Parenteral Anticoagulant Drugs

Heparin is a linear polysaccharide with alternating residues of glucosamine and either glucuronic or iduronic acid (Fig. 26-4) derived from animal sources. The amino group of glucosamine is either acetylated or sulfated, and there is a variable degree of sulfation (≤40%) on the hydroxyl groups, rendering heparin a heterogeneous compound. Heparin acts by increasing the activity of antithrombin, a plasma glycoprotein that inhibits serine protease clotting enzymes. Heparin binds to antithrombin, causing a conformational change that renders the reactive site on antithrombin more accessible to serine proteases, inactivating thrombin and factors IXa and Xa; low molecular weight heparin inhibits mainly factor Xa. After the binding of antithrombin to thrombin, the heparin molecule is released and can bind to another antithrombin molecule. Although low doses of heparin act primarily by neutralizing factor Xa, at high doses it acts by preventing thrombin-induced platelet activation and prolongs bleeding time. Although the heparin-antithrombin complex is a very efficient inhibitor of free thrombin, clot-bound thrombin is resistant to inhibition.

FIGURE 26–4 Structures of selected anticoagulants. Top left: Structure of a repeating unit in heparin. There is considerable variation in the extent of sulfation of different hydroxyl groups. Lower right: Abciximab is a Fab fragment of a chimeric human-murine monoclonal antibody. The variable (V) and constant (C) regions of the light (L) and heavy (H1) chains are shown.

Fondaparinux is a synthetic pentasaccharide that binds to antithrombin and selectively catalyzes inactivation of factor Xa. Because of its short chain length, it does not promote thrombin inhibition, making it an antithrombin-dependent selective factor Xa inhibitor. Fondaparinux is used to prevent and treat deep venous thrombosis and does not affect platelet function.

Several agents directly inhibit thrombin. Hirudin is a 65-amino-acid leech salivary gland protein that directly inhibits thrombin activity by blocking the active site of thrombin, as well as another site that mediates fibrinogen binding. Recombinant hirudins include desirudin and lepirudin, while analogs include bivalirudin. These compounds are used primarily in patients intolerant of heparin.

Argatroban is a synthetic, directly acting thrombin inhibitor derived from l-arginine that reversibly binds to active site of thrombin. Argatroban is used as an alternative to the hirudin analogs.

Oral Anticoagulant Drugs

The oral anticoagulants, typified by warfarin and the coumarins (Fig. 26-4), represent a very important class of agents whose action involves their ability to inhibit vitamin K. A subset of blood coagulation factors (II, VII, IX, X) and anticoagulant proteins C and S are activated via the γ-carboxylation of several glutamic acid residues, which mediate their Ca⁺⁺-dependent binding to phospholipid surfaces, critical for assembly of complexes necessary to generate thrombin. This activation requires vitamin K as a cofactor, and carboxylation of these vitamin K-dependent coagulation factors leads to the concomitant oxidation of vitamin K to its corresponding epoxide. The regeneration of vitamin K necessary to sustain the carboxylation reaction is mediated by vitamin K epoxide reductase, an enzyme inhibited by warfarin and the coumarins. Thus these compounds block recycling of the oxidized form of vitamin K to the reduced form required for cofactor function. Because these compounds inhibit the synthesis of clotting factors but have no direct effect on previously synthesized factors, plasma levels of preexisting vitamin K-dependent factors must decline before the anticoagulant effect of these agents becomes apparent, which requires several days. The first to decline is factor VII, followed by other factors with longer half-lives (see Table 26-2). The full anticoagulant effect of warfarin is typically reached within 4 to 7 days. Because of genetic variations in metabolism, drug interactions, and differences in vitamin K intake, significant variations between individuals exist in the time required for a maximal effect and in doses required for maintenance. Consequently, careful monitoring of prothrombin time (PT), a standard laboratory measure, is necessary.

TABLE 26–2 Rates of Disappearance (Half-Lives) of Vitamin K-dependent Proteins from Blood

Proteins C and S, two other vitamin K-dependent factors, also inhibit excessive coagulation in the activated state. This mechanism involves the binding of thrombin to thrombomodulin, an endothelial cell surface protein, which results in a different proteolytic specificity than free thrombin. In this state thrombin does not cleave fibrinogen or activate platelets. Rather, it activates protein C, which in combination with protein S proteolytically inactivates clotting factors Va and VIIIa (Fig. 26-5), thereby providing a feedback inhibition to down regulate blood clotting after vascular injury. Genetic deficiency of protein C or protein S can cause thromboembolic disease.

FIGURE 26–5 The anticoagulant protein C pathway. Thrombin bound to thrombomodulin on the surface of vascular endothelial cells has a proteolytic selectivity different from that of free thrombin. Rather than cleaving fibrinogen, it cleaves protein C to activated protein C, which then cleaves factors Va and VIIIa to give inactive products. This process is accelerated in the presence of protein S and platelets. Both protein C and protein S are vitamin K-dependent and affected by warfarin.

