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Normal haemostasis



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Hemostasis and Coagulation
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Normal haemostasis

  1. 1. Moderators Respected :- Ishwar Bihana Sir Respected :- Joseph Sir Respected :- S.K.Bose Sir Presented By Gaurav Kumar MLT Student Part II Department of Haematology Post Graduate Institute of Medical Education And Research, Chandigarh
  2. 2. • It is a biological or physiological phenomenon which is responsible to keep the blood in fluid state in the circulation as well as to arrest bleeding followed by an injury to blood vessel. Defination
  3. 3. Aims and Objective To understand how our vascular system keep our blood in its fluid state. To know how bleeding is arrested. To know the role of haemostasis components. To know the biology and mechanism of coagulation. To know how clot is removed from vascular system.
  4. 4.  To maintain the blood in fluid state while it remains circulating within vascular system.  To maintain the integrity of the vessels wall.  To arrest bleeding at the site of injury or blood loss by the formation of haemostatic plug.  Eventual removal of plug when healing is complete. Importance of Hemostasis
  5. 5. ADPase TM Thrombin Protein CaProtein C Destroys FVa,FVIIIa Protein S HS AT-III Inhibit FXa,FIXa,thrombin tPA plasminogen plasmin fibrinolysis endothelium Overview of Hemostasis Blood coagulation cascade Vessel injury Neurogenic Mayogenic endothelin vasoconstriction Reduce blood flow Collagen exposure+vWF Tissue factor Plt.adhen & activationserotonin TxA2,ADP plt.aggregation Primary hemostatic plug Stable 2ndry hemostatic plug healing fibrinolysis antiplasminogen Cell migration & proliferation PF thrombin fibrin
  6. 6. Blood vessels Platelets Plasma coagulation factors Inhibitors Fibrinolytic system Components of Hemostasis
  7. 7. Blood vessels Structure of blood vessels
  8. 8. Function of Blood Vessel Blood vessel with muscular coat help to reduce blood loss by vasoconstriction. Blood vessels with a pipe system transport nutrients , hormones , gases and other essential factors which are transported by blood.
  9. 9. Endothelial Cell Function Normal vascular endothelium is a thromboresistant surface. Non-thrombogenic- don’t react with plasma or cellular elements of the blood. It is antithrombotic. It activates antithrombin III. When injured (either biologically , chemically or mechanically) it can profoundly promote hemostasis.
  10. 10.  Antiplatelets Effect:  Inhibition of platelet adhesion, activation and aggregation.  PGI2 (Prostacyclin)-vasodilator and antiplatelet agent.  NO-Nitric Oxide/PGI2  Both bind with the receptors on the platelets and inactivate them. So inhibit platelet aggregation.  ADPase:  Enzymes that break down the ADP (strong proaggregating agent) released by the platelets and thus inhibit platelet aggregation. ADPase TM Thrombin Protein CaProtein C Destroys FVa,FVIIIa Protein S HS AT-III Inhibit Fxa,FIXa,thrombin tPA plasminogen plasmin fibrinolysis Endothelial Antithrombotic Mechanism
  11. 11. ADPase TM Thrombin Protein CaProtein C Destroys FVa,FVIIIa ProteinS HS AT-III Inhibit Fxa,FIXa,thrombin tPA plasminogen plasmin fibrinolysis Surface expressed integral membrane protein which binds thrombin & results in loss of the pro-coagulant properties of thrombin. Thrombin/thrombomodulin complex is a potent anticoagulant complex. Since it activates protein C to activated-Protein C. Activated protein C down regulates coagulation by inactivating important proteins (FVa, FVIIIa) Thrombomodulin
  12. 12. Heparan sulphate • Long unbranched polysaccharide expressed at the endothelial membrane surface. • Act as a cofactor for the plasma inhibitor antithrombin III. • Inhibits thrombin and other coagulation factors(FIXa,FXa) ADPase TM Thrombin Protein CaProtein C Destroys FVa,FVIIIa Protein S HS AT-III Inhibit Fxa,FIXa,thrombin tPA plasminogen plasmin fibrinolysis Binding and Inhibition of Thrombin
  13. 13. platelet GPIb receptor vWF Endothelial cell subendothelial collagen Subendothelial Cell Function Subendothelium consist of collagen ,elastic tissues, proteoglycans and non collagenous glycoproteins {fibronectin, vWF}. Exposure of this layer after damage of vessel wall is responsible for platelet adherence. vWF bind with collagen, vWF then undergoes a conformational change and platelets are captured via their surface membrane glycoprotein GpIb binding to vWF.
