Thrombin plays a central role in clot formation through platelet activation and fibrin generation at the sites of arterial disruption, such as that caused by angioplasty or plaque rupture.1–3 Thrombin also amplifies its own generation by activating factors VIII and V, key cofactors in the activation of factor X and pro-thrombin, respectively, as well as Factor XI. Anticoagulant strategies to inhibit arterial thrombogenesis have therefore focused on inhibiting thrombin or preventing thrombin generation.4 Heparin in combination with aspirin has been a cornerstone of treatment in conditions characterized by arterial plaque disruption, acute coronary syndromes and percutaneous coronary intervention (PCI). Heparin acts as an anticoagulant by activating anti-thrombin, which in turn inhibits thrombin and activated Factor X (Factor Xa). However, heparin has a number of limitations that are not observed with direct thrombin inhibitors. This article will review the role of thrombin in arterial thrombogenesis, discuss the mechanism of action and the limitations of heparin, an indirect thrombin inhibitor, provide an overview of the direct thrombin inhibitors and review the current literature on the use of direct thrombin antagonists in acute coronary syndromes and in the setting of percutaneous coronary intervention. Role of thrombin in arterial thrombogenesis. Disruption of atherosclerotic plaque exposes blood to thrombogenic substances in the lipid-rich core (Figure 1). Thereafter, platelets adhere to subendothelial matrix components, such as collagen and von Willebrand factor, become activated, and contribute to thrombus formation.5 Following activation, platelets express increased amounts of anionic phospholipids on their surface which promote coagulation by supporting the assembly of coagulation factor complexes (the so-called prothrombinase and tenase complexes). Upon activation, platelets, release agonists, such as adenosine diphosphate and thromboxane A2, which allow adherent platelets to recruit additional platelets to sites of arterial injury. Coincident with platelet adhesion and activation, contact of blood with tissue factor expressed by activated macrophages and smooth muscle cells found in the core of the disrupted plaque results in activation of the coagulation system.1-3 Exposed tissue factor binds to activated Factor VII, and this complex in turn activates Factors IX and X.5 In the presence of calcium, Factors IXa and Xa bind to their cofactors on the surface of activated platelets (Factors VIIIa and Va, respectively) to form intrinsic tenase and prothrombinase complexes, respectively. Activation of Factor X by intrinsic tenase, and subsequent conversion of prothrombin to thrombin by prothrombinase, results in a burst of thrombin generation. Thrombin converts fibrinogen to fibrin, which forms a matrix for thrombus. During this process, thrombin binds to fibrin where it remains enzymatically active and protected from inactivation by fluid-phase inhibitors.6 By serving as a reservoir of active thrombin, the clot is intensely thrombogenic because thrombin bound to fibrin locally activates platelets,7 amplifies coagulation by activating Factors V and VIII,8 and converts fibrinogen to fibrin.6 In addition, fibrin-bound thrombin activates Factor XIII, which renders the thrombus more resistant to lysis both by cross-linking fibrin and by cross-linking 2-antiplasmin, the major plasmin inhibitor, onto fibrin. Fibrin-bound thrombin may also activate a latent carboxypeptidase B, known as thrombin activatable fibrinolysis inhibitor (TAFI).9 Once activated, this enzyme attenuates fibrinolysis, presumably by removing carboxy-terminal lysine residues from fibrin, thereby preventing the binding of plasminogen and plasmin.10 Additional sources of thrombin are induced during coronary thrombolysis. Plasmin is generated during pharmacologic thrombolysis and it promotes clotting by activating contact factors,11 transiently activating Factor V,12 and directly convert prothrombin to thrombin.13,14 Furthermore, lysis of coronary thrombi exposes additional fibrin-bound thrombin,15 and thrombin remains bound to soluble fibrin degradation products where it also is protected from inactivation by fluid-phase inhibitors.16 Limitations of Unfractionated Heparin and LMWH A. Heterogenous mixture of active and inactive factors. Heparin inhibits arterial thrombosis by activating antithrombin, which then inhibits thrombin and Factor Xa and IIa.17 Binding of heparin to antithrombin produces conformational changes in antithrombin that accelerates the rate at which it inhibits Factor Xa. Heparin also enhances thrombin inhibition by antithrombin through an additional mechanism: heparin binds simultaneously to both thrombin and antithrombin, thereby maintaining physical proximity of the two molecules. Heparin induces the Xa inducibility property of antithrombin through a pentasaccaride sequence known as the “essential pentasaccaride.” However, heparin induced inactivation of thrombin requires the presence of an 18 saccharide sequence. Since almost