PHAR 423: Anticoagulants and Thrombolytic Agents

1. PHAR 423: Anticoagulants and Thrombolytic Agents Dr Thomas Abraham Spring 2004

2. Overview of the Coagulation Cascade • Damage to the vessel wall results in platelet adhesion and activation and activation of the coagulation cascade. • DVT, PE, myocardial ischemia, coronary vasospasm, atrial arrhythmia • Adherent platelets release autocoid mediators (TXs, ADP, etc.) which lead to aggregation of platelets. • Aspirin inhibits autocoid release; • Thienopyridines decrease aggregation step • The extrinsic coagulation cascade leads to production of thrombin which processes fibrinogen to fibrin to form a rigid meshwork. • Coumarins interfere with coagulation factor function while heparins inhibit thrombin activity.

3. Overview of the Coagulation Cascade • Adherence of activated platelets to the fibrin strands leads to thrombus formation. • Thrombin formation has multiple consequences but also serves to amplify the extrinsic pathway and enhance platelet release of mediators and aggregation. • A formed clot is eventually degraded by the fibrinolytic pathway. • Enhanced by thrombolytic agents e.g TPA, Urokinase

4. Overview of the Coagulation Cascade Extrinsic Coagulation Pathway • Binding of factor VII to injured blood vessels in the presence of tissue factor (TF), phospholipid and Ca2+ results in activation of factor VII. • Sequential conversion of factors X, V and prothrombin ultimately produce thrombin which has multiple functions e.g. vasoconstriction, mitogenesis of vascular tissue and cleavage of fibrinogen. • Va cofactor for Xa • Cleavage of fibrinogen to fibrin results in the polymerization of fibrin into dense strands that form a meshwork onto which platelets adhere.

5. Orally active Anticoagulants Coumarin and Indandione Derivatives • Bishydroxycoumarin originally identified from sweet clover. • Minimal structural requirements for activity of coumarin derivatives are substitutions at positions 3, 4. • Warfarin is weakly acidic, (sodium salt) and marketed as racemic mixture of R-(d) and S-(l)-forms. S-form has greater potency as anticoagulant. • 4X greater potency of S-isomer • Major anticoagulant effect due to ability of coumarin derivatives to antagonize vitamin K-mediated carboxylation of coagulation factors II, VII, IX, X, protein C and S. Wisconsin Alumni Research Foundation-arin

6. Orally active Anticoagulants Mechanism of coumarin action: • N-terminal glutamate residues are dicarboxylated by a carboxylase with vitamin K as a cofactor. Dicarboxylation of coagulation factors required for calcium binding properties of coagulation factors. • g-carboxylated glutamate residues have the divalency required for high affinity Ca2+ binding. • Cellular reductase regenerates the epoxide to the active vitamin K in the presence of NADH. • Warfarin competes with the epoxide form of vitamin K for binding to the reductase. • This results in build-up of the the epoxide form of vitamin K and decreased carboxylation of newly synthesized coagulation factors.

7. Orally active Anticoagulants Coumarin derivatives (contd.) • Since coumarin derivatives only affect the g-carboxylation of newly synthesized coagulant proteins there is a lag time of several days before optimal anticoagulation is achieved. • More rapidly acting anticoagulants need to be coadministered to the patient until warfarin’s effects become more overt. • Warfarin dosing is adjusted to decrease coagulation function by about 75%

8. Orally active Anticoagulants Coumarin derivatives (contd.) • Almost complete bioavailability from oral, IM, IV admin. of warfarin. Onset of action requires depletion of functional coagulation factors: full anticoagulant effect may take several days to achieve. • Very high plasma protein binding (mostly to albumin); may be a site of major drug interaction. • Caution with oral hypoglycemic agents and cardiac glycosides. • Warfarin will cross the placental barrier and cause birth defects and spontaneous abortion. These agents should be used with extreme caution in any stage of pregnancy. • Metabolized to inactive metabolites by liver (predominant) and kidneys and excreted in stool and urine (enterohepatic cycling); half-life of elimination 25-60 hours. R-form has more prolonged plasma half-life than S-isomer. • Patients with decreased liver function will have decreased coagulant activity – increased susceptibility to warfarin.

