HEMOSTASIS- PHYSIOLOGY {S1} - PowerPoint PPT Presentation

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  2. Four elements of hemostasis: vasoconstriction, platelet aggregation, coagulation cascade ​activation, and fibrin deposition. • Factor II = prothrombin (activated to IIa or thrombin) Factor III = tissue factor Serine proteases include XII, XI, X, IX, VII, and II. These all circulate as zymogens. • Serine protease cofactors include VIII, V, III (tissue factor), and high-mol-wt- kininogen (HMWK).

  3. These increase reaction kinetics by concentrating the serine proteases and their substrates. • ​Vitamin K is required to put the γ-carboxy glutamate side chain on X, XI, VII, II, protein C, ​and protein S. This side chain is required for binding to the platelet plug.

  4. Other clotting elements include fibrinogen and XIII. • ​Factor XIII covalently bonds fibrin strands together, strengthening the clot. It also ​incorporates a​plasmin inhibitor into the clot to resist fibrinolysis.

  5. The extrinsic pathway only uses Factor VII and tissue factor (the extrinsic protein). Extrinsic ​pathway is the most important coagulative pathway. • Tissue factor is never normally in contact with the bloodstream. Vascular damage exposes tissue ​factor, which contacts small amounts of VIIa in the blood. Together these activate Factor X. ​This initiates the common pathway.

  6. The intrinsic pathway uses no extrinsic blood components. Only Factors VIII, IX, and IX are ​important for coagulation. • Factor XII is activated by exposure to subendothelium. XIIa activates prekallikrein to kallikrein, ​using HMWK as a cofactor. Kallikrein activates more XII, starting a positive feedback loop.

  7. Factor XIIa also activates Factor XI, again using HMWK as a cofactor. • Factor XIa activates IX on the phospholipid-rich surface of activated platelets. • At this point, Factor VIII has already been activated by small amounts of IIa from extrinsic pathway.

  8. VIIIa and IXa together are the “tenase complex” and activate Factor X on the surface of the ​platelet plug. Factors IX and X are both Vitamin K dependent since they must bind the ​platelet plug. • This initiates the common pathway.

  9. Factor VIII is a critical cofactor for IXa during activation of Factor X. Deficiency of Factor VIII ​is called Hemophilia A. Deficiency of Factor IX (less common) is called Hemophilia B.

  10. The common pathway starts with activation of Factor X either by VIIa+tissue factor or by the ​VIIIa+IXa “tenase complex.” Both these reactions must occur on the platelet surface. • At this point, Factor V has already been activated by small amounts of IIa from extrinsic pathway.

  11. Factor Xa couples with its cofactor Va to form the “prothrombinase complex.” This activates ​Factor II (prothrombin) to IIa (thrombin). Again, this rxn occurs on the platelet surface. • Thrombin (IIa) then cleaves fibrinogen to fibrin monomers, which loosely polymerize. IIa also ​activates Factors XIII, VIII, V, and protein C. • Factor XIIIa “spot welds” fibrin strands together and incorporates α2-antiplasmin into the clot to ​protect against fibrinolysis by plasmin.

  12. Coagulation Screening tests (APTT and PT) The PT (prothrombin time) measures the extrinsic pathway (VII) and common pathway (X, V, ​II, and fibrinogen). • The APTT (activated partial thromboplastin time) measures the intrinsic pathway (XII, HMWK, ​prekallikrein, XI, VIII) and the common pathway.

  13. Neither test measures XIII. • Increased PT or APTT suggests deficiency in a coag factor, or an auto-antibody against a factor. • A mixing study (mix patient + normal plasma) is used to distinguish between deficiency and ​auto-antibody. • ​In a coag factor deficiency, the prolonged time will be corrected. • ​In autoimmunity against a coag factor, the prolonged time will not be corrected.

  14. Hemophilia A is a factor VIII defiency. X-linked recessive inheritance. • Characterized by deep tissue bleeding, especially in joints (hemarthrosis). • Treatment is injection of VIII. • Hemophilia B is a factor IX deficiency. X-linked recessive inheritance.

  15. vWD • The most common congenital bleeding disorder. Autosomal dominant inheritance. • vWF helps platelets adhere to sub endothelial collagen, and protects factor VIII from being ​degraded by Protein C. • Characterized by bleeding from mucosal surfaces, menorrhagia, and petechia.

