Acute Coronary Syndromes: A Focus on Thrombin

Acute coronary syndromes (ACS) are usually caused by thrombosis superimposed on disrupted atherosclerotic plaque.¹ Intracoronary thrombi, which form under high shear conditions, are composed of platelet aggregates held together by fibrin strands. Thrombin plays a central role in arterial thrombosis. Generated at sites of vascular injury, thrombin serves as a potent platelet agonist and is the enzyme responsible for fibrin formation and stabilization. Because of the importance of thrombin and platelets in arterial thrombosis, most regimens for treatment of ACS include drugs that inhibit thrombin or prevent its generation in addition to platelet inhibitors. Although heparin and aspirin, the cornerstones of therapy, decrease the risk of death or recurrent ischemia in patients with ACS,^2,3 both drugs have limitations. Despite treatment with these agents, patients remain at risk of new myocardial infarction or death, both during the acute phase and long-term.^4,5 The occurrence of breakthrough ischemic events during treatment suggests that neither aspirin nor heparin fully attenuates the arterial thrombotic process, whereas the observation that patients remain at risk of recurrent ischemia after treatment stops suggests that the triggers for intracoronary thrombosis persist long after the index event. This paper will 1) review the importance of thrombin in coagulation; 2) highlight the role of thrombin in platelet aggregation; 3) outline the mechanisms responsible for the limitations of heparin in the treatment of arterial thrombosis; 4) discuss how direct thrombin inhibitors overcome the limitations of heparin; and 5) provide clinical perspectives as to future directions in antithrombotic therapy for ACS. Role of thrombin in coagulation Thrombin, the main effector of the coagulation cascade, is generated at sites of vascular injury when circulating factor VII or activated factor VII (factor VIIa) binds tissue factor. An integral membrane protein, tissue factor is constitutively expressed by nonvascular cells of the vessel wall and is exposed when the integrity of the endothelial cell lining is compromised. Low levels of tissue factor are also expressed on circulating monocytes and leukocyte-derived microparticles.⁶ Tissue factor-bearing cells become tethered to platelets and endothelial cells at sites of vascular injury, thereby increasing local concentrations of tissue factor. Additional tissue factor is exposed when tethered leukocytes generate microparticles. The tissue factor:factor VIIa complex initiates the coagulation cascade by activating factor X (Figure 1). Factor Xa, together with its cofactor (factor Va), converts a small amount of prothrombin to thrombin.⁷ Coagulation is amplified by this small amount of thrombin because thrombin feeds back to promote its own generation by activating platelets and factors V and VIII. Platelet activation induces the exposure of anionic phospholipids on the platelet surface onto which clotting factors bind in a calcium-dependent fashion. Factor XI, which is activated by thrombin on the platelet surface, activates factor IX. Factor IXa, together with its cofactor (factor VIIIa), binds to the platelet surface where it activates factor X, thereby producing a burst of thrombin.⁷ Thrombin converts fibrinogen to fibrin and stabilizes the fibrin mesh by activating factor XIII, a cross-linking enzyme. Thrombin also plays a key role in platelet activation and aggregation at sites of vascular injury. The fibrin network serves to stabilize these platelet aggregates to form a platelet-rich thrombus. Role of thrombin in platelet activation Thrombin is a potent platelet agonist. It causes shape change in platelets and triggers them to release adenosine diphosphate (ADP), serotonin and thromboxane A2, substances that augment the formation of platelet aggregates by serving as agonists for ambient platelets. Thrombin also activates glycoprotein (GP) IIb/IIIa, an abundant integrin on the platelet surface. Once activated, GP IIb/IIIa links adjacent platelets together by binding fibrinogen and, under high shear, von Willebrand factor. Thrombin signaling in platelets is mediated, at least in part, by a family of G-protein-coupled protease-activated receptors (PARs).⁸ PAR1, the prototype of this receptor family, is activated when thrombin binds to its extracellular domain and cleaves a specific peptide bond (Figure 2). Hydrolysis of this bond creates a new amino-terminus that serves as a tethered ligand by folding back on itself to bind to the body of the receptor. This tethering process triggers a series of G-protein-coupled signaling pathways that mediate platelet shape change, secretion and integrin activation.