9. Orally active Anticoagulants Coumarin derivatives (contd.) • Significant drug interactions are likely with many different agents: • Cholestyramine –chelation of warfarin in the GI tract. • Increased vitamin K intake –decreased ability of warfarin to inhibit reductase function • Amiodarone, sulfinpyrazone, cimetidine, metronidazole,grapefruit juice – decreased warfarin metabolism or increased displacement from albumin binding • Phenytoin, barbiturates, rifampin –induction of warfarin metabolism. • Cephalosporins –heterocyclic side chains inhibit vit. K reductase function to enhance warfarin action. • Adverse effects: bleeding (intracranial, pericardial, nerve sheath, spinal cord, GI), skin necrosis, purple toes syndrome, alopecia, dermatitis, fever, NVD are less common. • Skin lesions, purple toe syndrome may be due to coagulation in microvessels due to the loss of protein C (natural anticoagulant). • Continuous serious bleeding can be treated with admin. of vitamin K (phytonadione); requirement of new functional coagulation factors results in delay of antidote effects (up to 24 h).

10. Orally active Anticoagulants Coumarin derivatives (contd.) • Warfarin and its derivatives may be used in cases of venous thromboembolism, arrhythmias, cerebral vascular emboli and after prosthetic valve placement. Also used in DVT and PE.

11. Orally active Anticoagulants • Interferes with platelet aggregation by: • 1. inhibiting phosphodiesterase in platelets which leads to increased cAMP that decreases platelet adhesion. • Inhibit PDE cAMP metabolism cAMP accumulation in platelets • 2. preventing uptake of adenosine into RBCs causing increased A2 receptor stimulation to increase cAMP in platelets. Dipyridamole (Persantine®) • Adverse effects are primarily due to vasodilator activity of the agent: headache, dizziness, GI intolerance, NVD, flushing. • Used in combination with warfarin for pts with prosthetic heart valves, and in pts. with thromboembolic disease (usually in combination with other agents).

12. Orally active Anticoagulants Aspirin • Metabolism of arachidonic acid results in formation of thromboxane A2 which is a potent platelet aggregator and vasoconstrictor. • Aspirin covalently inactivates cyclooxygenase to decrease TBX A2 production in platelets. • This action occurs at much lower concentrations than required for anti-inflammatory and anti-pyretic effects. • 80-160mg taken daily can inhibit platelet function. • More effective in preventing arterial clots than venous clots

13. Orally active Anticoagulants Thienopyridines: Ticlopidine (Ticlid®), clopidogrel (Plavix®) • Orally active agents. May be prodrugs since they cannot prevent platelet aggregation in vitro. • plasma from treated pts. inhibits platelet aggregation in vitro. • Mechanism of action: antagonizes adenosine diphosphate-mediated binding of activated platelets to fibrinogen. • ADP via the P2PLC receptor causes the activation of phospholipase C and elevation of intracellular calcium and change in shape. • shape change of platelets allows adhesion of fibrinogen • ADP via the P2AC receptor inhibits adenylate cyclase and decreases VAMP phosphorylation. • Gi inhibits adenylyl cyclase to decrease cAMP levels • VAMP involved in platelet aggregation and adhesion • Thienopyridines covalently bind to the P2 receptors to prevent ADP-mediated intracellular signaling mechanisms that promote the stickyness and aggregation of platelets.

14. Orally active Anticoagulants Thienopyridines: Ticlopidine (Ticlid®), clopidogrel (Plavix®) • Thienopyridines appear to be activated by CYP4503A4 to reactive metabolites that covalently interact with the P2 receptors • Will increase bleeding time and has been found to be useful in preventing thrombosis following coronary artery stenting. Often used in combination with aspirin for synergistic therapeutic benefit. May have beneficial effects when combined with GP IIb/IIIa inhibitors. • May be used in pts. who cannot tolerate aspirin, in combination with other anticoagulants. • May decrease metabolism of phenytoin, carbamazepine and theophylline to enhance toxicity. • Generally well tolerated compared to aspirin with the following adverse effects: diarrhea, rash, nausea, dyspepsia, neutropenia (rare but may be fatal), cholestatic jaundice. • In general clopidogrel has less toxic profile than ticlopidine.

