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Hemostasis/Coagulation Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653.

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Presentation on theme: "Hemostasis/Coagulation Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC 27709 919-541-0653."— Presentation transcript:

1 Hemostasis/Coagulation Gregory S. Travlos, DVM, DACVP National Institute of Environmental Health Sciences Research Triangle Park, NC

2 Hemostasis The process by which bleeding is arrested. It is a series of physiological and biochemical events which terminate in the formation of an insoluble fibrin clot Hemostatic Sequence: Interaction between vessel wall and platelets Blood coagulation Fibrinolysis

3 Hemostatic Component Interactions Thompson & Harker, 1983

4 Blood Vessels Intact endothelium forms a thromboresistant surface Required for the free flow of blood; does not promote platelet adherence or activate coagulation Passive mechanisms: Endothelial glycocalyx (negative charge - repels like-charged particles, e.g.,platelets). Presence of  2-macroglobulin at cell surface (protease inhibitor). Active mechanisms: Endothelial cells remove platelet aggregation promoters from circulation (e.g., PGF 1, bradykinin, serotonin, adenine nucleotides). Secretion of PGI 2 - potent inhibitor of platelet aggregation, induces vasodilation. Proteoglycan matrix of the vessel wall influences thrombogenicity. Heparin, heparan sulfate and dermatan sulfate have anticoagulant activity; other glycosaminoglycans and hyaluronic acid do not. Veins have the highest concentration.

5 Endothelium Besides their role in thromboresistance, endothelial cells have additional synthetic functions. Produce Von Willebrand’s factor Absorbed by platelets; needed for adherence to collagen Produce plasminogen activator (tPA) Mediates fibinolysis Injured cells release thromboplastin (factor III) Activates the “extrinsic” coagulation cascade Others (e.g., type III and IV collagens, elastin, fibronectin, etc.)

6 Blood Vessel Structure Thompson & Harker, 1983

7 Platelets Adhere to exposed collagen (platelet plug) Occurs in seconds; can control hemorrhage of minute injuries Secretory functions; mediators of coagulation and fibrinolysis Releases ADP; sticky and promotes platelet adherence ADP activates phospholipase A2 which stimulates thromboxane A2 synthesis Release of membrane fibrinogen, factor V, factor VIII and calcium Release of membrane platelet phospholipid.

8 Platelet - TEM mitochodrion microtublules OCS granules

9 Ultrastructural and Functional Platelet Anatomy

10 Platelets - cont. The role of platelets in hemostasis is as important as the coagulation mechanism. Thrombocytopenia, thrombasthenia or thromobopathia - impair hemostasis Thrombocytosis or thrombocythemia - may impair, but usually promotes clotting (predisposes to thrombosis). Platelets promote hemostasis by: Release of ADP and other agonists; promotes adherence. ADP activates phospholipase A 2 which stimulates thromboxane A 2 synthesis Thromboxane A2 - stimulates vasoconstriction and platelet aggregation Release of membrane fibrinogen, factor V, factor VIII and calcium Components of coagulation localized at site of injury Release of membrane platelet phospholipid. Accelerates the “intrinsic” and “common”pathways of coagulation

11 Prostaglandin Metabolism Harlan & Harker, 1981

12 Hemostatic Platelet Functions Thompson & Harker, 1983

13 Platelet Response When a vessel is injured or severed a brief, local, reflex vasoconstriction occurs. Reduces blood flow at site. Maintained by vasoactive compounds (platelets, surrounding tissues). Passing platelets adhere to exposed collagen. Occurs in seconds; initially adhere in a single layer and become activated. Severe injury - collagen serves as a potent platelet activator. Less severe injury - vWF and fibrinogen become the major activators. The adhered platelets undergo a conformational change. From discoid to development of long filopodia. Activation of GP receptors for fibrinogen and/or vWF (GPIIb/IIIa and GPIb/IX/V).

14 Structure of the GPIb-IX-V receptor Tablin, 2000

15 Platelet Response to Agonists Platelets - unstimulatedAddition of ADP (mild stimulation) Addition of thrombin (strong stimulation ) Characteristic discoid shapeShape change (elongation and crescents) and filaform process formation (arrows) Increased spreading, filaform process extension (arrows) and aggregate formation (stars) SEM plates; Gentry, 2000

16 Platelet Response cont. Activated platelets release their  -granule and dense body contents inducing additional platelet recruitment. Dense granules - ADP, serotonin and epinephrine. alpha-granules - fibrinogen (and vWF in human and pig). Synthesis and release of PAF and TxA 2. The agonists accelerate the development of an irreversible platelet aggregate (platelet plug). Reversible v. irreversible responses. Thrombocytes of birds and reptiles do not respond to ADP. Serotonin and epinephrine: Serotonin - shape change (rat, g. pig and dog); aggregation (human, rabbit, cow, horse, pig, sheep and cat). Epinephrine - only human, primate, cat and horse platelets appear responsive. Either serotonin or epinephrine combined with another agonist - strong response in all species.

