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Coagulation-From Cut to Clot

Coagulation-From Cut to Clot. Chandra Saravanan Anatomic Pathology Resident. HEMOSTASIS. Hemostasis is a dynamic process whereby blood coagulation is initiated and terminated in a rapid and tightly regulated fashion Imbalance may result in: Thrombosis Hemorrhage 3 components involved:

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Coagulation-From Cut to Clot

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  1. Coagulation-From Cut to Clot Chandra Saravanan Anatomic Pathology Resident

  2. HEMOSTASIS • Hemostasis is a dynamic process whereby blood coagulation is initiated and terminated in a rapid and tightly regulated fashion • Imbalance may result in: • Thrombosis • Hemorrhage • 3 components involved: • Vascular wall (endothelium) • Platelets • Coagulation cascade

  3. Steps in Hemostasis A. Vasoconstriction B. Primary hemostasis (Platelet plug formation) C. Secondary hemostasis (Fibrin clot formation) D. Clot Retraction E. Clot Dissolution

  4. A. VASOCONSTRICTION Robbins and Cotran, 8th edition Trauma > Release of Endothelin > Bind to Endothelin receptors > Elevated intracellular Ca2+ > Reflex vasocontriction

  5. B. Primary Hemostasis: Platelets latelet adhesion vWF Robbins and Cotran, 8th edition

  6. Platelet Adhesion to Vessel Wall Nieswandt et al . 2001 EMBO J • Integrin 21and glycoprotein (GP) VI (on Platelets) to exposed collagen (vessel wall) • GPIb–V–IX receptor complex (on Platelets) and its main ligand von Willebrand Factor (vWf) (vessel wall)

  7. von Willebrand’s factor (vWf) • Large, multimeric glycoprotein produced by endothelial cells and platelets • Monomers linked by disulfide bonds into multimers • Stored in endothelial cytoplasmic granules (Weibel-Palade bodies) • Some is bound to subendothelial collagen • Some circulates in complex with coagulation Factor VIII

  8. von Willebrand Disease • Most common inherited disorder of hemostasis • 50 breeds of dogs • Severity of bleeding is highly variable Kaneko. Clinical Biochemistry of Domestic Animals, 6th Edition

  9. Platelets: Aggregation • Aggregation = Binding to other platelets via fibrinogen • Mediated by Fibrinogen Receptors (GPIIb/IIIa) • Cluster • Undergo conformational change to active state

  10. Glanzmann's Thrombasthenia • Also called thrombasthenic thrombopathia • Autosomal recessive inherited platelet disorder in Otterhounds and Great Pyrenees dogs • Absent or dysfunctional GPIIb/IIIa on platelets (mutation in calcium binding domain) • Prolonged bleeding from minor wounds, spontaneous epistaxis and readily form hematomas at sites of injury or venipuncture • Normal to mildly decreased platelet count, increased mean platelet volume, prolonged buccal mucosa bleeding time, abnormal clot retraction http://ahdc.vet.cornell.edu/clinpath/modules/coags/Glanz.htm

  11. Platelets: Activation After binding or activation by exposure to certain substances, an activating signal is transduced from the platelet surface Activating factors include: ADP, thrombin, thromboxaneA2, thrombospondin, epinephrine, platelet-activating factor Results in release of calcium from the open canalicular system Secretion of granules

  12. Agonists, Receptors, and Effector Systems in Platelet Activation Davi and Patrono . 2007. N Engl J Med

  13. Canine Thrombopathia • Bassett hound: • Petechiae, aural hematomas and prolonged hemorrhage during estrus, shedding of deciduous teeth and after trauma or surgery • Decreased platelet retention and absent platelet aggregation to all agonists, except thrombin • Shape change does occur and clot retraction is normal • May involve defect in cAMP metabolism > increased cAMP> inhibit intracellular Ca2+ release and agonist-receptor binding and agonist-induced phospholipid hydrolysis • Spitz : • Chronic intermittent mucosal bleeding • Platelet secretion in response to ADP is absent

  14. Platelets: Secretion • Conformational change induced by adhesion/aggregation results in granule release • Platelet granules contain numerous preformed substances •  granules • P-selectin, fibrinogen, factors V and VIII, platelet factor 4 (a heparin-binding chemokine), PDGF and TGF- • Dense () granules bodies • ADP, ATP, ionized calcium, histamine, serotonin and epinephrine • Promotes platelet aggregation, fibrin generation, vasoconstriction, inflammation and repair

  15. Platelets: Secretion • Synthesize and release newly formed mediators • Thromboxane A2- stimulates further platelet aggregation, vasoconstriction • Undergo conformational change and expose platelet phospholipid complex (Platelet Factor 3) • Critical site for several steps in intrinsic and common coagulation cascades

  16. Platelets Storage Pool Diseases • Chediak-Higashi syndrome (CHS): • Autosomal recessive genetic disorder in Persian cats, human, mink, mice, cattle and killer whale • Abnormal granule formation in leukocytes, melanocytes, and platelets • No dense granules in platelets • Platelet delta-storage pool disease: • American Cocker Spaniels • Normal coagulation panels, platelet counts and vWf:Ag. • Dense bodies were visible on electron microscopy. • Defect in delta (dense) granule ADP transport with deficient ADP storage