Fibrinolytics

Fibrin clots are lysed mainly through the proteolytic action of plasmin, the enzyme produced by the proteolytic activation of plasminogen by plasminogen activators (Fig. 26-6). A minor aspect of clot lysis may result from release of proteolytic enzymes, such as elastase, from leukocytes. The two major classes of endogenous plasminogen activators are tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA). Recombinant forms of these proteins are used as clot-dissolving drugs.

FIGURE 26–6 Key components of the fibrinolytic system. Dotted lines depict inhibition of t-PA and u-PA by plasminogen activator inhibitor-1 (PAI-1) and the inhibition of plasmin by α₂-antiplasmin.

Streptokinase, a protein produced by streptococci, forms a complex with plasminogen that activates free plasminogen molecules to plasmin and is also used as a thrombolytic agent. Plasmin formed by the action of plasminogen activators attacks not only fibrin but also several other proteins, including fibrinogen, factor V, and factor VIII. The recombinant forms of u-PA and t-PA may have some advantages over streptokinase because of their selectivity in binding to the fibrin clot, but streptokinase is less expensive. Streptokinase, however, is highly immunogenic and cannot be used repeatedly. Modified forms of t-PA, such as reteplase and tenecteplase, have deletions or mutations in domains responsible for clearance from the circulation.

Antiplatelet Drugs

The activation and subsequent aggregation of platelets is a major component of arterial thrombosis and may be involved in initiation of venous thrombosis. Interaction of platelets with vessel wall collagen appears to be a key step. Activation of platelets leads to formation and release of thromboxane A₂ (TXA₂) from arachidonic acid in platelet membranes (see Chapter 15). TXA₂ is a potent aggregating agent and vasoconstrictor. Platelet activation also causes secretion of adenosine diphosphate from storage granules. Both TXA₂ and adenosine diphosphate, which act through specific receptors, cause activation of integrin α_IIbβ_IIIa receptors on the platelet surface for fibrinogen and for other adhesive proteins, including von Willebrand’s factor (vWF). Fibrinogen binding to its integrin receptor mediates aggregation, whereas vWF is involved primarily in adhesion of platelets to extracellular matrices in the vessel wall. Thrombin generated locally on the surface of activated platelets greatly amplifies the response by causing further activation and mediator secretion. Although TXA₂, adenosine diphosphate, and thrombin all increase cytoplasmic Ca⁺⁺, the mechanism by which thrombin activates its receptor is unique. Thrombin cleaves the N-terminal sequence of its platelet receptor (PAR1), forming a new N-terminus that serves as a “tethered ligand” and binds to and activates the receptor to induce transmembrane signaling (Fig. 26-7).

FIGURE 26–7 Predicted linear structure of the platelet thrombin receptor, protease activated receptor 1 (PAR1). It is thought to have a characteristic seven-transmembrane domain G-protein-coupled receptor structure (see Chapter 1). However, thrombin acts by cleaving the N-terminal extracellular tail of the receptor, releasing an activation peptide and leaving a new N-terminal sequence: Ser-Phe-Lys-Lys-Arg-. This sequence acts as a “tethered ligand,” which activates the receptor and is a powerful aggregating agent.

Platelet activation is inhibited by elevation of intracellular cyclic adenosine monophosphate (cAMP), and agents that increase this second messenger inhibit aggregation. The most active agent is prostacyclin, released by cells of the vessel wall (see Chapter 15). Other mediators from endothelial cells may also contribute.

The major therapeutic approach to reducing platelet aggregation is through inhibition of cyclooxygenase. Aspirin irreversibly inhibits platelet cyclooxygenase by acetylating a serine residue near the active site of the enzyme, thereby blocking TXA₂ formation (see Chapter 36). Aspirin also blocks the synthesis of the endogenous vasodilator and platelet inhibitor prostacyclin, although at standard doses this prothrombotic effect is insignificant.

Clopidogrel (see Fig. 26-4) irreversibly blocks activation of the platelet P2Y receptor by adenosine diphosphate. Clopidogrel is a prodrug that is metabolized by cytochrome CYP3A to an active metabolite. Clopidogrel has become a mainstay in the treatment of patients with coronary artery disease, and it is also commonly administered to patients with peripheral arterial occlusive disease and cerebrovascular disease. It is routinely administered with aspirin, particularly to patients who have received coronary artery stents.

Dipyridamole acts as an antiplatelet drug by stimulating prostacyclin synthesis, enhancing its inhibitory action, inhibiting phosphodiesterase, and blocking the uptake of adenosine into vascular and blood cells, leading to accumulation of this platelet-inhibitory and vasodilatory compound. At therapeutic doses dipyridamole does not prolong bleeding time or inhibit ex vivo platelet aggregation. Aggrenox is a combination antiplatelet agent consisting of dipyridamole and aspirin that has been found to be useful in secondary prevention of stroke.

