Hemostasis & Thrombosis Beth A. Bouchard BIOC 212: Biochemistry of Human Disease Spring 2006

1. Hemostasis & Thrombosis Beth A. Bouchard BIOC 212: Biochemistry of Human Disease Spring 2006

2. HEMOSTASIS Hemorrhage Thrombosis Hemostasis

3. HEMOSTASIS (CONT.) 1). INITIATION Vessel wall – endothelial cells and subendothelial components 2). LOCALIZATION Platelets – circulating cellular elements 3). PROPAGATION/AMPLIFICATION Plasma coagulation proteins (factors) 4). TERMINATION Plasma coagulation protein inhibitors 5). ELIMINATION Fibrinolytic system

4. Vessel Wall: Endothelial cells

5. Antithrombotic Attributes of Vascular Endothelium

7. Vessel Wall (cont.)

8. Response to Vessel Wall Injury: Platelet adhesion • Exposure of flowing blood and platelets to subendothelial components • Platelets bind to the subendothelial collagen bound tovon Willebrand factor (vWF), which is secreted from endothelial cells directly into the subendothelial space or adsorbed from plasma following endothelial cell secretion • vWf also binds directly to platelets via glycoprotein Ib-IX

9. PLATELETS

10. Platelets adhered to damaged endothelium

11. Platelet Plug Formation = 1° hemostasis

12. Platelet Plug Formation: Platelet activation • Activated via their interaction with subendothelial collagen • Additional platelet agonists include ADP, epinephrine, thrombin, immune complexes, and high shear stress – all of the compounds interact with specific platelet membrane receptors • Several platelet activation pathways are initiated

13. Platelet Plug Formation: Platelet activation events • Platelet shape change: extend pseudopodia, which facilitates aggregation and coagulant activity • Release of alpha and dense granule contents including a number of compounds involved in hemostasis (eg ADP, factor V and fibrinogen) • Aggregation

14. Response to Vessel Wall Injury: Vasoconstriction • Temporarily reduces local blood flow and hence, blood loss • Mediated in part by serotonin and thromboxane A2 (TXA2) from activated platelets Serotonin is released from platelet dense granules TXA2 is a product of platelet prostaglandin metabolism

15. Activated platelets

16. Platelet Plug Formation: Platelet aggregation • Platelet activation results in the functional expression of membrane receptors normally expressed in a non-functional state (glycoprotein IIb-IIIa) • Fibrinogen from the plasma or released from activated platelet alpha-granules binds to activated glycoprotein IIb-IIIa membrane receptors effectively bridging platelets to each other

17. Platelets adhered to and aggregated upon collagen

18. Platelet Plug Formation

19. HEMOSTASIS 1). INITIATION Vessel wall – endothelial cells and subendothelial components 2). LOCALIZATION Platelets – circulating cellular elements 3). PROPAGATION/AMPLIFICATION Plasma coagulation proteins (factors) 4). TERMINATION Plasma coagulation protein inhibitors 5). ELIMINATION Fibrinolytic system

22. BLOOD COAGULATION

23. BLOOD COAGULATION (CONT.) • Deficiencies in all of the factors, except factor XII, lead to a bleeding tendency in the affected individual • Described as a ‘waterfall’ or ‘cascade’ sequence of zymogen (pro-enzyme) to enzyme conversions, with each enzyme activating the next zymogen in the seqeunce • Activated factor enzymes are designated with an “a”, e.g. factor Xa

24. Common constituents of coagulation complexes Vitamin K-dependent (VKD) zymogen Ca2+ Protein cofactor Appropriate membrane surface - activated platelets (VIIIa/IXa complex, Va/Xa complex) • subendothelial cells, typically fibroblasts (TF/VIIa complex)

26. Common constituents of coagulation complexes Vitamin K-dependent (VKD) zymogen Ca2+ Protein cofactor Appropriate membrane surface - activated platelets (VIIIa/IXa complex, Va/Xa complex) • subendothelial cells, typically fibroblasts (TF/VIIa complex)

27. Functional Domains of the Vitamin K- dependent Zymogens

28. Gamma (g)-carboxyglutamic acid

29. Formation of Gla residues subsequent to protein synthesis (post-translational) H2 • Group of related, fat soluble compounds, which differ in the number of side-chain isoprenoid units • Plant derived (vitamin K1) and synthesized by intestinal bacteria (vitamin K2)

30. Common constituents of coagulation complexes Vitamin K-dependent (VKD) zymogen Ca2+ Protein cofactor Appropriate membrane surface - activated platelets (VIIIa/IXa complex, Va/Xa complex) • subendothelial cells, typically fibroblasts (TF/VIIa complex)

