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1

Secondary vs primary hemostasis

-Secondary: fibrin plug formation via coagulation cascade (joints and muscles)
-Primary: platelet plug formation via adhesion, activation, and aggregation (mucosa, ecchymosis, petechiae)

2

Defects in secondary hemostasis

-Factor defeciency (ex: factor VIII deficiency is hemophilia A- X linked)
-Increased fibrinolysis
-Decreased inhibition of coagulation
-Vit K deficiency (affects II, VII, IX, X; factor V will be normal)
-Liver disease (decreases all factor except factor VIII- made in endothelium)
-Use lab tests: PT, aPTT, thrombin time, fibrinogen level, 50/50, factor assays

3

Defects in primary hemostasis

-Thrombocytopenia (decreased production, ineffective production, increased destruction)
-The cause of thrombocytopenia parallels the cause of anemia (i.e. if the anemia is due to hemolysis, then thrombocytopenia is also due to increased destruction)
-Splenic sequestration (normally 30% of all platelets are in the spleen)
-Abnormally high amounts of platelets in spleen leads to splenomegaly
-Platelet function defects (i.e. missing or mutated receptors)
-vWD (vonWillibrand's disease) leads to primary hemostatic defects
-Dilution of platelets (many transfusions)
-Vascular defects
-Lab tests: platelet count, PFA100 (or bleeding time), vWF:Ag

4

Thrombin time

-Test to only look at conversion of fibrinogen to fibrin by thrombin
-Only tells you if there is low fibrinogen, abnormal fibrinogen, or an inhibitor of thrombin (heparin, Ig, paraproteins, FDPs, ect)
-Doesn't distinguish btwn these results, so must do fibrinogen level assay to see if there is a quantitative defect (hypofibrinogenemia)

5

Prothrombin time (PT)

-PT test only looks at the extrinsic + common coagulation pathways
-This test is insensitive to thrombin inhibitors like heparin, since you overwhelm the system w/ tissue factor (TF) to induce coagulation
-Tests for the availability/function of factors VII, V, X, and II
-Also will be prolonged w/ hypo/dysfibrinogenemia (qualitative defects in fibrinogen)
-If PT test and aPTT are both prolonged, then may have defect in common pathway
-If PT test is prolonged but aPTT is normal then think factor VII problem (deficiency or Ab)
-Do a 50/50 mix to identify if its deficiency or inhibitor (Ab)

6

Activated partial thromboplastin time (aPTT)

-Looks only at intrinsic + common coagulation pathways
-Is sensitive to heparin since there is no overwhelming stimulus, like in PT test
-The aPTT can be prolonged due to XII/HMWK/PK (prekallikrein) deficiencies, but these will not cause bleeding
-If PT and aPTT are both prolonged, think common pathway problem
-If PT is normal but aPTT is prolonged, think factor XI, IX, or VIII problem
-Deficiency vs inhibitor can be distinguished by doing 50/50
-In vWD there will be prolonged aPTT b/c lack of vWF leads to clearance of VIII (VIII requires vWF for stability)

7

Platelet plug formation 1

-Starts w/ adhesion: exposure of sub endothelial collagen and vWF release due to damaged vessel, vWF binds to the exposed collagen
-vWF binds to GPIb/IX receptor on platelet
-Collagen binds to the GPIa/IIa and GPVI receptors on the platelet
-These binding processes are energy independent
-Activation occurs upon collagen, vWF, or thrombin binding to platelet
-Thrombin binds to PARs (protease activated receptors) on platelet membrane and contributes to hemostasis

8

Platelet plug formation 2

-Activated platelets use energy dependent processes that result in: actin-induced shape change, rearrangement of cell membrane phospholipids (phosphatidyl serine to outer membrane), granule release, and adjustment of membrane receptors
-Platelets express receptor IIb/IIIa, which binds vWF (in the arteries) or fibrinogen (in the veins) and causes aggregation
-Platelets also express P-selectin to promote WBC migration to site of injury (binds to WBC PSGL-1), and facilitate the fusion of microvesicle w/ platelet membrane (lets platelets present TF on their membrane)
-Platelets release granules: dense (ADP, serotonin, Ca), alpha (fibrinogen, vWF, factor V, other coag and growth factors)
-Granules amplify aggregation, promote activation, or cause coagulation cascade

9

Platelet plug formation 3

-Platelet aggregation is caused by the binding of platelets to each other through the IIb/IIIa receptor
-IIb/IIIa binds either vWF (multimeric, best in high sheer arteries) or fibrinogen (dimeric, best in low sheer veins)
-Aggregation is amplified by generation of thromboxane A2 (TxA2)
-TxA2 is an eicosanoid generated from arachidonic acid (taken from membrane by phospholipase) through the COX pathway
-Aspirin irreversibly inhibits the COX nz, while other NSAIDs reversibly inhibit it
-TxA2 will cause other platelets to express IIb/IIIa, inducing activation and aggregation
-TxA2 is also a potent vasoconstrictor