17 Platelet Response cont. More about agonists. Platelet Activating Factor (PAF). Cow, horse, sheep, primate, dog, g. pig and rabbit respond to PAF. Human less sensitive and rat and mouse are insensitive to this agonist. Thromboxane A 2 (TxA 2 ). Strong agonist - human, g. pig and rabbit. Weak agonist - horse. Insensitive - rat, cow, pig. In real life, however, platelets are exposed to multiple agonists from platelets and other cells (e.g., red cells, ADP; white cells, PAF).

18 Platelet Aggregation to Thrombin Harlan & Harker, 1981

19 Hemostatic Plug Formation Baumgartner & Muggli, 1980

20 Coagulation System Consists of a cascading system of proteins Primarily originating from liver (except factor III) Circulate in inactive form (except, possibly, factor VII) System includes: Enzymatic factors Non-enzymatic factors Tissue thromboplastin (factor III) Calcium (factor IV) Platelet phospholipid (PF 3) - structural component; accelerates factor activation Anticoagulant factors The coagulation system consists of three pathways (intrinsic, extrinsic and common)

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22 Procoagulant Factors

23 Coagulation Systems - cont. Enzymatic factors Circulate as non-active zymogens - must be activated to function Activated enzymatic factors are not consumed during clotting (except factors II and XIII) Partial deficiency results in partial loss of clotting ability Activated enzymatic factors inhibited by antithrombin III (complexed with heparin) and some alpha-2-glycoproteins Enzymatic factors: XI and XII (contact factors) II, VII, IX and X (vitamin K-dependent factors) XIII (clot stabilizing factor or fibrin-stabilizing factor)

24 Coagulation Systems - cont. Non-enzymatic factors Originate from liver but associate with platelet membranes (also found in plasma) Normal clotting with partial deficiency; almost total absence needed to affect hemostasis or clotting Clotting consumes these factors - absent in serum No known natural inhibitors Considered reactive proteins - increased during inflammatory and neoplastic processes (except factor III) Non-enzymatic factors: Fibrinogen (factor I) Factor V Factor VIII:C (associated with Von Willebrand’s factor)

25 Coagulation Cascade Interactions

26 Does this turkey have factor XII? Of course, he does But, his feathered companion does not

27 Coagulation Systems - cont. Clot stabilization Fibrin stabilizing factor (factor XIII) forms fibrin strand cross-links. Synthesized by monocytes and hepatocytes. Zymogen is activated by thrombin (plus calcium). A very small amount of factor XIII (2 - 10%) is adequate for hemostasis. Converts soluble fibrin monomers (unstable) to a fibrin polymer (stable). Lead, silver, zinc and snake venoms are known inhibitors.

28 Coagulation Inhibitors The activity of coagulation system must be attenuated. Numerous inhibitors are found in blood. Coagulation is controlled by three types of actions. Inhibition of converting enzymes (e.g., AT III, C1 esterase inhibitor,  2 - macroglobulin,  2 -antiplasmin,  1 -antitrypsin, HC-II). Act on one or more of the converting enzymes (activated factors). AT III-heparin pathway: major system - 80% of the thrombin inhibitory action in plasma. Destruction of protein cofactors (e.g., TM-PC-PS system). TM-PC-PS system degrades cofactors V & VIII:C, inhibiting prothrombinase and tenase complexes, respectively. Blocking receptor availability needed for complex formation (e.g., Tissue factor pathway inhibitor (TFPI) and annexin V).

29 Proposed Mechanism of AT III-Heparin System Heparin Thrombin Antithrombin III Lysine sites Serine site Arginine site H Th H AT III Th

30 Proposed Mechanism of Thrombomodulin, Protein C and Protein S (TM-PC-PS) System Thrombin Prothrombin Protein C Thrombomodulin Thrombin F-Xa Activated platelet PS F-Va x Ca ++ Activated Protein C

31 Proposed Mechanism of Tissue Factor Pathway Inhibitor (TFPI) Activity F-Xa Endothelium Tissue factor F-VIIa TFPI F-Xa TFPI F-Xa

32 Anticoagulant Factors

33 Fibrinolytic System Method for removing clots and maintenance of a patent vascular system and fibrin deposited during inflammation and tissue injury must be removed. Plasmin (serine protease) primarily responsible for fibrinolysis. Produced in the liver and kidney, it circulates in an inactive form (plasminogen). Activators: tissue plasminogen activator (tPA), cytokinases-urokinases (urine, CSF, tears, saliva, milk, bile, synovial, prostatic and amniotic fluids), erythrocyte erythrokinase, neutropil activator and factor XII-dependent activator (XII-prekallikrien-hageman factor cofactor complex). In addition to fibrin and fibrinogen, plasmin will hydrolyse a variety of proteins. While plasminogen is normally found in blood and body fluids, plasmin is usually absent due to numerous antiplasmins. Inactivators: antithrombin III,  2-macroglobulin,  1-antitrypsin and C1 inactivator.