⁹ In addition to PAR1, human platelets also express PAR4, another thrombin-activated receptor. Antibody blockade of PAR1 prevents platelet activation in response to low, but not high, concentrations of thrombin.^10–12 In contrast, blockade of PAR4 has no effect on thrombin-mediated platelet activation.¹⁰ When PAR1- and PAR4-blocking antibodies are combined, however, there is profound inhibition of platelet activation in response to both low and high concentrations of thrombin.¹⁰ These findings suggest that PAR1 mediates platelet activation by low concentrations of thrombin, whereas PAR4 serves as a receptor for spillover activation by high concentrations of thrombin.¹³ Recent studies in mice highlight the importance of thrombin in platelet aggregation in vivo. In contrast to human platelets, PAR4 is the major thrombin receptor on mouse platelets.¹³ Platelets from PAR4-deficient mice fail to change shape, release ADP or aggregate in response to thrombin, but respond normally to other agonists.¹⁴ With an absent platelet response to thrombin, PAR4-deficient mice were used to explore the importance of thrombin-induced platelet aggregation in hemostasis and thrombosis. Tail bleeding times in PAR4-deficient mice are markedly prolonged compared to control mice, highlighting the importance of thrombin-induced platelet aggregation in hemostasis. PAR4-deficient mice also exhibit attenuated thrombosis after arterial injury, suggesting that other platelet agonists generated at the site of vascular injury cannot compensate for a lack of thrombin-induced platelet aggregation.¹⁴ If the findings in mice can be extrapolated to humans, thrombin plays a critical role in both platelet aggregation and coagulation. Consequently, inhibition of thrombin should be the goal of antithrombotic strategies. Although heparin is the agent most often used to inactivate thrombin, it has limitations. Limitations of heparin Heparin has both pharmacokinetic and biophysical limitations in the setting of arterial thrombosis (Table 1). Heparin acts as an anticoagulant by binding to antithrombin and enhancing the rate at which antithrombin inactivates factor Xa and thrombin.¹⁵ The pharmacokinetic limitations of heparin reflect its propensity to bind to plasma proteins other than antithrombin, thereby reducing the amount of heparin available to exert an anticoagulant effect. Plasma levels of heparin-binding proteins vary between patients because some of these proteins are acute-phase reactants, whereas others are proteins released from platelets or endothelial cells in response to thrombin. PF4, which is released from thrombin-activated platelets, may be a particularly important heparin:binding protein because it neutralizes heparin’s anticoagulant activity.¹⁵ Because the levels of heparin-binding proteins are so variable, heparin therapy must be monitored to ensure that a therapeutic anticoagulant effect is obtained. Biophysical limitations of heparin reflect the inability of the heparin-antithrombin complex to inactivate factor Xa bound to the activated platelet surface^16,17 or thrombin bound to fibrin.^18,19 Consequently, factor Xa bound to activated platelets trapped within the thrombus will generate thrombin even in the face of heparin, thereby increasing the amount of thrombin available to bind to fibrin.²⁰ Heparin promotes thrombin binding to fibrin by simultaneously binding to fibrin and to thrombin to form a ternary heparin:thrombin:fibrin complex.²¹ Thrombin within this ternary complex retains its catalytic activity, but is protected from inactivation by the heparin:antithrombin complex. Therefore, the thrombus serves as a sanctuary for active thrombin. This concept is supported by a recent analysis of thrombi removed at autopsy from patients with arterial or venous thrombosis that demonstrated relatively high levels of enzymatically active thrombin within such thrombi.²² Fibrin-bound thrombin contributes to the procoagulant and antifibrinolytic properties of the thrombus (Figure 3). Fibrin-bound thrombin acts as a procoagulant because of its capacity to locally convert fibrinogen to fibrin, activate platelets and amplify its own generation.^23,24 The antifibrinolytic properties of fibrin-bound thrombin reflect its capacity to activate factor XIII and a latent carboxypeptidase-B like enzyme known as thrombin activatable fibrinolysis inhibitor (TAFI). Factor XIIIa renders the thrombus resistant to lysis by cross-linking the fibrin strands and by cross-linking #a-antiplasmin, the major inhibitor of plasmin, onto fibrin. Thrombin-mediated activation of TAFI attenuates fibrinolysis because TAFI releases carboxy-terminal lysine residues from fibrin, thereby removing plasminogen and plasmin binding sites.