John M. Maraganore

Treatment with Bivalirudin (Hirulog) as Compared with Heparin during Coronary Angioplasty for Unstable or Postinfarction Angina

BACKGROUND Heparin is often administered during and after coronary angioplasty to prevent closure of the dilated vessel. However, ischemic or hemorrhagic complications occur in 5 to 10 percent of treated patients. We studied whether these complications could be prevented when the direct thrombin inhibitor bivalirudin (Hirulog) was used in place of heparin. METHODS We performed a double-blind, randomized trial in 4098 patients undergoing angioplasty for unstable or postinfarction angina. Patients were assigned to receive either heparin or bivalirudin immediately before angioplasty. The primary end point were death in the hospital, myocardial infarction, abrupt vessel closure, or rapid clinical deterioration of cardiac origin. RESULTS In the total study group, bivalirudin did not significantly reduce the incidence of the primary end point (11.4 percent, vs. 12.2 percent for heparin) but did result in a lower incidence of bleeding (3.8 percent vs. 9.8 percent, P < 0.001). In the prospectively stratified subgroup of 704 patients with postinfarction angina, bivalirudin therapy resulted in a lower incidence of the primary end point (9.1 percent vs. 14.2 percent, P = 0.04) and a lower incidence of bleeding (3.0 percent vs. 11.1 percent, P < 0.001), but in a similar cumulative rate of death, myocardial infarction, and repeated revascularization in the six months after angioplasty (20.5 percent vs. 25.1 percent, P = 0.17). CONCLUSIONS Bivalirudin was at least as effective as high-dose heparin in preventing ischemic complications in patients who underwent angioplasty for unstable angina, and it carried a lower risk of bleeding. Bivalirudin, as compared with heparin, reduced the risk of immediate ischemic complications in patients with postinfarction angina, but this difference was no longer apparent after six months.

Randomized, Double-blind Comparison of Hirulog Versus Heparin in Patients Receiving Streptokinase and Aspirin for Acute Myocardial Infarction (HERO)

Background Thrombolytic therapy improves survival after myocardial infarction through reperfusion of the infarct-related artery. Thrombin generated during thrombolytic administration may reduce the efficacy of thrombolysis. A direct thrombin inhibitor may improve early patency rates. Methods and Results Four hundred twelve patients presenting within 12 hours with ST-segment elevation were given aspirin and streptokinase and randomized in a double-blind manner to receive up to 60 hours of either heparin (5000 U bolus followed by 1000 to 1200 U/h), low-dose hirulog (0.125 mg/kg bolus followed by 0.25 mg · kg−1 · h−1 for 12 hours then 0.125 mg · kg−1 · h−1), or high-dose hirulog (0.25 mg/kg bolus followed by 0.5 mg · kg−1 · h−1 for 12 hours then 0.25 mg · kg−1 · h−1). The primary outcome was Thrombolysis In Myocardial Infarction trial (TIMI) grade 3 flow of the infarct-related artery at 90 to 120 minutes. TIMI 3 flow was 35% (95% CI, 28% to 44%) with heparin, 46% (95% CI, 38% to 55%) with low-dose hirulog, a...

Anticoagulant effects of Hirulog, a novel thrombin inhibitor, in patients with coronary artery disease

Selective thrombin inhibitors are a new class of antithrombotic drugs that, unlike heparin, can effectively inhibit clot-bound thrombin and escape neutralization by activated platelets. Hirulog is a 20 amino acid hirudin-based synthetic peptide that has shown promise in experimental models of thrombosis. Little information is available about the effects of hirulog in patients with coronary artery disease. Forty-five patients undergoing cardiac catheterization, who were taking aspirin, were randomized to receive either (1) hirulog, 0.05 mg/kg intravenous bolus followed by 0.2 mg/kg/hour intravenous infusion until the end of the catheterization; (2) hirulog, 0.15 mg/kg intravenous bolus followed by 0.6 mg/kg/hour intravenous infusion; or (3) heparin; 5,000 U intravenous bolus. Serial activated partial thromboplastin time (APTT), prothrombin time, activated clotting time and fibrinopeptide A were measured. Hirulog produced a dose-dependent prolongation of all coagulation parameters; the 0.6 mg/kg/hour dose prolonged the APTT to 218 +/- 50% of baseline after 2 minutes and 248 +/- 50% of baseline after 15 minutes. The half-life of the effect on APTT was 40 minutes. The hirulog blood level correlated well with the APTT, prothrombin time and activated clotting time (r = 0.77, 0.73, and 0.82 respectively, all p < 0.001). Both doses of hirulog potently suppressed the generation of fibrinopeptide A (p < 0.05). There were no major hemorrhagic, thrombotic or allergic complications in patients treated with hirulog or heparin. Thus, hirulog, a direct thrombin inhibitor, provides a predictable level of anticoagulation and appears to have a potent yet well-tolerated anticoagulant profile in patients with coronary artery disease.