  16. The amount of vWF is measured using ELISA. • The function of vWF is measured using ristocetin, an anitbiotic that aggregates platelets only in ​the presence of good vWF. • APTT is often normal in vWF. • Treated with DDAVP, which causes endothelial cells to release their vWF stores.

  17. Vitamin K deficiency • The most common acquired bleeding disorder. • Without Vitamin K, liver produces inactive X, IX, VII, and II (prothrombin). • Factor VII has the shortest half-life, so an increased PT is a good sign of Vitamin K deficiency.

  18. Liver disease causes similar problems, but will have increased PT and APTT. Liver disease will ​cause all factors except VIII to drop (because VIII is produced outside the hepatocytes).


  20. Blood circulation prevents accumulation of clotting factors. • Endothelial cells have Thrombomodulin (binds thrombin, activates Protein C) and tissue ​plasminogen activator to promote Fibrinolysis. • Antithrombin III inhibits serine proteases (IIa, IXa, Xa, XIa), and its effect is magnified 1000x ​by heparin.

  21. Proteins C and S are Vitamin K-dependent proteins that bind to platelets. Protein C is activated ​(“APC”) by endothelial Thrombomodulin complexed with IIa. Protein S floats free in the ​blood. Protein S and Protein C combine to inactivate Va and VIIIa. This only occurs on ​endothelium just adjacent to injury, because it requires thrombin + Thrombomodulin.

  22. Fibrinolytic system relies on plasminogen. Plasminogen is activated by tissue plasminogen ​activator (TPA) and Urokinase (UK). TPA is released from endothelium and converts ​plasminogen to plasmin. Plasmin cleaves fibrin clots into fragments. • α2-antiplasmin inhibits plasmin, and is incorporated into clots by Factor XIII to protect clots.

  23. Estrogen and OC increase synthesis of factor VIII, vWF, and fibrinogen, so they are pro-coagulants. • ​Pregnancy has the exact same effects, and also decreases free protein S.

  24. Factor V Leiden resists degradation by activated Protein C (APC). So this is a congenital pro-​thrombotic mutation. Heterozygotes have higher than normal chance of thromboembolism, ​especially if taking OC. Homozygotes have extremely high chance of thromboembolism. • ​Factor V Leiden increases risk of venous thrombosis but not arterial thrombosis!

  25. Diagnosis of Factor V Leiden is with the APC resistance assay. • ​Use patient plasma plus Factor V deficient plasma plus APC. The APTT time should ​increase a lot, but in Factor V Leiden it doesn’t increase. • ​Another similar mutation is 20210 mutation, which increases translation of prothrombin. ​As with Factor V Leiden, only the risk of venous thrombosis increases.

  26. Other mutations that predispose venous thrombosis --Antithrombin III (which normally inactivates serine proteases). • Heterozygous Protein C or Protein S deficiency. These can be congenital, but also acquired ​with Vitamin K or warfarin therapy. Protein S deficiency also occurs in pregnancy and ​with estrogen therapy or OC.

  27. Anti-phospholipid antibody syndrome • This is an acquired cause of both venous and arterial thrombosis. These antibodies are not ​directed against phospholipids (i.e. platelet surfaces), but rather against proteins that ​normally bind to phospholipids. So the platelet phospholipids have increased exposure ​to clotting proteins.

  28. This will cause heavy thromboses, fetal loss, and thrombocytopenia ​(due to platelet activation/consumption). • ​These antibodies are especially common in RA and SLE patients. • ​The APTT time will be prolonged because little phospholipid is used in the test.

  29. The dilute Russell viper venom time (dRVVT) can also detect these antibodies. The test ​will be prolonged in the presence of anti-phospholipid antibodies.

  30. Elevated homocysteine • Homocysteine is elevated in folate or B12 deficiencies. It increases the risk of both venous and ​​arterial thromboses. • Homocysteine induces tissue factor and damages endothelium, so it initiates coagulation. • Can be treated with folate and B12 supplements.

  31. DIC • DIC is characterized by microvascular thromboses and consumption of platelets and coag factors, ​which causes bleeding. So there is both clotting and bleeding. • DIC is caused by the release of coagulation activators (tissue factor, thrombin) into systemic ​circulation. Fibrinogen, platelets, and other factors get consumed in clots. • Plasmin will digest the fibrin clots into fragments called “D-dimers.”