10

Platelets and coagulation cascade 1

-While this is happening, the coagulation cascade is simultaneously being activated
-Basement membrane exposed TF leads to activation of both intrinsic and extrinsic pathways (via factor VIIa activation of factor IX)
-The fibrin polymers that are produced by the coagulation cascade are covalently cross-linked together and to platelets by factor XIII

11

Platelets and coagulation cascade 2

-Many of the coagulation reactions require a membrane surface (the ones that require PL)
-These membranes are usually on the platelet surface or endothelial cells
-Platelets that bind to the sub endothelial collagen make a surface for the coagulation cascade
-Other sources of TF: non-cell associated sub endothelial TF, TF on monocytes/macrophages, fibroblasts, glial cels, blood-borne micro-vesicles (released from monocytes/macrophages in BM)

12

Platelets and coagulation cascade 3

-TF can be upregulated on the surface of cells (endothelium and monos/macs) by a number of factors: cytokines, endotoxins, GFs (VEGF)
-P-selectin on activated platelets binds to PSGL-1 on the microvesicles released by monocytes
-The microvesicles also have TF/VII bound to the surface
-When P-selectin and PSGL-1 bind, the microvesicle fuses w/ platelet membrane and the TF/VII becomes displayed on the platelet, facilitating the coagulation cascade by more TF expression

13

Disorders of excessive TF expression

-Primarily can result in disseminated intravascular coagulation (DIC) or localized thrombosis
-Cytokines released during infection induces expression of TF on endothelial cells and monocytes/macrophages
-TF can also be released from cells due to trauma or expressed on tumor cells, both can result in DIC/thrombosis
-The ability of factor VII to bind to a specific site of injury (where TF is released/expressed) and selectively enhance its function, resulting in the coagulation cascade, is the underlying theme of hemostatic plug formation

14

Tissue factor pathway inhibitor (TPFI)

-TF/VIIa complex has the ability to activate both IX and X
-However, when it binds to X the complex is inactivated by TFPI
-This requires that hemostasis must proceed through IX and VIII tenase complex (allowing for more control of hemostasis)

15

Activation of IXa and VIIIa (tenase complex)

-Factor IX bound to its receptor on endothelial cells and/or platelets gets activated by the TF/VIIa complex
-IXa can now bind to its cofactor VIIIa (which is already complexed w/ vWF) to form the tenase complex
-The tenase complex activates X to Xa (process requires negatively charged surface, such as an activated platelet membrane or a monocyte membrane)

16

Disorders of IX, VIII, and vWF

-Functional or quantitative deficiency of IX is hemophilia B (severe hemorrhagic disorder)
-Functional or quantitative deficiency of VIII is hemophilia A (hemorrhagic disorder)
-A is more common but less severe than B
-VIII must be bound to vWF or else it is cleared, thus if vWF level increases VIII levels also increase
-vWF is acute phase reactant (APR) protein, meaning its levels increase w/ inflammation
-This is one reason why chronic inflammatory diseases are associated w/ high chance of thrombosis and stroke (more VIII)
-Quantitative or functional deficiency of vWF is vonWillebrand's disease, and has similar characteristics w/ hemophilia A (since lower vWF leads to lower VIII)

17

Vitamin K dependent proteins

-The vit K dependent proteins are II, VII, IX, X, and protein C/S
-Only C/S are anticoagulants
-Vit K used to make a g-carboxyglutamic acid on these proteins
-This altered residue is essential for Ca binding and thus conformation change to induce activation
-Therefore w/o vit K the proteins cannot bind Ca and cannot be activated
-The conformation change induced by Ca leads to exposure of a consensus sequence in the amino terminus, required for factor binding to its receptors

18

Disorders of vit K metabolism

-Vit K deficiency is rare in adults
-Only occurs in those who are very Ill, not eating and receiving antibios, or in those with impaired fat absorption (such as biliary obstruciton- vit K is fat soluble)
-Vit K deficiency can occur in premature newborns resulting in hemorrhagic disease of the newborn
-Lack of vit K leads to inability of II, VII, IX, and X to undergo activation, thus predisposes a person to bleeding
-Warfarin (coumadin) inhibits the action of one of the nzs needed in the g-carboxyglutamate rxn (warfarin is vit K antagonist)
-Therefore warfarin is an anticoagulant by blocking the function of factors II, VII, IX, and X
-Overdose of warfarin can lead to excessive bleeding

19

Factor X, V and the prothrombinase complex

-Activated Xa (by the tenase complex) requires the cofactor Va (activated by trace thrombin) to form the Xa/Va prothrombinase complex
-Coag nzs are essentially inactive unless they are bound to their cofactors and receptors (such as Va, Ca, PL)
-When all of the factors are assembled (Xa/Va/Ca/PL) the prothrombinase complex activates prothrombin to thrombin to begin fibrin production and polymerization

20

Disorders of factor X and V

-Disorders of these factors are very rare and generally result in severe hemorrhagic disorders
-A mutation in V (called V Leiden) exists in some people where an Arg is substituted for a Glu
-This mutation renders factor V resistant to inactivation by protein C
-This results in an inherited thrombotic disorder