  17. Inherited Thrombocytopenia • Cyclic Hematopoiesis of Gray Collies: • Autosomal recessive disorder • The peripheral cytopenias are the result of a defect in hematopoietic stem cells • The neutropenic and thrombocytopenic episodes occur approximately every 12 days • Mortality is high in affected puppies and most die of infection before six months of age

  18. Boudreaux 2008. JAVMA

  19. C. Secondary Hemostasis: The Clotting Mechanism Robbins and Cotran, 8th edition • Key reaction of the clotting system Fibrinogen (soluble) Fibrin (insoluble polymer) Thrombin

  20. Coagulation • A highly regulated cascade of reactions that form a variety of products involved in hemostasis • Participants include: • – Enzymatic coagulation factors • – Non-enzymatic co-factors

  21. Enzymatic Coagulation Factors • Proenzymes: Plasma proteins that circulate in inactive form; produced mainly in hepatocytes • Upon activation, they gain the suffix “a”; Prekallikrein is an exception, the activated form is called kallikrein • Production of some proenzyme factors is vitamin-K dependent – Factors II, VII, IX, X • These factors bind Ca+2 to allow critical interactions with phospholipid membranes

  22. Coagulation Disorders-Vitamin K • Inherited disorders involving the vitamin K-dependent enzymes have been reported in Devon Rex cats and Rambouillet sheep • Devon Rex cats: • Reduced activity for factor II, VII, IX and X • Defect is due to an abnormal gamma-glutamylcarboxylase (carboxylase-epoxidase) enzyme, which is required for activation of the vitamin K-dependent factors with vitamin K hydroquinone as a cofactor • Dietary deficiency • Rodenticide toxicity

  23. Non-enzymatic Coagulation Factors • Cofactors: necessary for enzymatic coagulation reactions • Cofactors include Factors I, III, V, VIII, High Molecular Weight Kininogen, Ca+2, and phospholipids

  24. Pathways of the “Classical” Coagulation Cascade Robbins and Cotran, 8th edition

  25. Cell-Based Model of Coagulation Schalm’s Veterinary Hematology 6th edition • Circulating FVIIa binds TF on surface of cell • Small amounts of Xa, IXa, thrombin are produced • Thrombin activates platelets, cleaves vWf-fVIII complex, generates Va, VIIIa and XIa • Activated coagulation factors aggregate on activated platelets and result in production of abundant Xa • Xa and Va then produce abundant THROMBIN • Xa is inhibited when separated from platelet surface complex

  26. THROMBIN • 36 kDa enzyme that results from cleavage of prothrombin • Binding sites for: • Na+- determines if thrombin is inactive (avidly binds to anticoagulant Protein C) or active • Anticoagulants • Proteins (protein C, antithrombin, naturally occurring compounds) • Sulfated polysaccharides (heparin and thrombomodulin) • Fibrinogen

  27. THROMBIN- coagulation • Converts soluble fibrinogen to fibrin • Also induces some vasoconstriction • Activates factors V, VII, VIII, XI-- generates more thrombin • Positive feedback/ amplification- Most thrombin generation occurs in association with platelet membranes • Activates factor XIII-- stabilizes clot • Recruits and activates platelets

  28. THROMBIN- inflammation and repair • Recruits and activates platelets • EXTREMELY potent! • Stimulates endothelial cells and macrophages • Via 7-transmembrane G-proteins • Increased expression of leukocyte adhesion molecules • e.g. P-selectin which binds circulating Tissue Factor-bearing membrane microparticles • Secretion/ expression of vasoactive substances and growth factors • VEGF and VEGF receptors • Histamine/ serotonin • Arachidonic acid metabolites

  29. Thrombin-Hemostasis and Cellular Activation Robbins and Cotran, 8th edition

  30. Fibrin Generation Proteolysis phase Polymerization phase Stabilization phase THROMBIN (IIa) FIBRINOGEN FIBRIN MONOMER (Spontaneous) FIBRIN MONOMER FIBRIN POLYMERS FIBRIN POLYMERS UREA-SOLUBLE FIBRIN FACTOR XIII UREA-SOLUBLE FIBRIN CROSS-LINKED FIBRIN Ca++

  31. Mechanism of Damage Due to M Protein Brown 2004. N Engl J Med

  32. D. Clot Retraction (Syneresis) • May toughen the hemostatic plug by squeezing out fluid • Platelet actin and myosin and interplatelet fibrinogen bridges are the major mediators • Lysis of fibrin formed by secondary hemostasis (fibrinolysis) will occur concurrently • Contraction and fibrinolysis minimize the size of the platelet/fibrin plug and initiates vascular repair

  33. E. Fibrinolysis • Plasmin • Derived from circulating plasminogen • General protease with wide variety of substrates, but has two major functions – Fibrinolysis and Inhibition of fibrin polymerization • Also activates Kinin and Complement pathways • Activated by Plasminogen Activators • Inactivated by α-2-antitrypsin and Plasminogen Activator Inhibitors (PAIs) Majno, Cells, Tissues, and Disease, 2nd Edition