²⁵ Because of its procoagulant and antifibrinolytic effects, fibrin-bound thrombin effects thrombus growth. Consequently, antithrombotic strategies have been devised to inhibit fibrin-bound thrombin.²⁶ Direct thrombin inhibitors represent a new class of antithrombotic drugs designed, at least in part, to overcome the limitations of heparin. Direct thrombin inhibitors In contrast to heparin, which requires antithrombin to exert its anticoagulant activity, direct thrombin inhibitors bind directly to thrombin and block the enzyme’s catalytic activity.²⁷ Direct thrombin inhibitors overcome the pharmacokinetic and biophysical limitations of heparin (Table 2). Because they do not bind to plasma proteins other than thrombin, direct thrombin inhibitors produce a more predictable anticoagulant response than heparin. Direct thrombin inhibitors inactivate both fluid-phase thrombin and thrombin bound to fibrin or fibrin degradation products.^19,28 Moreover, because the anticoagulant activity of direct thrombin inhibitors is unaffected by PF4, they retain their activity in the vicinity of platelet-rich thrombi.²⁷ Because they block the catalytic activity of thrombin, direct thrombin inhibitors attenuate thrombin-induced platelet aggregation (Figure 4). Thrombin docks on the extracellular portion of platelet PAR1 or PAR4 via exosite 1, its substrate-binding domain, whereas cleavage of these receptors is effected by the active site of the enzyme.^8–14 Bivalent direct thrombin inhibitors block both events because they bind to exosite 1 and the active site of thrombin. In contrast, active-site directed inhibitors only block receptor cleavage. Three parenteral direct thrombin inhibitors are licensed in North America; hirudin and argatroban are approved for treatment of heparin-induced thrombocytopenia, whereas bivalirudin is licensed as an alternative to heparin in patients undergoing percutaneous coronary interventions. The properties of each of these inhibitors will be briefly described. Hirudin. The prototypical direct thrombin inhibitor, hirudin is a 65-amino acid polypeptide originally isolated from the medicinal leech and now available through recombinant DNA technology.²⁹ Hirudin is a bivalent thrombin inhibitor that binds to two key domains on thrombin:exosite 1 (the substrate-binding domain); and the active site of the enzyme. The hirudin:thrombin complex is essentially irreversible, which can be a potential drawback should hemorrhagic complications arise. Hirudin has a plasma half-life of 60 minutes after intravenous administration. Because it is cleared by the kidney, the drug can accumulate in patients with renal insufficiency.³⁰Bivalirudin. A synthetic analog of hirudin, bivalirudin is composed of an amino-terminal Gly-Pro-Arg-Pro sequence linked via four Gly residues to a dodecapeptide analogue of the carboxy-terminal of hirudin.³¹ Like hirudin, bivalirudin binds to both exosite 1 and the active site of thrombin to form a bivalirudin:thrombin complex. Thrombin within this complex cleaves the Arg-Pro bond at the amino-terminal of bivalirudin, releasing the active-site directed portion of bivalirudin.³² Recovery of thrombin’s active site functions may allow the enzyme to participate in hemostasis, thereby endowing bivalirudin with a safety advantage over hirudin. Bivalirudin has a plasma half-life of 25 minutes after intravenous administration. Its clearance is mainly through extra-renal mechanisms, although bivalirudin dose adjustments are needed in patients with severe renal insufficiency.³³Argatroban. An arginine derivative, argatroban binds only to the active site of thrombin.³⁴ Argatroban is metabolized in the liver to generate at least three active species.³⁵ Because this process is impaired in patients with hepatic dysfunction, argatroban should be avoided in these individuals. Argatroban has a plasma half-life of 45 minutes after intravenous administration.³⁵ Clinical experience with direct thrombin inhibitors in arterial thrombosis A recent meta-analysis of 11 randomized clinical trials that compared direct thrombin inhibitors with heparin for treatment of ACS, including patients undergoing percutaneous coronary interventions, evaluated primary data from over 35,000 patients.³⁶ Compared with heparin, direct thrombin inhibitors produced a significant reduction in the combined endpoint of death and myocardial infarction at the end of therapy, an effect that was maintained at 7 and 30 days. Most of the benefit of direct thrombin inhibitors reflected a reduction in myocardial infarction.