all the heparin chains are at least 18 saccharides in length, heparin has equivalent inhibitory activity against thrombin and Factor Xa.17 In contrast, with a mean molecular mass of about 5,000 Da, fewer than half the low-molecular-weight heparin chains are long enough to bridge thrombin to antithrombin.18 Because heparin catalysis of Factor Xa does not require bridging between Factor Xa and antithrombin, these smaller chains retain their ability to promote Factor Xa inhibition. Consequently, low-molecular-weight heparins have greater inhibitory activity against Factor Xa than thrombin.18 B. Heparin is unable to inactivate fibrin-bound thrombin and platelet-bound Factor Xa. Although the heparins are capable of inactivating fluid-phase thrombin, they do not inhibit thrombin bound to fibrin5,19 or fibrin degradation products.15 In addition, Factor Xa bound to the surface of activated platelets is also resistant to inactivation by the heparin/antithrombin complex,20,21 as fibrin bound Xa remains active and continues to participate in thrombin generation, thereby increasing the amount of thrombin available to bind to fibrin. As a result, heparin is relatively an inefficient inhibitor of thrombus propagation. Thrombin binds to fibrin via exosite 1, a substrate-binding site that is distinct from its active site.22–24 Current evidence suggests that fibrin-bound thrombin is protected from inactivation by the heparin/antithrombin complex because heparin binds simultaneously to fibrin and to the heparin-binding domain on thrombin, so-called exosite 2, thereby approximating the two molecules and promoting thrombin’s activity within a clot.25 The formation of a ternary heparin/thrombin/fibrin complex increases the affinity of thrombin for fibrin and induces conformational changes in the active site of the enzyme.26,27 The resistance of thrombin within the ternary complex to inactivation by fluid-phase inhibitors may reflect allosteric modulation of its active site or spatial constraints that impair the reactivity of inhibitors with thrombin. Considerably less is known about the molecular basis for the resistance of platelet-bound Factor Xa to inactivation by the heparin/ antithrombin complex, but a similar mechanism is likely. C. Heparin binds to endothelium and to plasma proteins. The binding of heparin to cells and plasma proteins such as vitronectin, fibronectin, histidine-rich glycoprotein, platelet Factor IV, and high-molecular-weight multimers of von Willebrand factor, in a pentasaccharide-independent fashion28,29 decreases its anticoagulant effect by limiting its availability to interact with antithrombin.30 This effect differs between patients because some heparin-binding proteins, such as vitronectin and fibronectin, are acute-phase reactants and can increase to a variable extent in acute illness.31,32 In addition, platelet Factor 4, a highly cationic protein that binds heparin with high affinity,28 is released from platelets during the clotting process. High-molecular-weight multimers of von Willebrand factor also are released from both activated platelets and endothelial cells during clotting. Because of its nonspecific binding to endothelium and plasma proteins, the half-life of heparin is dose dependent and its anticoagulant effect varies from patient to patient. Consequently, careful laboratory monitoring is necessary to ensure that an adequate anticoagulant effect is obtained. D. Heparin causes thrombocytopenia and paradoxical thrombosis. Thrombocytopenia induced by heparin typically appears five or more days after the start of heparin therapy. The thrombocytopenia is caused by heparin-dependent IgG antibodies that activate platelets through their Fc receptors. Recently, several laboratories have shown that these antibodies recognize a complex of heparin with platelet Factor 4. Paradoxically, thrombotic complications develop in many patients with heparin-induced thrombocytopenia, because of both in vivo platelet activation and the generation of procoagulant rich platelet derived microparticles. When heparin-induced thrombocytopenia is defined as a decrease in the platelet count to below 150,000 per cubic millimeter, previous studies have noted heparin induced thrombocytopenia in as many as 2–7%33,34 of patients receiving unfractionated heparin with most of the patients having one or more thrombotic events (venous more arterial). Most patients with heparin-induced thrombocytopenia (HIT) require alternative anticoagulation. This is because HIT is highly prothrombotic and is characterized by markedly increased thrombin generation. Unfractionated heparins seem to induce HIT more often than low molecular weight heparins. There are three anticoagulants for which there is an emerging consensus for their efficacy in management of HIT, and which are currently approved for treatment of HIT in several countries: the recombinant hirudin, lepirudin, a direct thrombin inhibitor; the synthetic direct thrombin inhibitor, argatroban; and the heparinoid, danaparoid sodium, mainly exhibiting antifactor-Xa activity. Hirudin, the