  34. E. Fibrinolysis • Plasminogen Activators (PA’s) • Tissue Plasminogen Activator (tPA) • Synthesized by endothelial cells- e.g. upstream from blockage • Most active when bound to fibrin • Urokinase-type Plasminogen Activator (uPA) • Synthesized by many cells, esp. macrophages • Less susceptible to inhibition than tPA • Bacterial-derived Plasminogen Activators

  35. Fibrinolysis • Fibrin Degradation Products (FDPs) – Major fragments include X, D, Y, and E – They impair platelet function – They compete with fibrinogen for binding sites on thrombin and platelets – They interfere with fibrin polymerization – They increase during coagulation, DIC, inflammation, hemorrhage, or decreased clearance due to liver or kidney disease • Phagocytes • Also degrade/ digest clots after ingestion

  36. ENDOTHELIUM Majno, Cells, Tissues, and Disease, 2nd Edition

  37. Inhibition of Platelet Aggregation Prostacyclin (PGI2) • A product of arachidonic acid metabolism produced mainly by endothelium • Increases cyclic AMP concentrations via adenylatecyclaseactivation • Inhibition of platelet adhesion and aggregation • Vascular smooth muscle relaxation and vasodilation Ecto-ADPase • Endothelial cells produce adenosine diphosphatase that degrades ADP and inhibit platelet aggregation

  38. Inhibition of Platelet Aggregation • Nitric Oxide • Produced by endothelium, macrophages and neurons • These have many similarities to O2 metabolites • Inhibition of platelet adhesion and aggregation • Inhibition of leukocyte adhesion and chemotaxis • Vascular smooth muscle relaxation and vasodilation • Increases cyclic GMP concentrations via guanylate cyclase activation • Antimicrobial killing

  39. Inhibition of Coagulation Thrombomodulin Heparin-like Molecules www.medscape.com 2-Macroglobulin: • Activates Plasmin that in turn cleaves fibrin

  40. Endothelium-Derived Hyperpolarization Factor • A product of endothelium that induces hyperpolarization and relaxation of vascular smooth muscle • Its effect is most pronounced on small compared to large arteries • The precise mechanism of action is still unclear • – It involves activation of K+ channels

  41. Hemostasis Regulation • Coagulation inactivators/inhibitors include: • – Antithrombin (Antithrombin III) • – Protein S, Protein C, Thrombomodulin • – Tissue Factor Pathway Inhibitor • Fibrinolytic inactivators/inhibitors include: • – Plasminogen activator inhibitor -1 • – Antiplasmins • – C1 inhibitor

  42. Coagulation Inhibitors • Antithrombin III • -syn: Heparin cofactor (endothelium, liver and megakaryocytes) • - inhibit the activity of thrombin and others (factors IXa, Xa, XIa and XIIa) • - Antithrombin III is activated by binding to heparin-like molecules on endothelial cells • - Two opponents: Platelet factor 4 and adhesive proteins (vWf, thrombospondin, and laminin) • Tissue Factor Pathway Inhibitor: protein produced by endothelium that inactivates tissue factor-factor VIIa complexes

  43. Coagulation Inhibitors • Proteins C and S: • - Vit K-dependent proteins that act in a complex that proteolytically inactivates factors Va and VIIIa • - Protein C activation by thrombomodulin-thrombin complex on endothelial surface

  44. Fibrinolytic Inactivators • Plasminogen activator inhibitor - 1 • – Inhibits Tissue plasminogen activator and • urokinase to prevent conversion of plasminogen to plasmin • Antiplasmins • • −2-antiplasmin is the first to bind and neutralize plasmin • – There is rapid inhibition of circulating plasmin • so fibrinolysis remains localized • • −2-macroglobulin binds excess plasmin after α−2-antiplasmin becomes saturated • • -1 antitrypsin binds plasmin after α−2-macroglobulin becomes saturated

  45. Antiplasmin-Nonfibrinolytic Activity • Antiplasmins also havenon-fibrinolyticactivity • – α−2-macroglobulin can bind to certain • activated factors (eg: thrombin), and entraps but does not inactivate them • – α-1 antitrypsin is a potent inhibitor of Factor XIa • – α-1 antitrypsin and α−2-macroglobulin are the major plasma inhibitors of activated Protein C

  46. Fibrinolytic Inactivators • C1 Inhibitor • C1 inhibitor modulates the complement, coagulation, kinin, and fibrinolytic pathways • C1 inhibitor inhibits: • Activation of C1 • Cleavage of C2 and C4 • Coagulation factors XIIa and XIa • Activation of plasminogen • Activation of kallikrein

  47. Kaneko. Clinical Biochemistry of Domestic Animals, 6th Edition

  48. Examples of Inherited Coagulation Deficiency Disorders

  49. Examples of Inherited Coagulation Deficiency Disorders

  50. Examples of Inherited Coagulation Deficiency Disorders

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