  14. 14. The mechanism isDamage of vascular endothelium stimulates endothelial cells Endothelial cell Synthesize and secrete three Substances involved In haemostatic plug Formation vWF (VIII vWF) It help in adhesion Of platelet to sub endothelium PGI2 (synthesized from Arrachidonic acid And it inhibit platelet Aggregation Plasminogen Its release is Stimulated by Vascular damage
  15. 15. Stages of Hemostasis Fibrinolysis Formation of Platelet Plug Formation of blood clot Vascular Constriction 18 July 2015 15
  16. 16. Blood Vessels Injury
  17. 17. Vasoconstriction Endothelial cells produce vasoconstrictors such as angiotensin II and serotonin which help in vasoconstriction. Activated platelets produce thromboxane A2 (TXA2) which is a potent vasoconstrictor.
  18. 18. Function of platelets Coagulative function Haemostatic function
  19. 19. Platelets membrane glycoproteins: GPIb-IX: Constitute active receptor for vWF Mediates vWF dependent adhesion of platelets to subendothelial GPIIb/IIIa: On activation serve to bind fibrinogen Mediates aggregation Also receptor for vWF, fibronectin and thrombospondin GPIa-IIa: Constitutively active receptor for collagen. Mediates platelets adhesion independent of vWF. Hemostatic Function
  20. 20. Role of Platelets in Hemostasis With in 1-2 sec after injury to blood vessel, hemostatic process begins & proceed as out line bellow: 1. platelet adhesion 2. platelets activation 3. platelets release reaction 4. platelets aggregation
  21. 21.  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.  Binding via GpI b initiates activation of platelet. Platelets Adhesion
  22. 22. Platelet activation: The adhesion of platelets to the vessel wall activates them.  Platelets undergo a shape change from disc to tiny sphere with projecting pseudopodes.  Activation required for hemostatic plug formation.  Activators released or synthesized at the site of injury  Thrombin  Exposed collagen fibres  ADP, Adrenaline,serotonin,TxA2
  23. 23.  Immediately after adhesion & activation process of release reaction or secretion begins.  In this process content of platelets organelles are released to the exterior. Secretion of α granules a) PF4 b) β-Thrombomodulin c) Fibrinogen d) Factor V e) Fibronectin f) Thrombospondin g) PDGF h) PAI-1 Platelets Release Reaction
  24. 24. Secretion of dense granules:  ADP, GTP, GDP  Calcium, serotonin  Histamin, epinephrin  ADP released from dense granules promotes platelets aggregation. PF-4 release from alpha granules neutralize the anticoagulant activity of heparin PDGF – stimulate proliferation of vascular smooth muscle cell & skin fibroblast & plays a role in cut healing. TxA2 causes shape change & stimulates release reaction from alpha & dense granules, Also induce aggregation of other platelets & local vasoconstriction
  25. 25. Platelet aggregation Process by which platelets interact with one another to form a hemostatic plug. Chemical changes cause platelets to aggregate and stick to one another GPIIb-IIIa complex binds vWF, undergoes Ca++-dependent structural change, then acts as receptor for fibrinogen Fibrinogen + activated platelets serve as a bridge between two platelets .