Structure-function relationships of hirulog peptide interactions with thrombin

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095–7101). These peptides, designated ‘hirulogs’, interact with both thrombins catalytic center and its anion‐binding exosite for fibrinogen recognition. In order to investigate structure‐activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic‐site binding and anion‐binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with K 1s in the sub‐nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.

Structural elements for protein-phospholipid interactions may be shared in protein kinase C and phospholipases A2

Abstract A pattern of cystein residues that occurs in the phospholipases A 2 is also found in a repeated pattern of cysteine residues in the regulatory domain of protein kinase C. Within this cysteine pattern there is also homology for the active site of phospholipases A 2 . The similarities may represent structural elements involved in the interaction of both enzymes with phospholipid.

Hirudin Interactions with Thrombin

Hirudin was originally isolated from the salivary glands of the medicinal leech Hirudo medicinalis. The anticoagulant activity of leech saliva was first described over 100 years ago by Haycraft (1884) and a polypeptide with antithrombin activity was first isolated over 30 years ago by Markwardt (1957). The amino acid sequence of the major form of hirudin was determined by Bagdy et al. (1976) and Dodt et al. (1984) (see also Mao et al., 1987) and is given in Fig. 1. The most striking feature of the amino acid sequence is the high proportion of acidic residues (12 out of 65); seven of these occur in the C-terminal region from residues 53 to 65, including the sulfated tyrosine residue at position 63 that is formed by postranslational modification. Hirudin contains six cysteines that form three disulfide bridges (h-Cys-6—Cys-14, h-Cys-16—Cys-28, and h-Cys-22—Cys-391) that were elucidated by Dodt et al. (1985) and are also shown in Fig. 1. Subsequently, more than 20 different isoforms of hirudin have been isolated from H. medicinalis and sequenced (Dodt et al., 1986; Harvey et al., 1986; Tripier, 1988; Scharf et al.,1989) and all of these forms seem to have antithrombin activity. All of the isoforms are highly homologous with levels of identity usually greater than 80%; the conserved residues are indicated in Fig. 1. The disulfide bridges and the acidic nature of the molecule, including the sulfated tyrosine, are invariant.

Pre-Clinical and Clinical Studies on Hirulog: A Potent and Specific Direct Thrombin Inhibitor

Despite the use of heparin and aspirin, acute thrombotic events are frequent complications in a broad range of clinical settings including following surgery, during acute episodes of unstable angina, and during both mechanical and pharmacologic revascularization therapies. New antithrombotic drugs are required to interrupt thrombus formation in those clinical indications where heparin, aspirin, or their combination are of limited or insufficient benefit. The enzyme thrombin has emerged as a key target for the development of more effective antithrombotic drugs. Our studies have addressed the following questions: 1) Is direct antithrombin therapy more effective than heparin in prevention of thrombosis in animal models? 2) What is the basis for the improved actions of direct antithrombins? 3) Can a direct antithrombin be administered to humans at levels required to obtain optimal effects? Is it well-tolerated?

