Hemostasis and Coagulation

1. Hemostasis and Coagulation Miklós Molnár

2. Definition of HEMOSTASIS • The arrest of bleeding by repair of vessel wall • Maintaining a balance • Coagulation • Fibrinolysis • Hypocoagulation: excessive bleeding (inherited or acquired) • Hypercoagulation (thrombosis) inadequate activation of the fibrinolytic system

3. Systems Involved in Hemostasis • Vascular system • Injured vessel initiates vasoconstriction • Platelet System • Injured vessel exposes collagen that initiates platelet aggregation and help form plug • Coagulation System • protein factors of intrinsic and extrinsic pathways produce a permanent fibrin plug

4. HEMOSTASISPrimary vs. Secondary vs. Tertiary • Primary Hemostasis • Platelet Plug Formation • Dependent on normal platelet number & function • Initial Manifestation of Clot Formation • Secondary Hemostasis • Activation of Clotting Cascade  Deposition & Stabilization of Fibrin • Tertiary Hemostasis • Dissolution of Fibrin Clot • Dependent on Plasminogen Activation

5. Primary Hemostasis • vasoconstriction (vascular system) • platelet exposure to subendothelial connective tissue of blood vessels • Platelet release of ADP, ATP, Thromboxane A2 (promotes vasoconstriction) • Platelet aggregation, phospholipid provides site for fibrin formation

6. Platelet Origin & Development • Endomitosis • Multiple mitotic division of DNA without cytoplasmic division • Largest cell in the BM low power • Stem cell to mature platelet = 5 days • Each megakaryocyte can shed 500-4000 platelets • Cytoplasm breaks apart along demarcation membranes

7. Megakaryocyte

8. Platelets forming from cytoplasm

9. Normal platelets and one giant platelet

10. Platelet Structure • Three zones • Peripheral zone (adhesion & aggregation) • glycocalyx, plasma membrane • Sol-gel zone (structure & support) • microfilaments, thrombosthenin, open canalicular system, dense tubular system • Organelle zone (Secretion & storage) • granules: alpha, dense, glycogen • mitochondria, lysosomes

11. Glycocalyx • Glycoproteins • Ib (GPIb) • Receptor site for vWF • IIb, IIIa (GPIIb/IIIa) • Complex becomes receptor site for fibrinogen

13. Granular content • Dense granules • ATP • ADP • Calcium • Magnesium • Serotonin • epinephrine

14. Hemostatic proteins Fibrinogen Factor V vWF Plasminogen Plasminogen activator inhibitor (PAI-1) α2-antiplasmin Nonhemostatic proteins β-thromboglobulin, Platelet factor 4 Platelet derived growth factor (PDGF) Albumin fibronectin, Granular content (Alpha granules)

15. Formation of primary hemostatic plug • Platelets converted from inactive to active state • Adhesion to collagen • Triggers platelet activation • Tromboxane A2 is synthesized from arachidonic acid and stimulates secretion • Aggregation of platelets to each other • prostacyclin (PGI2) inhibits platelet aggregation • Secretion (discharge of granule contents)

16. VON WILLEBRAND FACTOR • Large Adhesive Glycoprotein • Polypeptide chain: 220,000 MW • Base structure: Dimer; Can have as many as 20 linked dimers • Multimers linked by disulfide bridges • Synthesized in endothelial cells & megakaryocytes • Constitutive & stimulated secretion • Large multimers stored in Weibel-Palade bodies • Functions:1) Stabilizes Factor VIII2) Essential for platelet adhesion

17. Stable adhesion Platelet activation/aggregation Platelet Rolling Platelet adhesion Blood Flow VWF VWF VWF VWF collagen collagen VWF collagen collagen VWF

18. Inactive Active

20. Secondary hemostasis • Intrinsic Pathway • All components required for initiating this pathway are circulating in the blood • triggered by contact with collagen or glass • Extrinsic Pathway • Initiated by the release of tissue thromboplastin and calcium from damaged tissue • Common Pathway • Leads to clot formation including the platelet plug and fibrin produced

21. Coagulation Proteins • Zymogens • enzyme precursors II, VII, IX, X, XI, XII, Prekallkrein • When activated become serine proteases • Cofactors • Nonenzymatic V, VIII, HMWK, Tissue factor(thromboplastin) • Kinin factors prekallikrein, kallikrein, HMWK • Roles include coag activation as well as fibrinolytic activation

22. Coag factors (by group) • Fibrinogen group: I,V,VIII,XIII • most labile, are consumed in coagulation, found on platelets • Prothrombin group: II,VII,IX,X • Vitamin K dependent, may be affected by coumarin,diet, antibiotics • Contact group: XI,XII,HMWK, Prekallikrein • initiate intrinsic path and fibrinolysis

23. Positive feedback Tissue factor pathway inhibitor X Xa II (prothrombin) IIa (thrombin) VIIa + Tissue Factor (TF) Tissue Damage Vessel wall Cell particles Initial Tissue Factor Pathway Activation of Hemostasis

24. XI XIa VIII VIIIa V Va Fibrinogen Fibrin IIa Thrombin Pro-coagulant effects

25. Precursor Enzyme Reaction on Activated Platelets XIa IX IXa FVIIIa/Ca2+/Phospholipid X Xa FVa/Ca2+/Phospholipid II IIa Fibrinogen Fibrin