31. Relative Rate of Prothrombin Activation Prothrombinase Components -Thrombin Prothrombin 1 FXa FXa Ca2+ FVa HC 300 FXa Ca2+ Ca2+ FVa LC FXa Ca2+ 30 Ca2+ Prothrombinase FXa 300,000 Ca2+ FVa HC FVa HC Ca2+ FVa LC FVa LC Relevance of complex formation and its constituents

32. Common constituents of coagulation complexes Vitamin K-dependent (VKD) zymogen Ca2+ Protein cofactor Appropriate membrane surface - activated platelets (VIIIa/IXa complex, Va/Xa complex) • subendothelial cells, typically fibroblasts (TF/VIIa complex) ** Express anionic phospholipids and membrane receptors for coagulation proteins. In platelets, the expression of this membrane surface is activation-dependent.

33. Activate platelets

35. HEMOSTASIS (CONT.) 1). INITIATION Vessel wall – endothelial cells and subendothelial components 2). LOCALIZATION Platelets – circulating cellular elements 3). PROPAGATION/AMPLIFICATION Plasma coagulation proteins (factors) 4). TERMINATION Plasma coagulation protein inhibitors 5). ELIMINATION Fibrinolytic system

36. INHIBITORS

37. INHIBITORS (cont.)

38. FIBRINOLYSIS

39. FIBRINOLYSIS (CONT.)

41. Platelet Plug Formation • Measured clinically as the bleeding time • Normal bleeding time is from 2 – 10 min • Usually the bleeding time is sufficient to detect defects of platelet adhesion and aggregation, in which it is prolonged

43. Intrinsic Pathway of Blood Coagulation • No factors extrinsic to the blood are involved • Clinical test to assess the functionality of this pathway is the activated partial thromboplastin time (aPTT) • Kaolin and cephalin are added to the test plasma sample • The normal range is ~30 – 50 seconds (varies slightly depending on the laboratory) • Prolongations in the aPTT are observed in deficiencies of factors XI, IX, VIII, X, and V, prothrombin, or fibrinogen. • Used to test for common congenital hemophilias (deficiencies in IX, VIII, or XI) and to monitor heparin treatment

45. Extrinsic Pathway of Blood Coagulation • Extrinsic refers to tissue factor, which is expressed on subendothelial cells • Clinical test to assess the functionality of this pathway is the prothrombin time (PT) • Lipidated tissue factor is added to test plasma sample • The normal range is ~10-15 seconds (varies slightly depending on the laboratory) • Prolongations in the PT are observed in deficiencies of factors VII, X, V, prothrombin, or fibrinogen. • Used to test for the rare congenital deficiencies in these factors: More often it is used to diagnose acquired bleeding disorders resulting from vitamin K deficiency, oral anticoagulants (e.g. warfarin), and liver disease

46. Thrombin Time (TT) In this test, thrombin is added to plasma • The normal range is ~10-15 seconds (varies slightly depending on the laboratory) • Prolongations in the TT are observed in congenital fibrinogen deficiency or acquired fibrinogen deficiency resulting from consumption of fibrinogen in DIC (disseminated intravascular coagulation), or may occur following treatment with fibrinolytic drugs

47. Hemorrhage Bleeding disorders can span the spectrum from weeping blood vessels to full-fledged internal and external hemorrhage Genetic defects: platelet abnormalities blood vessel wall abnormalities clotting factor deficiencies (hemophilias) excess clot breakdown (fibrinolysis) Acquired defects: liver disease (site of clotting factor synthesis) vitamin K deficiency autoimmune disease (platelet destruction) trauma

48. Disorders of Platelet Adhesion or Aggregation • Affecting constituents of the vessel wall • Affecting the ability of the platelet to interact with the subendothelium at sites of blood vessel injury • Affecting the ability of the platelet to interact with other platelets

49. Vessel Wall Defects • von Willebrand’s disease: a group of autosomal dominant disorders that result in reduced or abnormal synthesis of vWF • Defects in collagen synthesis Ehlers-Danlos Syndrome: congenital defect in collagen synthesis Scurvy: results from vitamin C (ascorbic acid) deficiency, which is involved in collagen synthesis Excess exogenous or endogenous corticosteroids: also leads to acquired deficiency in collagen synthesis

50. Platelet Defects • Bernard-Soulier Syndrome: expression of low levels of or defective glycoprotein Ib-IX on the platelet surface • Glanzmann’s thrombasthenia: expression of low levels of or defective glycoprotein IIb-IIIa on the platelet surface