Inhibitors of glycoprotein IIb/IIIa (Fig. 26-8) bind to α_IIbβ_IIIa, or glycoprotein IIb/IIIa, the platelet integrin receptor for fibrinogen, vWF, and other adhesive ligands. These inhibitors prevent fibrinogen cross-linking of platelets, which is the final common pathway of aggregation. These agents are administered by intravenous infusion, primarily to prevent platelet-dependent thrombosis during treatment of acute coronary artery syndromes and after implantation of intracoronary stents. Abciximab is the Fab fragment of a chimeric human-murine monoclonal antibody that binds to the glycoprotein IIb/IIIa receptor of human platelets (see Fig. 26-4). Abciximab also binds to the vitronectin receptor (α_Vβ₃) present on platelets, vascular endothelial cells, and vascular smooth muscle cells. Platelet function gradually recovers after abciximab infusion is stopped. Plasma drug levels fall quickly, though platelet-bound abciximab can be detected for up to 15 days.

FIGURE 26–8 Inhibition of platelet aggregation by glycoprotein (GP) IIb/IIIa receptor antagonists. The GP IIb/IIIa receptors on unstimulated platelets exist in an inactive conformation that does not support fibrinogen binding. Activation of platelets by agonists, such as adenosine diphosphate (ADP), epinephrine, thrombin, and thromboxane A₂, converts the GP IIb/IIIa receptors to an active conformation capable of binding fibrinogen, which leads to the formation of platelet aggregates. Blocking of the GP IIb/IIIa receptors with antagonists, such as abciximab, eptifibatide, and tirofiban, prevents fibrinogen from cross-linking activated platelets, thereby inhibiting thrombus growth.

Eptifibatide is a cyclic heptapeptide containing six amino acids and one mercaptopropionyl (des-amino cysteinyl) residue. Eptifibatide inhibits platelet aggregation by blocking the binding of fibrinogen to glycoprotein IIb/IIIa. Inhibition of platelet aggregation by eptifibatide is reversible after the drug is stopped due to dissociation from the platelet surface.

Tirofiban is a nonpeptide antagonist of the platelet glycoprotein IIb/IIIa receptor. Platelet inhibition after tirofiban is reversible after infusion is stopped.

Anticoagulants

Anticoagulants and antiplatelet compounds are commonly used to prevent thromboembolic disease. Heparin is effective in prevention and treatment of venous thrombosis and pulmonary embolism and related events and can be used either for prophylaxis or treatment. For prophylaxis, it is given by subcutaneous injection once or twice daily at a dose that does not affect in vitro clotting times, such as the activated partial thromboplastin time (APTT). A higher dose is required for treatment of ongoing thrombotic processes. If unfractionated heparin is used, its anticoagulant effect must be monitored, such as by APTT. The anticoagulant response varies significantly among patients with thromboembolic disease. The risk of bleeding is increased as the dose increases. For this reason the APTT is used to monitor the degree of anticoagulation. Because of their shorter chain lengths, low molecular weight heparins have a greater capacity to inhibit factor Xa than thrombin. Consequently, at standard clinical doses they produce only a mild prolonging of the APTT. Self-administered low molecular weight heparins can be used to provide full anticoagulation in the outpatient setting without monitoring. Because they are cleared from the blood by the kidneys, their dosing must be adjusted in patients with renal insufficiency.

Oral coumarin anticoagulants are effective in primary and secondary prevention of arterial and venous thromboembolism. The one-stage PT) is used to measure their anticoagulant effects. Because different clinical laboratories use different thromboplastins, a formula was developed to transform the PT to an index that allows results from different laboratories to be compared. This index, the international normalized ratio (INR), is used routinely to report PT results. Warfarin therapy prolongs the PT and the INR.

Platelet function is assessed by measurement of bleeding time, which involves incising the forearm skin under standardized conditions and measuring the time required for bleeding to stop. Platelet aggregation in vitro can be monitored optically using an aggregometer.

If a patient has an acute thrombus or is at high risk of forming one within the next few days, heparin is used, because its antithrombotic effect is immediate, whereas that of warfarin is delayed. If long-term anticoagulation is necessary, warfarin can be started as soon as therapeutic anticoagulation with heparin is achieved. After therapeutic anticoagulation with warfarin is achieved, heparin is often continued for 1 to 2 days. This overlap is commonly used because the antithrombotic effect of warfarin can lag behind laboratory measurements of warfarin anticoagulation, which is largely affected by a reduction in the level of factor VII, which has a short t_1/2 (Table 26-3). If warfarin is started for prophylaxis of thrombosis, and the short-term risk is not high (e.g., a patient with atrial fibrillation being started on warfarin to lower stroke risk), heparin may be unnecessary. Most experts recommend that loading doses of warfarin should not be used, that is, the patient be started on the anticipated maintenance dose. During the first week of warfarin therapy, the coagulation response should be checked at least twice. Depending on the rapidity and stability of this measure, the time interval between subsequent determinations is gradually increased.

Antiplatelet Drugs

Clinical trials have shown that aspirin reduces the incidence of myocardial infarction and death from cardiac causes by 30% to 50% in patients with unstable angina. Aspirin also significantly reduces the incidence of a first myocardial infarction in men with stable angina and is effective as an antithrombotic agent after coronary angioplasty/stenting or bypass grafting. Aspirin is effective in secondary prevention of myocardial infarction. Aspirin is also recommended for use in patients with transient ischemic attacks. There are, of course, adverse effects of long-term aspirin therapy (see Chapter 36).