Hemostasis & Coagulation Ahmad Sh. Silmi Msc Hematology,FIBMS

1. Hemostasis & CoagulationAhmad Sh. Silmi Msc Hematology,FIBMS Unit 1: Primary Hemostasis

2. Vessels Coagulation Proteins Platelets Fibrinolysis/Inhibitors Homeostasis State of fluid equilibrium within the blood vessels

4. Hemostasis • Arrest of bleeding • Events preventing excessive blood loss • Vascular spasm: Vasoconstriction of damaged blood vessels • Platelet plug formation • Coagulation or blood clotting

5. Haemostasis: • Vasoconstriction • Platelet activation • Haemostatic plug • Coagulation • Stable clot formation • Clot dissolution

7. Primary hemostasis • Involves: • Vasoconstriction • Platelet adhesion • Platelet secretion • Platelet aggregation

8. Vascular System • Blood Vessels • Arteries • Carry blood from the heart to capillaries • Thickest walls of the vasculature • Veins • Return blood from capillaries to the heart • Thinnest walls of vasculature • Capillaries • No vessel wall • Do not contribute to hemostasis

9. Vascular System: Blood Vessels • Construction • Endothelium • Single layer of endothelial cells, lining vessels • Coated by glycocalyx • Protects basement membrane • Produces Von Willebrand's factor (vWF), a part of Factor VIII • Secretes prostaglandins, plasminogen activators • Negatively charged, repels circulating proteins and platelets • Subendothelium • Smooth muscle and connective tissue with collagen fibers

10. Vascular System: Blood Vessels • Basement membrane • Collagen material – stimulates platelets • Connective tissue • Elastic fibers- provide support around vessels

12. Vascular System: Blood Vessels • Function • Endothelium • Controls vessel permeability • Controls blood flow rate • Produces and releases substances that inhibit OR stimulate platelets, coagulation and fibrinolysis • Subendothelium • Collagen within is whats exposed upon injury

13. Vascular Endothelium Products:Stimulators • Produces vonWillebrand factor (vWF) • Helps in platelet adhesion to collagen • Carries factor VIII • Tissue factor (TF) activates secondary hemostasis via extrinsic pathway • Tissue plasminogen activator (tPA) is released activating fibrinolysis

14. Vascular Endothelium Products:Inhibitors • Release of tPA activates release of plasminogen activator inhibitor (PAI-1) to inhibit fibrinolysis • Thromomodulin forms a complex with thrombin • Platelet aggregation via prostacyclin production

15. Vascular System: Function Following Injury • Initiate hemostasis • Vasoconstriction of the arterioles • Minimizes blood flow to injured area • Prevents blood loss • Immediate • Short-lived

16. Vasoconstriction • Mechanism • Neurogenic factors • Regulatory substances • Prolong vasoconstriction • Serotonin ( made by platelet activation & endothelium) • Thromboxane A2 ( made by platelet activation & endothelium) • Endothelin-1 (made by damaged endothelial cells)

17. Vasoconstriction • Vasodilation Counteracts Vasoconstriction • Endothelial cells • Prostaglandin (PGI2)/ Prostacyclin • Vasodilates to increase blood flow to bring fresh supplies of clotting substances • Inhibits platelet aggregation • Contraction of venules • Causes gaps between them which pushes fluids causing edema or swelling

18. Thought question… • Think about the last time you cut your finger with a piece of paper. Did your finger bleed immediately? • If not, what might have prevented the bleeding?

19. Answer.. • No, the finger probably did not bleed immediately, due to vasoconstriction of the blood vessels

20. Discussion • What actions of the endothelial cells prevent clotting from occurring within the blood vessels?

21. Answers.. • Since the endothelial lining has a negative charge, it normally repels coagulation proteins and platelets in the circulation. It synthesizes products that help to inhibit fibrin formation.

22. All About Platelets… • Second major component of the hemostatic system

23. Platelets • What is a platelet? • Small 2-3 µm • Anuclear • Reddish-purple granules • Fragments of megakaryocyte cytoplasm

24. Platelets • Life span • 9-10 days • Normal Range • 150-440 x 109 /L

25. Platelet: Side noteSeen in conditions with increased need and/or destruction Giant platelets Micromegakaryocytes= Dwarf Megs • May Hegglin anomaly, Bernard-Soulier syndrome, pregnancy, malignancy • Seen in malignant disorders such as CML and MDS

26. Anatomy of a Platelet • Peripheral zone: Responsible for platelet adhesion and aggregation • Glycocalyx: • Contains glycoprotein receptors: • GPIb binds von Willebrand’s factor needed for platelet adhesion to collagen • GPIIb/IIIa bind fibrinogen needed for aggregation • Bind ADP and thrombin, promoting aggregation • Factors I, V, VIII on surface, involved in 2o hemostasis • Plasma membrane: • Exposed on platelet activation • Layer called PF3 (platelet factor) surface for interaction of plasma coagulation factors • Initiation of formation of thromboxane A2. This stimulates aggregation and vasoconstriction