³⁶ Subgroup analysis revealed that bivalent direct thrombin inhibitors, such as hirudin and bivalirudin, but not univalent inhibitors (including argatroban), were superior to heparin. Hirudin produced more bleeding than heparin, whereas bivalirudin produced less bleeding.³⁶ These findings suggest that the various direct thrombin inhibitors have distinct benefit-to-risk profiles. The benefit-to-risk profile of bivalirudin appears to be better than that of hirudin or argatroban. Conclusions and future directions Direct thrombin inhibitors have mechanistic advantages over heparin.²⁸ In addition to inactivating fluid-phase thrombin, direct thrombin inhibitors block fibrin-bound thrombin, an important effector of thrombus growth. Direct thrombin inhibitors also prevent thrombin-mediated platelet activation and aggregation. In contrast, high concentrations of heparin may activate platelets,³⁷ thereby promoting platelet aggregation in patients undergoing percutaneous coronary interventions. Based on a recent meta-analysis,³⁶ direct thrombin inhibitors are superior to heparin at reducing recurrent ischemia in patients with ACS, likely reflecting the mechanistic advantages of direct thrombin inhibitors over heparin. Of the approved direct thrombin inhibitors, bivalirudin appears to have a better benefit-to-risk profile than hirudin.^36,38,39 When compared with heparin in patients undergoing coronary angioplasty for unstable angina, bivalirudin produced a greater reduction in recurrent ischemic events and caused less bleeding.³⁸ Because bivalirudin inhibits both coagulation and thrombin-induced platelet aggregation, studies are underway to determine whether bivalirudin obviates the need for GP IIb/IIIa antagonists in all but the highest-risk patients undergoing percutaneous coronary interventions. There is mounting interest in agents that block thrombin generation by targeting clotting factors higher in the coagulation cascade. The availability of parenteral drugs that inhibit factor Xa or factor VIIa provides an opportunity to explore the utility of this approach in patients with ACS. DX9065a, a direct factor Xa inhibitor,⁴⁰ is currently undergoing phase-II testing in patients with coronary artery disease. A recombinant version of a protein originally isolated from the canine hookworm is also being evaluated in patients with unstable angina. This protein, which is designated nematode anticoagulant protein (NAP)c2, inhibits factor VIIa in a two-step fashion. NAPc2 first binds to a noncatalytic site on factor X or factor Xa and the NAPc2:factor Xa complex, then inactivates factor VIIa bound to tissue factor.⁴⁰ Finally, a synthetic analogue of the pentasaccharide sequence on heparin that mediates its interaction with antithrombin is also entering phase-III clinical trials in patients with ACS. This drug, which is known as fondaparinux, catalyzes factor Xa (but not thrombin) inhibition by antithrombin.⁴⁰ Fondaparinux has already proven to be more effective than low molecular weight heparin for thromboprophylaxis after major orthopedic surgery.^41,42 Additional trials will be needed to determine whether fondaparinux, or other agents that block thrombin generation, are more effective than direct thrombin inhibitors for treatment of ACS. The observation that patients with ACS remain at risk for recurrent ischemia after initial treatment of their index event sets the stage for long-term therapy. Recently developed orally active anticoagulants may be useful for this indication. Ximelagatran, a pro-drug of melagatran (an active-site directed thrombin inhibitor) and DPC906 (an orally active direct inhibitor of factor Xa) are two such agents.⁴⁰ In summary, parenteral direct thrombin inhibitors are only now finding their niche for treatment of ACS. Bivalirudin, a dual-acting inhibitor of coagulation and thrombin-induced platelet activation, is the most promising of this class of drugs. With the availability of parenteral agents that block specific coagulation enzymes above the level of thrombin and orally active inhibitors of thrombin and factor Xa, we can identify the optimal targets for attenuation of thrombosis. This information will be essential when designing new antithrombotic strategies. Acknowledgments. This work was supported by grants from the Heart and Stroke Foundation of Ontario (HSFO), Canadian Institutes of Health Research and the Ontario Research and Development Challenge Fund. Dr. Weitz is a Career Investigator of the Heart and Stroke Foundation of Canada and holds the HSFO/J. Fraser Mustard Chair in Cardiovascular Research and the Canada Research Chair in Thrombosis from the Government of Canada. Dr. Bates, from the Canadian Institutes of Health Research/University Industry (bioMerieux), is the recipient of a New Investigator Award.