Hemostasis & Coagulation Ahmad Sh. Silmi

1. Hemostasis & CoagulationAhmad Sh. Silmi Unit 1 : Primary Hemostasis

2. Hemostasis • Heme= blood • stasis= to halt • Process of retaining blood within the vascular system • Repairs injury to blood vessels • Stops or prevents blood loss

3. Fibrinogen Procoagulant Factors RegulatoryFactors Fibrin Balance of Hemostasis *Balance of bleeding (hemorrhaging) and clotting (thrombosis) *Imbalance in one direction can lead to: bleeding : hypocoagulable state OR thrombosis: hypercoagulable state

4. Hemostasis • Components • Vascular System • Controls rate of blood flow • Platelet System • Interaction of vasculature and platelets form a temporary plug • Coagulation System • (i.e) fibrin forming • Fibrinolytic System • Fibrin lysing • Coagulation Inhibition System • Natural inhibitors • Control fibrin formation and fibrin lysis

5. Failure or deficiencies in any of these five systems can leads to varying degrees of uncontrolled hemorrhaging or clotting

6. Hemostasis The hemostatic components remain inert in the presence of intact vascular tissue or endothelium Following injury, each component must function optimally.

7. Hemostasis: OverviewConsists of three stages • Primary Hemostasis • Process of blood clotting in response to injury where blood vessels (vasculature) and platelets are the main “players.” • Primary Hemostatic plug is formed • Platelet plug temporarily arrests bleeding. Insoluble fibrin strands deposit on the initial plug to reinforce and stabilize. The fibrin originates from soluble plasma proteins. • Secondary Hemostasis • Actions of the protein coagulation factors form fibrin in response to injury • At this time, blood has changed into a solid state • Fibrinolysis • Clot is removed following healing of wound

8. http://health.howstuffworks.com/adam-200077.htm

9. 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

10. 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

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

13. 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

14. 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

15. 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

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

17. 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)

18. 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

19. 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?

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

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

22. 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.

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

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

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

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

27. 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

28. 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

30. 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)

31. 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

33. Diagrammatic Representation of the Platelet

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

35. 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

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

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

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

39. 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.

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

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

42. 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

43. Formation of Primary Hemostatic Plug Once the platelets “normal” environment is changed, they become activated or adhesive Three stages of plug formation

44. Stage 1: Platelet Adhesion • Platelets attach to non-platelet surfaces, suchascollagen fibers in the subendothelium • Platelets move from the blood vessels and into the tissues. • Exposure to surfaces in the tissues causes them to bind to collagen with the presence of von Willebrand factor ( vWF) and Glycoprotein IbIX, making a bridge formation, which triggers a shape change • Reversible • No ADP released

45. 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

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

47. Stage 2: Platelet aggregation • Chemical changes cause platelets to aggregate and stick to one another • Newly arriving platelets become activated by agonists • Exposure of GPIIb/IIIa sites bind fibrinogen • Fibrinogen + activated platelets serves as a bridge between two platelets • Calcium must be present

48. Activated platelet membrane generates TXA2 TXA2 stimulates release

49. Arachidonic acid Cyclo-oxygenase Cyclic Endoperoxides PLATELET ENDOTHELIAL CELL Thromboxane synthetase Prostacyclin synthetase Prostacyclin (PGI2) Thromboxane (TxA2) Inhibits plt aggregation Vasodilator Enhances plt aggregation Vasoconstrictor

50. e.g. ADP TXA2 thrombin Aggregated Platelets Resting Platelet GPIIb/IIIa (inactive) GPIIb/IIIa Agonist (activated) Fibrinogen high shear: von Willebrand factor substitutes for fibrinogen