  26. 26.  Primary haemostasis involves the binding of platelets to exposed collagen in the sub endothelium of damaged vessels.  Secondary haemostasis is the process of activation of coagulation factors leading to the production of thrombin. 18 July 2015 26
  27. 27. Primary Hemostasis First physiological response to vascular injury, which is mediated by platelets, in order to arrest bleeding Mechanism: – Activation of platelets via stimulators such as thrombin – Adhesion of platelets to subendothelium via interaction between GPIb and von Willebrand Factor (VWF) – Release of platelet granule products in order to recruit more platelets to the injured site – Aggregation of platelets via interaction between GPIIb/IIIa (aIIb3) and fibrinogen to form the initial plug.  Triggers secondary hemostasis (coagulation proteins)
  28. 28. • Process of blood coagulation Mechanism:  Coagulation proteins work in concern to generate thrombin  Thrombin converts fibrinogen to fibrin  Fibrin consolidates the platelet plug made in primary hemostasis such that a thrombus (secondary hemostatic plug) is formed • Prevents further blood loss from the injury site Secondary Hemostasis
  29. 29. Platelet Aggregation 18 July 2015 29
  31. 31. COAGULATION of blood take place with the help of some proteins called clotting factors present in plasma. Clotting factor act as zymogens and under the influence of enzyme are themselves converted into active enzymes Role of Clotting Factors
  32. 32. Coagulation factors in the form of zymogens precursors. • Ca++ as a co-factor and organizing surface,  Provided by platelets in vivo  Provided by a phospholipids emulsion in vitro The coagulation process is initiated when tissue factor - bearing cells are exposed to blood at a site of injury. Requirements for Coagulation
  33. 33. FX FXa Ca++ PL FVIIIa Prekallinokerin kallinokerin FXII FXIIa FXI FXIa FIX FIXa Ca++ FVIIa FVIIa Ca++ Intrinsic pathway Expose collagen Tissue factor FVIIFVIIa Extrinsic pathway Prothrombin Thrombin Fibrinogen Fibrin monomer Cross linked Fibrin clot FXIII FXIIIa Ca++ PL FVa Ca++ FIIa Common pathway
  34. 34.  Fibrinolytic system keep the vascular system free of deposited fibrin clots.  Essential purpose of fibrinolysis is to digest and solublize the fibrin, thus restoring potency to occluded vessel. Fibrinolytic system
  35. 35. Plasminogen activator Plasminogen and plasmin Inhibitors of fibrinolysis: Antiactivators Antiplasmins COMPONENTS OF FIBRINOLYTIC SYSTEM
  36. 36. FIBRINOLYTIC PATHWAY It involves-conversion of plasminogen to plasmin Plasmin break down the fibrinogen or fibrin in their degradation products
  37. 37. Conversion of Plasminogen To Plasmin Plasminogen
  38. 38. Fibrinogen degradation product • Plasmin initially attacks alpha chain of the fibrinogen molecule & removes small fragments designed as A,B & C from the C terminal of the Aα chains. • Followed by degradation of Bβ chains with removal of first 42 amino acids. • Lead to the formation of a large fragment X that still remains fibrinopeptide A. • Next cleavage involves all the three chains in an asymmetrical manner with the release of fragment Y & D. • Fragment Y is rapidly degraded by plasmin liberating two D&E.
  39. 39. Fibrin Degradation Product Degradation of cross linked fibrin is different from fibrinogen. The fibrin degradation products are different because of the presence of covalant bonding. Thus the characteristic fragments are oligomers of X and Y D-dimer. D-E complex and Y-D complex. Fibrin degradation is slower due to the presence of crosslinkages. Normally the FDPs are cleared from the circulation by macrophages.
  40. 40. INHIBITORS Inhibitors of Serine Proteases Antithrombin Inhibitor of coagulation having antithrombin activities Types:  Antithrombin I Show thrombin adsorbing effect of fibrin  Antithrombin II Act jointly with heparin act as heparin cofactor  Antithrombin III(alpha 2 glycoprotein) Antithrombin is a serin protein inhibitor that degrades the serine proteases: thrombin, FIXa, FXa, FXIa, and FXIIa. It is constantly active, but its adhesion to these factors is increased by the presence of heparan sulfate.
