HEMOSTASIS- PHYSIOLOGY {S1}

HEMOSTASIS- PHYSIOLOGY {S1} BY RANJEET RAMAN

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).

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

THROMBOSIS

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.

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.

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.

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.

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!

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.

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.

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.

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.

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

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.

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.”

TARGETING COAGULATION CASCADE

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.

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

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

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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

HEMOSTASIS- PHYSIOLOGY {S1}