drug for which most data from prospective trials exists, can be safely and effectively used in patients with HIT, its dramatically increased elimination half-life in patients with renal failure being the most important drawback. Argatroban, which is mainly eliminated by the liver, could be used preferentially in such patients with renal impairment. Interference with the international normalized ratio makes oral anticoagulation, which is necessary in many patients with HIT, problematic. Activated partial thromboplastin time is sufficient to monitor lepirudin and argatroban treatment in most cases. Danaparoid sodium, with an antifactor-X activity half-life of about 24 hours seems to be best suited for thrombosis prophylaxis in patients with HIT. In some patients monitoring by determining antifactor-Xa activity is necessary. No antidote is available for any of the drugs discussed, and bleeding complications are the most important adverse effects. In situations such as hemodialysis or cardiopulmonary bypass, not only the characteristics of the drug in use itself, but also availability of monitoring methods play an important role. Adjunctive treatments have not been systematically evaluated and should be used cautiously. Recent data suggest that re-exposure of patients with a history of HIT with heparin, for example during cardiopulmonary bypass, can be well tolerated provided no circulating HIT antibodies are detectable at the time of re-exposure, and heparin is strictly avoided pre- and post-operatively. These limitations of heparin in the treatment of arterial thrombosis have prompted the development of new antithrombotic agents that are capable of inactivating fibrin-bound thrombin. Of these new drugs, the most extensively studied to date are direct thrombin inhibitors. The Different Direct Thrombin Antagonists Direct thrombin inhibitors include hirudin, semisynthetic hirudin fragments (bivalirudin), small molecules that react with the active-site of thrombin (including PPACK and its derivatives, argatroban and others) and thrombin-binding DNA aptamers (Figure 2). Although all of these inhibitors bind directly to thrombin, their sites and modes of interaction are different (Figure 2). Two different sites on thrombin mediate substrate interactions: the active site and the exosite 1.35 Cleavage of the sessile bond in substrates occurs at the active site, whereas exosite 1 serves to dock substrates in the proper orientation. Direct thrombin inhibitors interact with one or both of these sites and inactivate thrombin by preventing it from interacting with its substrates. A. Hirudin and its derivatives. Hirudin is the prototype of direct thrombin antagonists. Hirudin is a 65 amino acid polypeptide originally isolated from the salivary glands of the medicinal leech, Hirudo medicinalis, and is now produced through recombinant DNA technology.36 Unlike native hirudin, the recombinant forms are not sulfated at the Tyr 63 site and exhibit at least a 10-fold reduced affinity for thrombin. Hirudin is a potent and specific inhibitor of thrombin which forms a 1:1 stoichiometric, slowly reversible complex with the enzyme. Analysis of the crystal structure of the thrombin-hirudin complex demonstrates the extensive contact that hirudin makes with thrombin with its globular amino-terminal “head” region interacting with the active site of thrombin and the carboxy-terminal “tail” binding to exosite 1 on the enzyme.37 Hirugen is a synthetic dodecapeptide comprising residues 53 to 64 of the carboxy-terminal region of hirudin.38 Sulfation of the tyrosine residue at position 63 increases the affinity of hirugen for thrombin. By binding to thrombin on exosite 1, hirugen blocks the enzyme’s interaction with its substrates, including fibrinogen and the thrombin receptor on platelets. Because it does not interact with the active site of thrombin, however, hirugen does not block hydrolysis of low-molecular-weight thrombin-directed substrates. Bivalirudin was formed by adding D-Phe-Pro-Arg-Pro-(Gly)4 to the amino-terminal of hirugen. This converted a weak competitive inhibitor of thrombin (hirugen) to a potent bivalent inhibitor, bivalirudin. Like hirugen, bivalirudin interacts with both the active site and exosite 1 on thrombin.39 However, bivalirudin differs from hirudin in that it produces only transient inhibition of the active site of thrombin. Once complexed with thrombin, the Arg-Pro bond on the amino-terminal extension of bivalirudin is cleaved, thereby converting bivalirudin into a lower affinity, hirugen-like inhibitor.40,41 This lower affinity binding may confer a better safety profile upon bivalirudin. B. Covalent inhibitors (D-Phe-Pro-ArgCH2Cl (PPACK) and its derivatives).42–44 PPACK is the prototype of a class of inhibitors that form covalent complexes with thrombin. PPACK interacts with the active site of thrombin and inhibits the enzyme by alkylating the active center histidine. Recent crystallographic studies confirm the tight interaction of this molecule with thrombin and its covalent derivatization of His-57 and Ser-195 