15. Parenteral Anticoagulants Heparins • Endogenous heparins are linear polysaccharides of 60-100kDa, complexed to a core protein and found in mast cells. Acidic characteristic and supplied as sodium salt. • mucopolysaccharide • Purified from pig intestinal mucosa or bovine lung; core protein removed. Polysaccharide chain degraded during purification to fragments of 2-30 kDa. Low molecular weight heparins (e.g. enoxaparin, less than 7 kDa) are purified from this fraction by gel filtration. • Heparins not absorbed through GI tract and given parenterally (i.v. or deep s.c., but not i.m.); Subcutaneous has variable bioavailability and delayed onset time. Low mol. wt. heparins have longer biological t1/2. • IM injection can result in intramuscular bleeding, necrosis. • Longer t1/2 of the LMWHs allows less frequent dosing.

16. Parenteral Anticoagulants Heparins (contd.) Heparin Factor Xa or Thrombin Antithrombin III • Mechanism of action: standard heparins catalyze the inhibition of Factor Xa and thrombin by antithrombin III. • Low mol. wt. heparins (enoxaparin, dalteparin) predominantly catalyze the inhibition of Xa. • Used in DVTs, PE, unstable angina, acute MI, during/after angioplasty or coronary stent placement, cardiac bypass surgery • Heparins are contraindicated in bleeding disorders, hypersensitivity to heparins, severe hypertension, renal dysfunction.

17. Parenteral Anticoagulants Heparins (contd.) • Adverse effects include bleeding, thrombocytopenia, hair loss, osteoporosis. • HIT: heparin-induced thrombocytopenia is severe form of platelet destruction that can result in uncontrolled bleeding or thrombus formation. • HIT: due to antibody formation against complex of heparin and platelet factor 4 which results in platelet activation, severe coagulation. • Thrombocytopenia may be less severe with porcine heparin than bovine heparins. • Combined use of standard and LMWHs can lead to excessive bleeding and death. • In case of life-threatening hemorrhage due to heparin the anticoagulant effects can be reversed by administration of protamine sulfate. • Ionic interaction between heparin and protamine inactivates heparin action. • The amount of protamine needs to be titrated to just neutralize the amount of heparin to prevent non-specific effects.

18. Parenteral Anticoagulants • Danaparoid (Orgaran®) • Non-heparin glycosaminoglycans which inhibit factor Xa via antithrombin III • Administered s.c. for DVT prophylaxis and HIT. • Lepirudin (Refludan®) • Recombinant derivative of hirudin. • Hirudin originally isolated from the salivary gland of medicinal leech • A direct inhibitor of thrombin activity. • Primarily used in HIT.

19. Parenteral Anticoagulants Inhibitors of Platelet Glycoprotein IIb/IIIa Role of Glycoprotein IIb/IIIa in thrombus formation • On unstimulated platelets, GP IIb/IIIa is in a conformation that has low affinity for soluble fibrinogen. • When platelets are activated, they undergo morphologic and physiologic changes, and GP IIb/IIIa molecule alters its conformation, becoming a high-affinity receptor for fibrinogen. • Each fibrinogen molecule can bind to 2 GP IIb/IIIa molecules and therefore cross-link receptors on adjacent activated platelets and ultimately lead to formation of platelet-rich thrombi.

20. Parenteral Anticoagulants (GPIIb/IIIa Inhibitors) Abciximab (ReoPro®) • Is the Fab fragment of a chimeric monoclonal antibody. Administered by i.v injection or infusion. • Used in pts. with unstable angina, post-MI and following angioplasty • Binds to platelet glycoprotein IIb/IIIa receptors to prevent adhesion of platelets to fibrinogen. • High affinity binding of the mAb to GPIIb/IIIa leads to prolonged platelet inhibition. • Also binds other integrin receptors on vascular endothelial cells • Adverse effects include bleeding, nausea, vomiting, hypotension, atrial fibrillation, edema. • Hypersensitivity reactions due to antibody formation Platelet Collagen ADP RheoPro Thrombin

21. Parenteral Anticoagulants (GPIIb/IIIa Inhibitors) Eptifibatide (Integrilin®) • A heptapeptide cyclized by a disulfide bridge that mimics carpet viper venom toxin in preventing coagulation. • Eptifibatide binds to GP IIb/IIIa with high affinity and prevents the interaction with von Willebrand factor and fibrinogen to prevent the formation of stable platelet aggregates. • Indicated for pts. suffering from acute MI or undergoing PTCA to prevent the formation of new clots. • Used in unstable angina • Generally used in combination with aspirin and heparin • Due to the relatively specific interaction of eptifibatide with GP IIb/IIIa no significant adverse effects result apart from potential for severe bleeding.