  41. 41. Other Inhibitors of Coagulation  α2 macroglobulin – It act as antithrombin, antiplasmin and inactivate kallikrein.  α2 antitrypsin ( alpha globulin) – Inhibitor of factor XIa and antiplasmin and is weakly antithrombin.  α2 antiplasmin – Inactivate plasmin.  Heparin – inhibit the action of thrombin. delay the interaction of thrombin and fibrinogen.
  42. 42. Protein C: • It is a vitamin K dependent glycoprotein synthesized in the liver. • Protein circulate as an inert zymogen and is activated by thrombin in the presence of thrombomodulin on the surface of vascular endothelial cell. • Protein C proteolytic destruction of activated FV & FVIII. Protein S : • It is also vitamin k dependent protein. • Act as a cofactor in protein C reaction and enhance the action of protein C Tissue Factor Pathway Inhibitor(TFPI): • (TFPI) limits the action of tissue factor (TF). It also inhibits excessive TF-mediated activation of FIX and FX. INHIBITION OF COAGULATION CO FACTORS
  43. 43. • Endothelial cell produced Nitric oxide, PGI2 and ADPase. • They act as a antiplatelets agents (inactivate the platelets receptor by binding with them) • ADP produced by platelets is favour to bind with endothelial cell but ADPase inactivate or digest ADP. • Healthy endothelium does not tolerate the presence of activated coagulation factor. • When anti thrombin III binds with heparan sulfate. AT-III is activated & cut down the thromin molecules & some activated coagulation factors ( FIXa, Fxa) SUMMARY How blood is kept in fluid state in circulation ????
  44. 44.  Normally thrombin helps in coagulation but when binds with thrombomodulin (TM) it modulate the function of thrombin which activate the protein C.  Activated protein C digest the activated FVa & FVIIIa.  Endothelial cells not only prevent the platelets aggregation but also inhibit the proteins that involve coagulation.  Endothelial cells also produce tissue plasminogen activator(tPA) which activate the plasminogen to plasmin  Plasmin cleaves fibrin strand in to the FDPs.  FDPs are removed from the circulation by macrophages and some eosinophill.  By this entire process blood is kept in fluid state .
  45. 45. • When the blood vessels are injured mechanically, chemically & physically the first step is vasoconstriction. • After the vasoconstriction platelets are adhere to the endothelial cell membrane. And under goes activation, release reaction and aggregation. • Which forms the primary hemostatic plug and stop the blood leakage from injured site. • Simultaneously activate the coagulation cascade. (extrinsic pathway, intrinsic pathway and common pathway) and forms the fibrin mesh in the clot which establish the plug and form secondary hemostasic plug Which is stable and irreversible. So that bleeding is arrested permanently. How bleeding is arrested???
  46. 46. Essential haematology: A. V. Hoffbrand, P. A. H. Moss, J. E. Pettit - 2006 - 380 pages Hemostasis and thrombosis: basic principles and clinical practice - Page 1569 Robert W. Colman - 2006 - 1827 pages Clinical laboratory medicine: Volume 2001 - Page 987 Kenneth D. McClatchey - 2002 - 1693 pages Consultative hemostasis and thrombosis - Page xxi Craig S. Kitchens, Barbara M. Alving, Craig M. Kessler - 2002 - 617 page Hemostasis and thrombosis protocols: Volume 489 - Page 3 David J. Perry, K. John Pasi - 1999 - 368 pages Rodak, BF, Fritsma, GA & Doig, K (2008). Hematology Clinical Principles & Applications. Saunders Elsevier. Chapters 40 & 45. McKenzie, Shirlyn B (2004). Clinical Laboratory Hematology. Pearson Prentice Hall. Chapter 35 Beckman Coulter Webinars: Fundamentals of Hemostasis at URL Hemostasis Basics: Programmed Learner Part I (Provided by Siemens Healthcare Diagnostics Hemostasis Technical Services at URL References