26. FIBRINOLYTIC SYSTEM • Definition: temporary fibrin clot systematically and gradually dissolved as the vessel heals • Key components • Plasminogen (inactive form) • Plasminogen activators • Plasmin • Fibrin • Fibrin Degradation Products (FDP) • Inhibitors of plasminogen activators and plasmin

27. Activators of Fibrinolysis • Intrinsic activators • Factor XIIa, XIa, kallikrein • Extrinsic activators • Tissue type plasminogen activator (t-PA) • Urokinase type plasminogen acitvator (u-PA) • Exogenous activators • Streptokinase (derived from beta strep)

28. Protein S Protein C Protein S IIa/Thrombomodulin TFPI interaction XII XIIa XI XIa IX IXa VIIIa+Ca+Pl X Xa Va+Ca+Pl II IIa TF / VIIa Fibrinogen Fibrin Fibrinolysis

29. VIIIa Va Protein S Activated Protein C Protein C IIa IIa Thrombin Anti-coagulant effects thrombomodulin Protein C Anticoagulant Pathway

30. Coagulation Regulatory Mechanisms • Naturally Occurring Anticoagulants rapidly interact with components of coag cascade to avoid unabated clot formation • Protein C (PC) and Protein S (PS) • deficiencies may be congenital or acquired • Antithrombin (AT) and Heparin Cofactor II • serine protease inhibitors (serpins) • Deficiency of inhibitors cause increased risk of thrombosis

31. Inhibitors of Fibrinolysis • Plasminogen Activator Inhibitors (PAI) • α2 –antiplasmin • α2 -macroglobulin

32. Bleeding disorders

33. Disorders of Hemostasis • Vascular disorders • Scurvy, easy bruising, Henoch-Schönlein purpura. • Platelet disorders • Quantitative - Thrombocytopenia • Qualitative - Platelet function disorders – Glanzmans • Coagulation disorders • Congenital - Haemophilia (A, B), Von-Willebrands • Acquired - Vitamin-K deficiency, Liver disease • Mixed/Consumption: DIC

34. Tests of Hemostasis: • Screening tests: • Bleeding.T  10m. Platelet & BV function • Prothrombin.T  Extrinsic, aPTT  Instrinsic • Thrombin.T  common path. (DIC) • Specific tests: • Factor assays – hemophilia. • Tests of thrombosis – TT, FDP, DDA, • Platelet function studies: • Adhesion, Aggregation, Release tests. • Bone Marrow study

35. Bleeding: Clinical Features • Local-vs- General, spontaneous… • Hematoma / Joint Bleeds- Coag • Skin / Mucosal Bleeds – PLT • wound / surgical bleeding – • Immediate - PLT • Delayed - Coagulation

36. Platelet Coagulation Petechiae, Purpura Hematoma, Joint bl.

37. Vascular disorders: • Petechiae, purpura, ecchymoses • senile purpura • vitamin C deficiency (scurvy) • Connective tissue disorders • Infections – Meningococcus • Henoch-Schonlein Purpura-Immu

38. Senile Purpura

39. Petechiae in Vasculitis (Rocky Mountain Spotted Fever)

40. Henoch-Schölein purpura • Immune disorder • Children • Follows infection • Petechiae with edema and itching.

41. Henoch-Schönlein purpura 20y Male, fever, painful symmetric polyarthritis for a day. During the next two days, edema and palpable purpura developed.

42. Platelet Disorders - Features: • Mucocutaneous bleeding • Petechiae, Purpura, Ecchymosis. • Spontaneous bleeding after trauma • CNS bleeding (severe  plt) • Prolonged bleeding time (BT)

43. BLEEDING TIME vs. PLATELET COUNT

44. REDUCED PLATELET NUMBER:THROMBOCYTOPENIA Normal platelet count 140-400 Increased bleeding time <100 Spontaneous bleeding <20

46. PLATELET FUNCTION DEFECTSProlonged Bleeding Time • Congenital • Drugs • Alcohol • Uremia • Hyperglobulinemias • Fibrin/fibrinogen split products • Thrombocythemia • Cardiac Surgery

47. PLATELET FUNCTION DEFECTSPlatelet Adhesion • Bernard Soulier Disease • Abnormal GPIb-IX Complex • Receptor for von Willebrand factor • Only adhesion mediator @ high shear stress • Tested by ability to aggregate platelets in presence of ristocetin • Von Willebrand disease • Reduced or dysfunctional von Willebrand factor

48. Von-Willebrand Disease: • Coagulation + PLT disorder: • Congenital disorder • Deficiency of vWF molecule • Part of FVIII, • Mediates platelet adhesion • Prolonged Bleeding time • Low Factor VIII & long aPTT • Mucocutaneous bleeding

49. Von-Willebrand Disease • vWF: F-VIII & PLT function. • Defective Platelet Adhesion • Skin Bleeding • Prolonged Bleeding time. • Low Factor VIII levels. • Prevalence: 0.8–2% (probable underestimate)

50. PLATELET FUNCTION DEFECTSPlatelet Release Defects - Congenital • -storage pool disease • Failure to form dense granules (Hermansky-Pudlak syndrome) • Do not release ADP, serotonin, calcium on activation • Fail to recruit platelets for aggregation • Gray platelet syndrome • Failure of packaging of α-granules • Do not release protein mediators of platelet aggregation • Decreased platelet aggregation • Mild bleeding disorder