within the catalytic triad. Boroarginine derivative of PPACK, D-Phe-Pro-Arg-borate, has been developed and appears to be a slightly more specific inhibitor of thrombin than the parent molecule. Although this agent has antithrombotic activity in laboratory animal models and has the potential for oral bioavailability, crossreactivity with other serine proteases has stopped further development. C. Non-covalent inhibitors or active site inhibitors.42-44 A number of low-molecular-weight active site inhibitors of thrombin have been developed. The prototype of these noncovalent inhibitors is argatroban.45 Others include napsagatran,46 inogatran,47 melagatran,48 and L372,460. Argatroban is a small (527 Da) DTI that binds reversibly to the active site of the thrombin molecule. It is selective for thrombin (inhibition constant 0.04 µmol/L), and has little effect on related serine proteases. Argatroban is intravenously administered and has an elimination half-life of 40 to 50 minutes. Monitoring of the aPTT is required to assess its anticoagulant activity. Dose adjustments may be required to attain a steady-state aPTT of 1.5 to 3 times the mean normal value. Argatroban is hepatically metabolized via hydroxylation and aromatization reactions, so dosing reductions and careful monitoring are recommended in patients with hepatic dysfunction. The metabolized products are removed via biliary excretion. Renal impairment has no influence on the elimination half-life of argatroban and dose adjustments therefore are unnecessary in patients with renal dysfunction. Argatroban has no known antidote. Although it is a potent inhibitor of thrombin in vitro, results with argatroban in laboratory animals and in humans with acute coronary syndromes have been mixed. Argatroban has been used successfully in patients with heparin-induced thrombocytopenia. Napsagatran, inogatran, melagatran, and Ximelagatran are other examples of low-molecular-weight noncovalent thrombin inhibitors. All are potent and selective inhibitors of thrombin. There are at least 10 oral direct thrombin inhibitors (DTIs) in clinical development, out of them Ximelagatran (Exanta™) and BIBR 1048 are most promising. Both are prodrugs with two protecting groups that are eliminated after absorption from the gastrointestinal tract. Their main active substances, melagatran and BIBR 953, are both potent and selective DTIs. Following oral administration, Ximelagatran is rapidly absorbed and converted to melagatran, the active form, achieving peak plasma concentrations in 1.6 to 1.9 hours. Melagatran is a potent, reversibly binding, active-site inhibitor of thrombin. After ximelagatran administration, melagatran has been shown to inhibit the generation of thrombin, probably because of reduced thrombin-mediated positive feedback to the coagulation system. In experimental models of thrombosis, melagatran has been shown to have a shallower dose-response curve than warfarin and, therefore, a better separation between efficacy and bleeding. Oral bioavailability, measured as the plasma concentration of the active metabolite, seems to be higher for ximelagatran (20%) than for BIBR 1048 (estimated to 5%). BIBR 953 has a longer half-life (about 12 hours) than does melagatran (3 to 5 hours) after oral administration of BIBR 1048 and ximelagatran, respectively. Both melagatran and BIBR 953 are mainly eliminated via the renal route. The variability of the plasma concentration of melagatran after oral administration of ximelagatran is low. There are no clinically relevant interactions with food or cytochrome P450 metabolized drugs and ximelagatran. In clinical studies, ximelagatran has been administered in a twice daily fixed-dose regimen without coagulation monitoring. Results of published clinical studies are encouraging, both with regard to efficacy and bleeding. Major indications in Phase III studies with ximelagatran are the prevention of venous thromboembolism (VTE) in hip and knee replacement surgery, treatment and long-term secondary prevention of VTE and prevention of stroke in patients with nonvalvular atrial fibrillation. It is anticipated that with a favorable outcome of the Phase III clinical studies new oral DTIs, with the oral fixed-dose regimen without routine coagulation monitoring, will ease the use of today’s anticoagulant therapy. D. DNA aptamers. A single-stranded, 15-nucleotide DNA aptamer49 has been developed that binds to exosite 1 on thrombin with high affinity. Like hirugen, the aptamer blocks thrombin’s interaction with its substrates but does not inhibit thrombin-mediated hydrolysis of low-molecular-weight synthetic substrates. The DNA aptamer is a potent anticoagulant in vitro and is an effective antithrombotic in laboratory animals. However, with a half-life of minutes, its clinical utility is limited. Recently, DNA aptamers that bind to exosite 2 on thrombin also have been identified. Because there is allosteric linkage between the two exosites on thrombin, DNA aptamer binding to exosite 2 can influence ligand binding to exosite 1. Exosite 2 aptamers have yet to