with highly avid receptors. Others make immunosuppressive factors like TGFb, or convince invading macrophages to become immunosuppressive. Some shed tumor antigens to “decoy” CTLs. And almost all tumors, as they progress, reduce the expression of MHC Class I so there is less and less for CTL to recognize.

Answer 239

All tumor cells can be shown to have antigens that are not readily found on the corresponding normal cell. Often they are found on normal cells, but in much lower quantities; they are overexpressed or abnormally expressed by the tumor. Such antigens are called tumor-associated antigens (TAA). A subclass of TAA are those that can be recognized by the immune system, in a way that could lead to the destruction of the tumor. Such antigens are called tumor rejection antigens.

Answer 240

Many tumors are known to be caused by tumor viruses; in humans about 20% of tumors are caused directly or indirectly by viruses. Especially noteworthy are HTLV-1 and -2 that have been strongly implicated in Sézary syndrome/mycosis fungoides as well as the similar epidemic lymphoma in Japan and the Caribbean. Cervical cancer (human papilloma virus) is currently the best-known virally-induced tumor in humans; one hopes the HPV vaccine will make it less well known. Quite a lot of liver cancer in the developing world follows a hepatitis virus infection. Epstein Barr Virus can induce Burkitt lymphoma and nasopharyngeal carcinoma.

Answer 241

Chemical and physical carcinogens lead to cellular transformation. Mutated proteins will be processed and presented to the immune system. Since the mutations contributing to the development of tumors are not always identical from patient to patient, immunotherapy designed against these antigens may not be as generalizable as might be with viral or normal gene products. These antigens are called tumor-specific antigens, TSA.

Answer 242

are made in normal fetal tissues. They are not found in the normal tissues of adults, but can be re-expressed in the tumor. The most familiar is carcinoembryonic antigen (CEA), found in the blood of patients with colon carcinoma and other cancers. There are commercially available kits to detect CEA in blood. They should not be used as a routine screening test because there are too many false positives. The proper use of CEA measurement comes when you have a high index of suspicion of colon cancer; or, when such a cancer has been removed, to confirm complete excision (levels fall to 0 and remain there) or to warn of recurrence.

Answer 243

These lineage-specific tumor-associated antigens can be greatly overexpressed in tumors, and they represent the most frequently identified TAA. The best studied are those from malignant melanoma (tyrosinase, gp100, MelanA/MART-1). In 30% of breast and ovarian cancers overexpression of the human EGFR-2 gene product (HER-2/neu) is observed. Therapeutic antibody and T cell responses to HER-2/neu can be induced. Prostate-specific antigen (PSA) appears in the blood of many men with prostate cancer, and its detection is used in screening programs, though its utility as a guide for treatment has come into question.

Answer 244

Expressed uniquely on the malignant clone. The most familiar example would be the idiotype of the surface immunoglobulin in B cell malignancies, or of the TCR in T cell malignancies. These are attractive prospects for therapeutic vaccine development.

Answer 245

These CD8+ T cells (CTL) are probably the most important cells in tumor resistance. CTL can recognize TAA presented by MHC class I. Naive T cells are activated in the lymph nodes, not at the tumor site, via interactions with antigen-presenting cells such as dendritic cells. Following the initial activating event, the CD8+ T cells undergo clonal expansion and acquire lytic function. Activated TAA-specific T cells leave the lymph node and migrate to the tumor. CTL can kill tumor cells by inducing apoptosis via either perforin or Fas-mediated pathways. The CTL also secrete IFNg upon engagement of their TCR, which attracts and stimulates macrophages. However, patient-derived T cells that recognize TAAs are often ineffective in controlling tumor growth. A seminal study by Lee et al. examined the biological properties of T cells specific for melanoma antigens from patients. Although the T cells divided in the presence of tumor cells, they did not produce cytokines nor were they cytotoxic. This abnormal behavior is not inherent in the T cell, but is induced by the tumor.

Answer 246

These CD4+ T cells recognize the tumor antigens, make lymphokines, and attract angry M1 macrophages. How can we make people with tumors get this system going better than it is? Th1 stimulation may be part of the goal for the development of therapeutic cancer vaccines (remember, Th1 also help CTL get activated.) Tumors frequently protect themselves by creating an environment in which M2-like, not M1, macrophages are favored. M2 seem to protect tumors, even encourage their growth.