22. Parenteral Anticoagulants (GPIIb/IIIa Inhibitors) Tirofiban (Aggrastat®) • Synthetic, nonpeptide antagonist of the GPIIb/IIIa receptor that prevents fibrinogen binding to platelets. • Poor oral bioavailability requires parenteral administration of tirofiban but other agents in this class are being devloped for oral adminstration e.g. sibrafiban, xemilofiban and orbofiban. • Major adverse effects observed are potential for bleeding and thrombocytopenia especially when administered in combination with heparin. • Anticoagulant effect is more reversible after withdrawal than that seen with RheoPro.

23. Thrombolytic Agents • Physiological Fibrinolysis • Fibrinolytic system dissolves intravascular clots as a result of plasmin activation. • Targets pathological rather than physiological clots • Plasmin cleaves fibrin strands and coagulation factors to dissolve clots. t-PA Fibrin Plasminogen • Regulation of plasmin activation: • removes unwanted fibrin thrombi but fibrin in wounds is maintained. • endothelial cells release tissue plasminogen activator (t-PA) in response to hemostasis. • t-PA rapidly inactivated by plasminogen activator inhibitor 1 and 2 (unless t-PA is bound to fibrin) to prevent systemic activation of plasminogen to plasmin. • t-PA binds to fibrin and converts plasminogen, that is also bound to fibrin, to plasmin. • activated plasmin bound to fibrin is protected from the negative actions of a2-antiplasmin • pharmacological application of plasminogen activators overwhelms the inhibitory controls on fibrinolysis.

24. Thrombolytic Agents • Streptokinase (Kabbikinase®, Streptase®) • 47 kDa protein produced by b-hemolytic streptococci; not an enzyme but forms stable 1:1 complex with plasminogen. • Alters the configuration of plasminogen to expose the proteolytic catalytic site which autocatalyzes its own activation to free plasmin. • Will activate plasminogen that is not bound to fibrin – less specific than t-PA • High loading doses are required to overcome antibody inhibition; has half-life of 40-80 min; streptokinase:plasminogen complex not inhibited by anti-plasminogen inhibitors. • Adverse rxns. include bleeding, allergic rxns., anaphylaxis and fever (rare). • Allergic rxns. in pts. who have had b-hemolytic streptococcal infections • Anistraplase (Eminase®) is a plasminogen-streptokinase complex that is more fibrin specific than streptokinase alone. Streptokinase

25. Thrombolytic Agents Tissue plasminogen activator (Alteplase®, Activase®) • Serine protease of ~ 500kDa, produced by recombinant DNA technology. Metabolized by liver with half-life of 5-10 min. • Requires the presence of fibrin to convert plasminogen to plasmin. Effective in lysing clots during acute MI, pulmonary embolism and DVT. • Reteplase is modified by the removal of several amino acids from the t-PA sequence. • Urokinase (Abbokinase®) • A serine protease isolated from cultured human kidney cells . • Used in the clearing of clots from IV lines. • Metabolized by liver with half-life of 15-20 min. • Is not fibrin specific and can cause systemic lytic state. • Current supply is inconsistent due to production problems

26. Thrombolytic Agents • Adverse effects of thrombolytic agents • Hemorrhagic toxicity is the main adverse effect in thrombolytic therapy and is due to: • Lysis of fibrin in physiological thrombi • Systemic lytic state • After inhibitory protein have been overwhelmed and destruction of coagulant proteins results • Can lead to intracranial hemorrhage • Excessive fibrinolysis due to t-PA can be limited with aminocaproic acid • Contraindications: GI ulcers, bleeding disorders, hypertension, recent surgery, aortic dissection.