MLAB 1227- Coagulation Keri Brophy-Martinez

1. MLAB 1227- CoagulationKeri Brophy-Martinez 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. 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 May Hegglin anomaly, Bernard-Soulier syndrome, pregnancy, malignancy Micromegakaryocytes= Dwarf Megs Seen in malignant disorders such as CML and MDS

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

29. Anatomy of a Platelet http://www.platelet-research.org/1/function_morpho.htm#

30. 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

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

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

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

34. 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.

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

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

37. 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

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

39. Stage 1: Platelet Adhesion Platelets attach to non-platelet surfaces, such as collagen 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

40. 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

41. Platelet Shape Change http://www.platelet-research.org/1/function_morpho.htm

42. 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

43. Activated platelet membrane generates TXA2 TXA2 stimulates release

44. Adhesion & Aggregation http://www.platelet-research.org

45. Stage 3: Platelet Secretion & Release Requires ATP Platelets release contents of their granules, causing vasoconstriction Granules trigger a secondary aggregation which is irreversible Granules consist of Alpha granules: Factor V, Factor VIII:vWF, Fibrinogen, a2-antiplasmin, platelet factor 4 Dense bodies: ATP, ADP, serotonin, Ca

46. Granules con’t Factor V: receptor on platelet surface for factor Xa & prothrombin PF4: heparin neutralizing factor ADP: agonist, continues to recruit and stimulate platelets by increasing cytoplasmic calcium

47. Side note Heparin is used on patients who clot excessively. Endothelial cells make heparin-like molecules and expose them on their surface. PF4 binds these substances. Heparin can complex with bound PF4 and heparin will be neutralized.

48. Final Stage : Stabilization of Clot AKA: primary hemostatic plug formation Thrombus formation Platelets release Factor V Expose factor III, accelerating coagulation cascade Promote activation of clotting factors

49. Platelet System: Additional Functions Provides the reaction surface for some coagulation system reactions, as well as platelet factor 3 (PF3) which is platelet phospholipid Supports and maintains endothelial lining Defective hemostasis can occur due to decreased number of platelets (quantitative) abnormally functioning platelets (qualitative)

50. Blood clot

51. Coagulation System Composed of 14 coagulation factors (serine proteases) which are interdependent (Factors I through XIII – there is no Factor VI – and PK and HMWK) Inactive form of each is an enzyme precursor which is usually designated by a Roman numeral but also given a name – Ex. Factor I fibrinogen. Numbers correspond to order of discovery NOT order in cascade. Active forms are usually designated by the letter “a” after the Roman numeral and may also have a different name – Ex. Ia Fibrin Cofactors are needed for many reactions in the cascade – Ex. Calcium, platelet factor 3 (PF3) Each molecule must be present in sufficient quantity as well as functioning normally Final product is fibrin mesh or clot which completely stops bleeding Secondary hemostasis Slow contraction and lysis of the clot occurs

52. Fibrinolytic System Plasminogen is converted to plasmin Plasmin enzymatically attacks the fibrin molecule producing fibrin degradation products (FDPs, sometimes called FSPs) that are cleared from the circulation by macrophages Fibrin is a product formed during hemostasis, tissue repair or inflammation Fibrin plays a temporary role Once injury heals, the fibrin clot is lysed

53. Coagulation Inhibition System Provides balance and control of clotting mechanisms Natural inhibitors and anticoagulants circulate in the plasma to: Prevent clotting when it’s not needed Limit or localize the clotting that is needed Examples: Protein C and S, antithrombin III