27. Anatomy of a Platelet • Structural or Sol-Gel zone: Responsible for platelet retraction/contraction functions and platelet shape • Microtubules • Cytoskeleton • Binding protein • Organelle zone: Responsible for storage and platelet release functions • Granules • Dense bodies, alpha granules, lysosomal granules and microperoxisomes • Mitochondria • Glycogen

29. Platelet Receptors • GPIb/IX – vWF • Required for PLT adhesion • GPIIb/IIIa – Fibrinogen • Required for PLT aggregation • Phospholipid (Pl) • Bind vitamin K dependent proteins , Ca++ dependent • Bind Va and VIIIa (called “PF3” in this context)

30. Platelet Granules DENSE • Plasma Derived Fibrinogen – PLT aggrgation Ig Albumin • Megakaryocyte Derived vWF – PLT adhesion, VIII carrier Coagulation Factor V • Plasminogen – source of plasmin • PAI-1 – inhibits activation of fibrinolysis • a2-antiplasmin – plasmin inhibitor • PF4 – inactivates heparin • PLT-derived Growth Factor (PDGF) – tissue repair • Thrombospondin – PLT aggregate stabilizer • Fibronectin – PLT binding • ALPHA • ATP • ADP • Serotonin • Calcium Ions

32. Diagrammatic Representation of the Platelet

33. PLATELET STRUCTURE PF4, ß-TG, PDGF, TGF-ß, fibrinogen, VWF, etc. ADP, ATP, Ca2+, serotonin, etc.

34. Production of Platelets • Made in Bone marrow • Need dictates the amount of platelets produced. • Stimulus for production is the platelet mass in circulating blood ~ 80 % and megakaryocyte mass in bone marrow • Originate from CFU-GEMM to form CFU-Meg • Cytokines and growth factors such as IL-3 and GM-CSF influences progenitor stages

35. Platelet Development • Megakaryoblast • 10-15 µm • Increased nuclear: cytoplasmic ratio • Promegakaryocyte • 80 µm • Dense alpha and lysosomal granules • Basophilic megakaryocyte • Megakaryocyte

36. Production of Platelets • Precursor Cell= Megakaryocyte • Produces about 2000-4000 platelets • Platelets are released via sinuses of bone marrow

37. Production of Platelets • Thrombopoietin (TPO) • Regulates platelet development • Influences all stages of megakaryocyte production • Produced in the liver, kidney and spleen

38. Production of Platelets • How does TPO work? • Maintains a constant number of platelets in peripheral blood by binding Mp1 (platelet receptor). Bound TPO can not stimulate proliferation of bone marrow progenitor cells • The higher the platelet count, the more TPO is bound and stimulation of bone marrow is decreased.

39. Thought question… • If a patient had a low platelet count what will happen?

40. Answer… • TPO increases the number of megakaryocytes in the bone marrow, increases size and DNA count of megakaryocytes and increases maturation rate

41. Function of Platelets • Surveillance of blood vessel continuity • Checks endothelial lining for gaps and breaks • Fill-in small gaps caused by separation of endothelial cells • Formation of primary hemostatic plug • Surface for coagulation factors to make secondary hemostatic plug • Aid in healing injured tissue

42. Formation of Primary Hemostatic Plug • Once the platelets “normal” environment is changed, they become activated or adhesive • Three stages of plug formation: • Adhesion • Release reaction • Aggregation

43. Stage 1: Platelet Adhesion • It is the binding of platelet to non platelet surface: sub endothelial collagen • involves changes from a disc shape to a slightly broader, plate like form to increase surface area • a number of plasma proteins are required for normal platelet adhesion. • Thrombin • Fibronectin • vWF • vWF is the largest component of factor VIII and secreted by platelets and by vascular endothelial cells.

44. Adhesion cont’d Collagen – vWF –Platelet • vWF bridge physical distance between platelate and sub endothelial collagen • Increase bond that seal platelet to the vessel wall • reversible

45. Adhesion is Reversible & No ADP is released.

46. Stage 1: Platelet Activation • Platelets undergo a shape change from disc to spiny sphere with projections • Activation required for 1O hemostatic plug formation • Activation continues until Ca ++ threshold met • Outcome • Activation of GPIIb/IIIa receptors for fibrinogen • Secretion of granules within platelets into tissues

47. Platelet Shape Change George Lancet 2000; 355:1531

48. Stage 2: Platelet Secretion & Release • Requires ATP • It is release of contents of the granules of platelet • Primarily ADP  stimulates aggregation • Cathecolamine (especially epinephrine) and serotonin enhance vasoconstriction • Granules trigger a secondary aggregation which is irreversible

49. Platelets contain 3 types of secretary granules: • Lysosome containing acid hydrolyses • α-granules containing platelate specific proteins (Plt factor 4, β- thromboglobulin, as well as other proteins such as Platelet derived growth factor and coagulation proteins found in plasma (fibrinogen & von Willebrand’s factor) • -granules containing ATP, ADP, Calcium & serotonin

50. Steps in platelet plug formation: Extension