34 Fibrinolytic System and Factors Regulating Fibrinolysis (Fibrinogenolysis) PlasminogenActivationInhibition Damaged endothelium Kallikrein Plasminogen activator inhibitor  -aminocaproic acid Prekallikrein Streptokinase Urokinase FHIIa tPA Plasmin  2 -Antiplasmin  2 -Macroglobulin Complement activation Fibrin/fibrinogen Degradation products Biodegradation of FV, FVIII, FIX, FXI fibrinogen Firbrinogen/fibrin

35 Degradation of Fibrin/Fibrinogen Fibrinogen or Fibrin Fragment XSmall Peptides Fragment YFragment DSmall Peptides Fragment EFragment DSmall Peptides Plasmin

36 Evaluation of Hemostasis Fundamental physiology and pathophysiology of hemostasis is similar in mammalian species. Variables identical for laboratory animals and human patients Platelets Platelet count - detection of thrombocytopenia Clot retraction - non-anticoagulated blood Failure to separate - platelet function defect or thrombocytopenia Bleeding time (BT)- in vivo test; simple; low sensitivity Used to evaluate platelet function defects Thrombocytopenia - prolongs BT Clotting factor deficiency does not alter BT Vascular disease (eg., scurvy) can prolong BT (humans, guinea pigs)

37 Considerations for Blood Collection Clean/smooth surfaces Want to avoid platelet clumping or activation of factor XII Use plastic or siliconized glass for sample collection Animal blood clots faster than human blood - prime needle with anticoagulant Collect sample from an endothelial-lined vessel and careful venipuncture Want avoid contamination with tissue juice (factor III) Small clot activates coagulation system invalidating results Samples from indwelling catheters are usually unacceptable

38 Sample Handling/Anticoagulants Plasma samples separated from cells within 30 minutes Perform analyses immediately Plasma samples may be quickly frozen (dry ice/alcohol or liquid nitrogen) and stored at -70 o for analysis at a later date Activity of factors V and VIII is lost rapidly in samples held at room temperature Citrate (trisodium salt) is the anticoagulant of choice. Oxalate anticoagulants are acceptable - not commonly used Heparin - unacceptable EDTA - unacceptable (except for indirect evaluation of fibrinogen concentration by heat precipitation and refractometry)

39 Evaluation -cont. Activated Coagulation Time (ACT) - in vivo test Measures (seconds) time to clot formation in fresh whole blood Careful attention to sample collection/handling Platelet counts <10,000 cause slight increase in ACT Results from lack of platelet phospholipid for test Increased ACT suggests factor deficiency in intrinsic or common pathways Deficiency must be 5% of normal to prolong ACT Activated Partial Thromboplastin Time (APTT) Measures (seconds) time to clot formation in citrated plasma Increased APTT - factor deficiency in intrinsic or common pathways Deficiency must be 30% of normal to prolong APTT Fibrinogen <50 mg/dL will prolong APTT; inflammation may shorten APTT Sensitivity increased with saline-diluted plasma Heparin therapy prolongs APTT - differentiate using a 1:1 dilution with normal plasma

40 Evaluation -cont. One-Stage Prothrombin Time (OSPT, PT) Measures (seconds) time to clot formation in citrated plasma Rabbit or synthetic tissue thromboplastin preferred; human origin reagent gives longer PT times Increased PT - factor deficiency in factor VII or common pathway Deficiency must be 30% of normal to prolong PT Fibrinogen <50 mg/dL will prolong PT Sensitivity increased with saline-diluted plasma Russel’s Viper Venom Time (RVVT) Measures (seconds) time to clot formation in citrated plasma Increased RVVT - in or common pathway but insensitive to factor VII deficiency Deficiency must be 30% of normal to prolong RVVT Fibrinogen <50 mg/dL will prolong RVVT Sensitivity increased with saline-diluted plasma

41 Evaluation -cont. Thrombin Clotting Time (TCT) Measures (seconds) time to clot formation in citrated plasma Increased TCT - decreased fibrinogen concentration (<100 mg/dL), dysfibrinogenemia, increased FDP concentration, heparin therapy Fibrinogen Concentration (factor I) In most species, fibrinogen is mg/dL Fibrinogen decreases in DIC, severe liver insufficiency and hereditary hypofibrinogenemia Inflammation can increase fibrinogen concentration

42 Evaluation -cont. Fibrin-Fibrinogen Degradation Products (FDP) Measures, by latex agglutination, the concentration of products of fibrinolysis; D-dimer assay is another method for measuring FDP Increased FDP - occurs with disseminated intravascular coagulation or severe internal bleeding In most species, normal FDP is <10 micrograms/mL

43 Example Acute oral study in dogs Animals given 3 X LD 50 in food Brodifacoum Bromadiolone Diphacinone Coagulation studies ACT RVVT PT

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