  33. The intrinsic system is activated by platelet phospholipids, and is measured by APTT. • The extrinsic system is activated by tissue factor and thromboplastin, and is measured by PT.

  34. Endogenous anticoagulants include: • ​prostacyclin (PGI2), which inhibits platelet aggregation ​--antithrombin, which acts like α1-antitrypsin to interfere with intrinsic/common pathways ​--heparin sulfate, which is on endothelial cells and acts like heparin ​--Protein C, which works with Protein S to degrade Factors Va and VIIIa

  35. Exogenous anticoagulant therapies: • ​heparin and coumarins (such as warfarin) are used to prevent and treat thrombus ​--thrombolytics are used to dissolve thrombus

  36. Coumarin anticoagulants • Warfarin is one of the Coumarin anticoagulants. • The mechanism of action is to antagonize Vitamin K. • ​Vitamin K is required for synthesis of Factors II, VII, IX, X, as well as Protein C and S. • ​Vitamin K helps put γ-carboxy amino acids on these factors, which they use to bind to ​platelet phospholipids.

  37. Coumarin specifically block the reduction of Vitamin K, inducing a Vitamin K deficiency. • This decreases synthesis of Vitamin K-dependent coag factors, and also reduces the activity of ​those that are produced. • 99% of warfarin is bound to albumin and isn’t active.

  38. Coumadin anticoagulants affect both the intrinsic and extrinsic pathway, and affects both APTT ​and PT. Most labs measure the drug effect using the International Normalized Ratio (INR). • Achieving steady state takes a long time so dose escalation must be conservative.

  39. Warfarin’s effects are antagonized by induction of drug metabolism (by barbiturates) and ​increased clotting factor production (by Vitamin K and oral contraceptives). • Warfarin’s effects are enhanced by low Vitamin K absorption (by antibiotics) and displacement ​of drug from albumin (by salicylates).

  40. Toxicities of Warfarin: • 1. Bleeding. • ​Antidotes include transfusing fresh frozen plasma (to replace coag factors) or high-dose ​Vitamin K supplementation. • 2. Teratogenicity. • ​Warfarin strictly not allowed during pregnancy. • Heparin • Heparin has variable molecular weights. • Its main mechanism of action is to greatly augment antithrombin activity. • Heparin mostly inhibits the intrinsic pathway, so APTT is prolonged much more than the PT.

  41. The T1/2 depends on the loading dose. This is weird. The greater the loading, the longer the T1/2. ​This is called dose-dependent pharmacokinetics. • Therapeutic monitoring is necessary, due to the risk of bleeding, thrombocytopenia, and alopecia.

  42. To counteract bleeding from heparin: • 1. Stop therapy. • 2. Protamine will reverse the effects of heparin. But protamine can cause anaphylaxis and ​hyper-coagulation. Protamine often induces anaphylaxis in diabetics on NPH insulin.

  43. Low-molecular-weight heparin • Low-molecular-weight heparin inhibits Factor Xa but not IIa. It does not prolong the APTT. • It is better at preventing DVT but equivalent for treating DVT. • LMW heparin therapy does not require therapeutic monitoring.

  44. Thrombolytic drugs • The endogenous system uses plasmin to digest fibrin clots. Plasminogen is activated by tissue ​plasminogen activator (tPA) to plasmin. α2-antiplasmin will inactivate plasmin, ​preventing systemic clot lysis. tPA is also tightly regulated to prevent a lytic state. • 1. Streptokinase activates plasminogen, creating lots of plasmin to digest clots.

  45. The best drug ​to try first, but can’t be used repeatedly because it is immunogenic. • 2. Urokinase activates plasminogen as well. It acts much like tPA. • 3. Recombinant tPA. Much more active on fibrin-bound plasminogen than free plasminogen. ​This provides good specificity for thrombi, but bleeding and systemic lysis is still a ​dangerous side effect. Very short T1/2 and severe toxicities including bleeding, increased ​circulating vWF, and thrombocytopenia. • ​Also, rtPA is associated with a small risk of intracranial bleeding.

  46. Procoagulant drugs • 1. Aminocaproic acid prevents plasminogen and plasmin from binding to fibrin. One obvious ​side effect is the thrombus formation. • 2. Desmopressin (DDAVP) promotes release of Factor VIII and vWF by endothelial cells. ​Often used to treat vWD, mild hemophilia, or platelet.