BL.2.2: Hemostasis

Question 1

What is hemostasis?

Answer 1

Hemostasis is the process whereby an injury to a blood vessel triggers a series of enzymatic reactions resulting in the formation of platelet and fibrin plugs at the site of the injury which then stems the loss of blood.

Question 2

Identify the elements that compose the hemostatic system. Understand the basic paradigm for coagulation factor activation.

Answer 2

Components of Hemostasis1. Coagulation factors2. Platelets3. Endothelium–vessel wall.Basic paradigm: Clotting factors circulate in inactivated form until needed. The procoagulant proteins circulate in excess concentration in the plasma in an inactive form or zymogen. Only a small percentage of the pro-enzymes need to be activated by proteolytic cleavage to form the clot.

Question 3

Describe the blood clotting pathway.

Answer 3

General idea: Endothelial wall becomes damaged, exposing collagen, tissue factor, and von Willebrand factor. Platelets bind to the site of injury and change configuration, exposing certain proteins that attract fibrinogen and vWF (thus binding more platelets). They also release granules containing thromboxane (leading to vasoconstriction of damaged vessel), vWF and ADP (to bind and activate other platelets), various clotting factors, etc. When a platelet integrin A2bB3 is activated, it binds vWF, fibronectin, and fibrinogen to accumulate with other platelets. Fibrinogen gets converted to fibrin and forms a network that is the clot.

Question 4

List which components of the system are vitamin K dependent factors.

Answer 4

Vitamin K-dependent factors: Factors II, VII, IX, and X.

Question 5

Distinguish the extrinsic and intrinsic pathways.

Answer 5

Extrinsic: tissue factor + VIIa -> Xa.Intrinsic: XIIa -> XIa -> IXa + VIII -> Xa.Common: Xa -> IIa -> Ia + XIII -> clot.

Question 6

Describe the screening tests to measure PT.

Answer 6

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.

Question 7

Describe the screening tests to describe aPTT.

Answer 7

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. Useful to detect Factor VIII, IX, XI, XII deficiencies

Question 8

Explain the function of platelets in hemostasis. Describe the process of platelet aggregation.

Answer 8

Platelets adhere to the site of vessel injury where collagen is exposed. A plasma protein, von Willebrand factor (and endothelial protein) and a platelet membrane protein (glycoprotein 6), are involved with the adherence. The platelets become activated by the generation of thrombin at the site of the injury and as the platelets are stimulated receptors for fibrinogen are exposed. These activated platelets change shape and release ADP, vasoactive amines and form thromboxane A2. Thromboxane contracts smooth muscle and causes vasoconstriction of the vessel at the site of the injury which also helps stop the leakage of blood. The platelet has receptors for some of the coagulation proteins and forms a surface for the coagulation cascade with the end result in increasing the production of thrombin.

Question 9

Explain how thrombin acts as the central regulation point for coagulation. Describe normal homeostasis in the coagulation system.

Answer 9

The goal of the series of reactions known as the coagulation cascade is to produce thrombin (Factor IIa). The thrombin converts fibrinogen, a soluble protein, to fibrin which makes a fibrous network in the clot. Thrombin also promotes platelet aggregation so that at the same time fibrin is being formed a firm mass of aggregated platelets and fibrin together make a plug that seals the injury in the vessel. Thrombin is also important in activating the cofactors in the coagulation cascade (Factors V and VIII), as well as activating the factors responsible for lysing the clot (fibrinolytic factors) and activates protein C which helps prevent uncontrolled thrombosis.

Question 10

Explain the contribution and influence of endothelial cells on coagulation.

Answer 10

An in-tact endothelium inhibits coagulation, prevents platelet aggregation, promotes clot breakdown, and provides a barrier to the reactive elements in the vessel wall.An injured endothelium exposes collagen, tissue factor or negatively charged surfaces which bind platelets and initiate the clotting cascade.

Question 11

Describe the regulatory mechanisms of coagulation, i.e., protein C pathway, antithrombin, fibrinolytic pathway.