21

Protein C/S regulation of coagulation

-Factor VIIIa is required for activation of the tenase complex, and factor Va is required for activation of the prothrombinase complex
-A proteins complex (activated protein C/S: APC) is responsible for proteolysis of both factor VIIIa and Va
-APC is made from the complex of thrombin and thrombomodulin on the endothelial cell surface
-This complex binds to protein C (which is bound to its endothelial cell receptor, EPRC) and activates it to APC
-APC binds to platelets and endothelial cells in complex w/ protein S to destroy endothelial and platelet bound VIIIa and Va
-Both protein C and S are vit K dependent proteins

22

Disorders of protein C and S

-Quantitative or functional deficiencies of protein C or S can result in a congenital thrombotic disorder due to inability to inhibit coagulation
-Children born w/ complete C/S deficiency have the fatal disease purpura fulminans
-Patients w/ C/S disorders who are placed on coumadin and do not receive concomitant heparin can become transiently hyper coagulable and develop cutaneous thrombosis (coumadin necrosis)

23

Prothrombin and thrombin

-Thrombin has many functions, it is an enzyme and a mitogen (induces mitosis)
-A lack of thrombin is not compatible w/ life
-Thrombin can initiate hemostatic plug, initiate tissue repair, and maintains vascular fluidity by limiting hemostatic plug extension
-Thrombin can amplify itself by activating XI, VIII, and V
-It also is them most potent activator of platelets, causing them to aggregate and form a surface for coagulation
-These activated platelets express IIb/IIIa to bind vWF/fibrinogen (leading to aggregation) and also express receptors for factors IX and X

24

Thrombin enzymatic function

-Proteolytically cleaves fibrinogen into fibrin, releasing fibrinopeptides that polymerize by hydrogen bonding
-This is the primary (or unstable) fibrin clot
-The clot is stabilized by cross linking of fibrin polymers to each other and platelets by factor XIII
-Disorders of quantity of fibrinogen result in bleeding disorders (usually after trauma)
-Disorders of quality of fibrinogen result in either bleeding or thrombotic disorders

25

Limiting coagulation

-Mechanisms associated w/ thrombin inhibition are concentrated on surface of endothelial cells
-Antithrombin is the most important, it is only active when associated w/ heparin-like molecules (glycosaminoglycans, GAGs)
-GAGs are present on the endothelium and subendothelium, thus active antithrombin is present at site of thrombin generation
-Quantitative or qualitative deficiencies in antithrombin results in inherited thrombotic disorder

26

Factor XIII

-XIII is activated by thrombin, and covalently cross-links fibrin polymers together
-It also covalently links fibrin to platelet fibrinogen/surface proteins
-This forms the stable clot
-Disorders of XIII: quantitative or functional disorders result in delayed bleeding, that is a lesion will stop bleeding but then rebelled due to instability of the clot (wounds slow to heal, women have difficulty maintaining pregnancy)

27

Thrombin and vascular repair

-Thrombin stimulates platelets to release platelet derived growth factor (PDGF), and platelet derive endothelial growth factor (PDEGF)
-Thrombin induces monocyte/macrophage chemotaxis and activates them
-These activated cells release many nzs and factors for tissue remodeling
-Thrombin is a mitogen for smooth muscle cells

28

Thrombin and vascular fluidity 1

-Thrombin also maintains fluidity by limiting fibrin amount
-Thrombin induces endothelial cells to release protaglandins (inhibit platelet aggregation) and nitric oxide (NO; to dilate vessels and inhibit platelet aggregation)
-Thrombin induces release of tPA and uPA (plasminogen activators), which induce the formation of plasmin and thus fibrin digestion
-Thrombin induces endothelial cells and monos/macs to express uPA receptor (uPAR) increasing plasmin generation
-Thrombin induces expression of tPA receptor (annexin) and produces fibrin (which tPA binds to), both of these can activate plasmin when tPA is bound

29

Thrombin and vascular fluidity 2

-Thrombin bound to thrombomodulin is directly inhibited, but this complex also can activate protein C
-Activated protein C degrades the cofactors of coagulation cascade, VIIIa and Va
-APC also enhances fibrinolysis by inactivating plasminogen activating inhibitor (PAI1, which degrades tPA and uPA), leading to an increase in plasmin
-Heparin cofactor II (HC) directly inhibits thrombin
-Thrombin induces platelet mediated clot retraction

30

Intrinsic pathway details

-Severe factor XI deficiency can cause mild-moderate bleeding, its not required for basal maintenance of coagulation but plays a role when system is stressed (trauma)
-XIa can activate IX w/o cofactors, and XI is activated by thrombin, not XIIa
-XI provides an amplification loop when higher levels of thrombin are needed

31

Central role of thrombin

-Activates factors XI, VIII, V (procoagulation)
-Induces tPA release and receptor/cofactor binding, as well as APC activation (anticoagulation)
-PAR activation (activates platelets, regulates smooth muscle cells and vascular tone), VEGF release, metaloprotease activation (angiogenesis)
-Induction of IL6 and IL8, promotes chemotaxis and activation of monocytes (inflammation)