Answer 247

NK cells look like large lymphocytes, but have peculiar granules in their cytoplasm, so they are usually called LGLs (large granular lymphocytes). They do not need to come from an immunized host to recognize and destroy quite a wide range of tumors, mostly of hematopoietic origin. Thus they are part of innate immunity. They recognize a small number of “stress-related” markers on tumor cells, using a small number of NK receptors which are neither immunoglobulin nor TCR. NK cells are down-regulated if the target cell expresses Class I MHC. It’s like the body knows that if there’s a lot of Class I, that make a CTL target, so NK needn’t bother; but if the tumor decreases Class I, thinking to evade CTL, then it becomes a NK target.

Answer 248

can be activated in vitro with foreign products (e.g., bacteria) to kill tumor cells. Much of this antitumor activity can be attributed to the cytokine TNF. However, tumors frequently learn to subvert macrophages and even recruit them to support tumor growth (turning M1 into M2, for example).

Answer 249

An antibody response is commonly made in tumor-bearing hosts, but it is not commonly effective. Opsonization of tumor cells by antibody and complement can kill some leukemias in vitro, but a strong B cell response to tumor antigens does not seem to correlate with resistance to the tumor. The cells of the tumor that survive immunoediting are likely to have downregulated antigen expression as much as they can. Others have become resistant to complement, or can inactivate it.

Answer 250

a.k.a., a tumor vaccine. There hasn’t been much success in this area but as more defined TAAs are being identified, keep an eye out for breakthroughs. Initially the vaccines will be therapeutic, not preventative. The most interesting vaccine experiments use the patient’s own dendritic cells mixed with tumor extracts or purified antigens as highly potent immunogens. One such treatment is the first to be approved (April 2010) by the FDA; it combines the patient’s own dendritic cells with a proprietary fusion protein containing the prostate cancer TAA prostatic acid phosphatase. Called Provenge (sipuleucel-T), it extended survival in phase III tests, but a series of immunizations costs $93,000. Some people are designing improved epitopes with higher affinity to MHC or to the TCR, or both, than the ones that the tumor itself chooses to use. With costly drugs like this, the Incremental Cost Effectiveness Ratio is unfavorable, but if biomarker studies can identify the subset of patients who are most likely to get real benefits, the ICER becomes much more attractive.

Answer 251

BCG (the tuberculosis vaccine) is injected directly into the tumor. A ferocious delayed-type hypersensitivity reaction to BCG ensues, and the tumor cells are killed as innocent bystanders. This is used to some degree in cutaneous tumors like melanoma, and BCG instilled directly into the bladder is the treatment of choice for superficial bladder carcinoma.

Answer 252

Antibody to TAAs should be useful, and quite a few monoclonal antibodies (mAb) are already available (see the Immunomodulators notes). They can be used as-is (possibly they activate complement, and the tumor is lysed or phagocytized; more likely they invoke ADCC), or they can be tagged with a poison such as ricin, or diphtheria toxin, or a radioisotope (such modified antibodies are called immunotoxins). At least one mAb is available coupled with a chelator that allows the attachment of either an imaging or a therapeutic radioisotope. Another approach is to use antibodies to growth factors or their receptors to try to inhibit autocrine (self-stimulating) tumors; one example is anti-IL-2 receptor in T lymphomas; and widely used is Herceptin, a mAb to the HER2 surface growth stimulatory molecule on some breast cancers. An mAb to VEGF (vascular endothelial growth factor) is available too.

Answer 253

An exciting development in tumor immunotherap, a human monoclonal Ab against PD-1 which binds and blocks CTL inactivation by tumors that express PD-1 ligands. There is also ipilimumab (approved 2011), an mAb blocker of CTLA-4, another downregulator of CTL activity. It would be interesting if a trial of these two together could be arranged. As more companies are developing their own versions of these mAbs.

Answer 254

This technology utilizes cells from the patient’s immune system to destroy cancerous cells that cannot be surgically removed. Cells from the immune system that have potential to fight the tumor are isolated from the patient’s blood, tumor, or lymph nodes. Cells directly from the tumor are called tumor-infiltrating lymphocytes (TIL). The T cells are expanded greatly in culture using cytokines such as IL-2. The patient’s immune system may then be partially destroyed by irradiation to make “room” for the expanded anti-tumor clones. They are reintroduced into the immune-depleted patient to kill remaining tumor cells.

blood and lymph3 Flashcards

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