Answer 11

The activation of the clotting cascade and of platelets is inhibited by an intact vascular endothelium and by continuous blood flow which washes away platelets and any activated factors.In addition, there are a number of natural anticoagulant proteins. Anti-thrombin III is a protein that binds with high affinity to heparin and to thrombin and inactivates it. It also inactivates Factors 2,9,10,11,12.Thrombin can activate another regulatory protein called protein C, which cleaves Factors Va and VIIIa, which are the cofactors of coagulation Fibrin is degraded by an enzyme called plasmin.The pro-enzyme plasminogen is activated by tissue plasminogen activator (TPA). Fibrin split or degradation products and D-dimer are formed. Fibrinolysis is inhibited by plasminogen activator inhibitors (PAIs) and alpha 2-antiplasmin.

Question 12

List some of the major congenital or acquired disease states causing bleeding and/or clotting.

Answer 12

Congenital: Hemophilia A & B- X linked disorders, Factor VIII and Factor IX deficiency, respectively. Factor XI deficiency, Factor VII deficiency, von Willebrand disease are other congenital disordersAcquired: Liver disease, Vitamin K deficiency, warfarin or rat poison ingestion

Question 13

What is aPTT?What is it testing?

Answer 13

Activated Partial Thromboplastin Time (aPTT or PTT): A surface activating agent (proclotting), calcium, and platelet phospholipid are added to citrated (anticoagulant) plasma– measure how many seconds until a clot forms.Generally about 30 seconds is normal.Mainly measures intrinsic/common pathway; thus measures activity of the entire clotting cascade, except Factor VII (VII is extrinsic pathway).Sensitive to inhibition by heparin and fibrin split products (which are also anticoagulants).

Question 14

What is TT?What is it testing?

Answer 14

Thrombin time: Add thrombin to plasma.Measures common pathways conversion of fibrinogen to fibrin.Sensitive to inhibitory effects of fibrin split products and heparin.

Question 15

What is PFA?What is it testing?

Answer 15

Platelet function analyzer: can determine an in vitro bleeding time with agonists. Faster.

Question 16

Provide a differential diagnosis of an abnormal PT/INR, APTT, TT, bleeding time, and PFA.

Answer 16

Causes of a Prolonged PTTHeparin in Sample, Hemophilia A and B (Factor VIII or IX Deficiency), Factor XI Deficiency, Factor XII Deficiency*, Acquired Hemophilia, von Willebrand Disease, Lupus Anticoagulant**Patients do not bleedProlonged Protime ± PTTProtime relatively more prolonged than PTT Liver disease, Vitamin K deficiency, Warfarin or rat poison ingestionPTT more prolonged than protime Disseminated intravascular coagulation (DIC)Prolonged Bleeding Time/ PFA Von Willebrand Disease and ThrombocytopeniasProlonged TTabnormal fibrinogen, deficient fibrinogen, and Heparin contamination

Question 17

What are the clinical and molecular factors of vonWillebrand disease?

Answer 17

Most common mild bleeding disorder. VWF helps platelets adhere to collagen. Lots of bleeding.

Question 18

Describe the clinical features and molecular basis for hemophilia A and B

Answer 18

Both A + B are X-linked.Prolonged PTT. 1/3 are new mutations. Both have excess bleeding / identical issues thus only way to diagnose is w/ specific factor testing.A: factor VIII deficiency. 1/5000 male births.B: Factor IX deficiency. 1/50k male births.Classify the hemophilia patients as to the residual percentage of factor activity they have. < 1% = severe hemophilia (spontaneous joint bleeding), 2-5% moderate (require trauma), >10% Mild (Trauma too).All require factor therapy. Need 50% for surgery.

Question 19

Describe the clinical and molecular basis for factor VII disease.

Answer 19

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.Autosomal: both men and women affected.

Question 20

Describe the role of liver disease in coagulopathy.

Answer 20

Lots of coagulation factors are made by the liver. So, factors made with Vitamin K (2,7,9,10). Also protein C, S, and antithrombin 3 are sometimes made there. Clotting problems ensue.

Question 21

Describe disseminated intravascular coagulation (DIC) with its associated conditions. Understand diagnostic testing for DIC and associated conditions

Answer 21

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, both of these tests will be markedly prolonged.

Question 22

Explain what a lupus anticoagulant is, how it affects coagulation, and ways to test for it.

Answer 22

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.Also try the viper venom test.

Question 23

Explain how a 1:1 mixing study can distinguish a clotting factor deficiency from an inhibitor of coagulation.

Answer 23

Mixing will fix a deficiency, but not an inhibitor.