be tested in laboratory animals, but are likely to have short half-lives in vivo. Despite their different mechanisms of action, all direct thrombin inhibitors have potential advantages over heparin. Consequently, researchers have assumed that results with one agent will be obtained with any drug in this class. This concept is problematic because even agents that interact with the same sites on thrombin may have different benefit-to-risk profiles. For example, closer inspection of clinical trials results suggests that bivalirudin is safer than hirudin, a phenomenon that may be explained by differences in the pharmacokinetic profiles of the 2 drugs. Advantages of Direct Thrombin Inhibitors Over Heparin Although unfractionated heparin is widely used for thrombin inhibition in the management of unstable coronary artery disease, clinical and experimental evidence suggests that it is suboptimal (Table 1). Recent pharmaceutical strategies to improve upon unfractionated heparin’s efficacy profile have centered on the development of 2 major classifications of thrombin inhibition medications: the naturally occurring leech protein hirudin (and synthetic analogs) and low-molecular-weight (LMW) heparins. Direct thrombin inhibitors have both biologic and pharmacokinetic advantages over heparin. The biologic advantage of direct thrombin inhibitors reflects their capacity to inactivate fibrin-bound thrombin. Because thrombin binds to fibrin via exosite 1, the activity of agents that interact with the active site is not compromised when the enzyme binds to fibrin. Hirudin and bivalirudin bind to exosite 1, as well as the active site of thrombin, and consequently compete with fibrin for access to exosite 1. However, in the absence of heparin, thrombin has only weak affinity for fibrin25 so that these agents readily interact with fibrin-bound thrombin. Unlike heparin, direct thrombin inhibitors do not bind to plasma proteins or endothelial cells. Hirudin forms a slowly reversible complex with thrombin and has a plasma half-life of 40 minutes after intravenous administration, and approximately 120 minutes after subcutaneous injection. Bivalirudin has a plasma half-life of 24 minutes after intravenous infusion. Unlike hirudin, bivalirudin produces only transient inhibition of the active site of thrombin and may, therefore, be safer. Furthermore, they produce a more predictable anticoagulant response than unfractionated heparin.50,51 Theoretically, this pharmacokinetic advantage of direct thrombin inhibitors could obviate the need for laboratory monitoring. The narrow therapeutic window for hirudin, however, makes monitoring necessary.52–54 Comparison of Direct Thrombin Inhibitors (Bivalirudin Versus Hirudin) Bivalirudin has both safety and potential efficacy advantages over hirudin. Bivalirudin appears to have a wider therapeutic window than hirudin, possibly reflecting the fact that bivalirudin produces only transient inhibition of the active site of thrombin. The wider safety margin of bivalirudin relative to hirudin permits administration of higher doses. This may provide bivalirudin with an efficacy advantage over hirudin because, as stoichiometric inhibitors of thrombin, high doses of these drugs must be given to inactivate the large amounts of thrombin generated in the vicinity of an arterial thrombus. Bivalirudin complexes and inhibits thrombin generated at sites of arterial injury. By the time these enzyme/inhibitor complexes reach the microcirculation, where thrombomodulin is more abundant,58 release of the amino-terminal domain of bivalirudin leaves only the carboxy-terminal segment bound to exosite 1 on thrombin. In contrast to bivalirudin, hirudin forms a very slowly reversible complex with thrombin and prevents the enzyme from binding to thrombomodulin. Despite the promise of direct thrombin inhibitors, clinical trials with hirudin as an adjunct to thrombolytic therapy61,62 or in patients undergoing coronary angioplasty63 have failed to show durable advantages over heparin. This likely reflects the fact that the high doses of hirudin needed for the treatment of arterial thrombosis cannot be given safely, particularly when hirudin is used in conjunction with thrombolytic agents. This concept is supported by the fact that hirudin is better than low-molecular-weight heparin for the prevention of venous thrombosis in common high-risk orthopedic patients,64 a setting where lower, and safer, doses of hirudin are effective. With its wider therapeutic window, bivalirudin may be better than hirudin because it can be given in the doses necessary to block arterial thrombogenesis. This possibility deserves testing in well-designed phase III trials, as does bivalirudin’s potential for promoting protein C activation. Differences between bivalirudin and hirudin, as well as other direct thrombin inhibitors, highlight the pitfalls of considering all direct thrombin inhibitors to have equivalent risk-benefit profiles. Part 2 of this article will be continued in the February 2005 issue.
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