Hirudin and Hirulog: Advances in antithrombotic therapy

Over the past two decades, advances in the prevention and treatment of coronary artery disease, e.g., coronary angioplasty and coronary thrombolysis, have been coupled with the need for improved antithrombotic therapy to prevent acute, life-threatening thrombotic complications. Concurrently, studies on the pathophysiologic mechanisms for the arterial thrombotic process have demonstrated a central, mediating role for thrombin. Brought forward from its identification in the late 19th century, the leech protein hirudin emerged as a model for recombinant DNA engineering and ‘protein-mimetic’ drug design. Recombinant hirudin (r-hirudin) and Hirulog are the resulting drug candidates which, as direct thrombin inhibitors, offer several potential advances in the management of acute thrombotic disorders. Clinical trials of these drug candidates have already provided evidence for clinical activity and tolerability in thromboembolic disease. Definitive evidence for efficacy is currently sought in large, controlled studies in the settings of coronary angioplasty, coronary thrombolysis and unstable angina.

Systemic Thrombin Inhibition by Hirulog Does Not Alter Medial Smooth Muscle Cell Proliferation and Inflammatory Activation after Vascular Injury in the Rabbit

Summary. The study evaluated the role of thrombin in activation of vascular smooth muscle cells early after vascular injury. The direct thrombin inhibitor Hirulog (10 mg/kg SQ tid) or vehicle was administered to rabbits over 3 days following balloon injury to the abdominal aorta and the right iliac artery. Hirulog treatment yielded marked systemic anticoagulation as evidenced by an about 3.5-fold prolongation of quantitative thrombin time one hour after an injection, but with a reduction to almost baseline levels at the end of the dosing interval. After 3 days, proliferating cells in the right iliac artery were enumerated. The expression of intercellular adhesion molecule 1, macrophage-colony stimulating factor, tumor necrosis factor α, and interleukin-1β as markers for inflammatory activation of the vessel wall was examined by immunohistochemistry and graded semiquantitatively. Mitotic indices did not differ between control and Hirulog-treated animals. There was also no difference in the expression of markers of inflammatory activation between both groups. In conclusion, thrombin inhibition by Hirulog administration does not reduce acutely (within 3 days) vascular smooth muscle cell proliferation or inflammatory activation after angioplasty. Thrombin inhibitors may therefore limit restenosis in the rabbit by acting later or via other, unknown pathways. The lack of effect of the thrombin inhibitor on the cellular events during the early phase of the response to balloon injury may explain the failure of such strategies to reduce restenosis in recent clinical trials despite effects towards acute thrombotic complications. Together, these results suggest that acute thrombin generation is not a crucial stimulus for early smooth muscle cell proliferation and inflammatory activation after vascular injury.

Inhibition by hirulog-1 of generation of plasminogen activator inhibitor-1 from vascular smooth-muscle cells induced by thrombin.

Hirulog-1 effectively prevents thrombosis in coronary artery disease and is associated with a low incidence of bleeding complications. Our study characterized the effect of Hirulog-1 on thrombin-induced production of plasminogen activator inhibitor-1 (PAI-1) in cultured baboon aortic smooth-muscle cells (BASMCs). Thrombin increased the steady-state levels of PAI-1 messenger RNA (mRNA) and the release of PAI-1 antigen from BASMCs. Treatments with 10-20 mg/L of Hirulog-1 inhibited >80% of thrombin-induced PAI-1 generation from BASMCs. Hirulog-1 alone did not significantly alter PAI-1 production in the absence of thrombin. Significant reduction of thrombin-induced PAI-1 release was observed in cultures treated with Hirulog-1 for 1 h. The maximal effect of Hirulog-1 on thrombin-induced PAI-1 release was achieved in cultures treated with thrombin plus Hirulog-1 for 3 to 6 h, associated with the normalization of PAI-1 mRNA levels induced by thrombin treatment. Strong inhibition by Hirulog-1 on thrombin-induced PAI-1 release remained in cultures with 8 h of the treatment, but the effect was attenuated 16 h after a single addition of the inhibitor. Our study demonstrates that Hirulog-1 effectively inhibited thrombin-induced PAI-1 production in cultured vascular SMCs at mRNA and protein levels. Vascular SMCs may be exposed to high concentrations of thrombin when endothelium is injured. The information generated from this study suggests that Hirulog-1 potentially prevents intravascular thrombogenesis through inhibiting thrombin-induced PAI-1 production in vascular SMCs, especially when hypercoagulation and endothelial injury occurs.