Systemic thrombin inhibition by Hirulog does not alter medial smooth muscle cell proliferation and inflammatory activation after vascular injury in the rabbit.

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 alpha, and interleukin-1beta 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.

Effectiveness of hirulog in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits.

BACKGROUND: Thrombin may play an important role in restenosis after balloon angioplasty (BA). Angiographic and pathologic restenosis have been shown to be reduced after BA in an atherosclerotic rabbit model using recombinant desulfatohirudin, a selective and direct thrombin inhibitor. We hypothesized that potent and specific thrombin inhibition with the synthetic peptide hirulog given intravenously at the time of angioplasty would reduce restenosis in rabbits, confirming a specific role of thrombin in restenosis. METHODS AND RESULTS: Focal femoral atherosclerosis was induced in 27 rabbits by air desiccation endothelial injury followed by a 2% cholesterol diet for 1 month. Rabbits received either heparin (150 units/kg bolus, n = 14) or hirulog (5 mg/kg bolus followed by 5 mg/kg/h for 2 h, n = 13) at the time of BA (2.5-mm balloon with three 60-second, 10-atm inflations 60 s apart). Angiograms performed before and after BA and before sacrifice were analyzed quantitatively. Rabbits were sacrificed 28 days after BA for quantitative histopathologic analysis. Minimum luminal diameter (mm) did not differ between treatment groups before (1.1 +/- 0.2 vs. 1.2 +/- 0.1 mm) or after (1.5 +/- 0.2 vs. 1.6 +/- 0.1) BA in arteries from heparin-versus hirulog-treated rabbits, respectively. At 28 days, however, minimum luminal diameter was significantly less (1.0 +/- 0.4 vs. 1.5 +/- 0.2, p = 0.0001) and percent stenosis was greater (0.46 +/- 0.25 vs. 0.22 +/- 0.08, p = 0.0002) in arteries from heparin- versus hirulog-treated rabbits, respectively. Similarly, quantitative histopathology showed less cross-sectional area narrowing by plaque in the hirulog group (56 +/- 24 vs. 42 +/- 21%, p = 0.04). CONCLUSION: A 2-hour infusion of hirulog at the time of angioplasty improved late angiographic luminal dimensions and reduced cross-sectional area narrowing by plaque in rabbits compared with heparin controls. Together with previous studies, this confirms a specific role for thrombin in restenosis after angioplasty.

Hirulog: a direct thrombin inhibitor for management of acute coronary syndromes.

Hirulog therapy has been studied extensively in numerous settings including prevention of DVT, treatment of unstable angina, treatment of acute myocardial infarction during thrombolysis, and prevention of acute complications of PTCA. Being one of the first direct thrombin inhibitors in clinical development, it has had to 'test the waters', so to speak, of the relationship between pathophysiology and clinical trial design: what are the correct indications, patient entry criteria, endopoints, frequency and duration of dosing, and so on? Our findings validate a role for thrombin in treating arterial thromboembolism and demonstrate clinical activity and tolerability of Hirulog.

Thrombin functions as an inflammatory mediator through activation of its receptor.

A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP-induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells.

Hirulog effect in rat endotoxin shock.

Hirulog is a thrombin catalytic site inhibitor which exhibits specificity for the anionic binding exosite of alpha thrombin. Here, we have evaluated the effect of Hirulog (1, 5 and 10 mg/kg, 30 min pretreatment) in a rat model of endotoxemia. Intravenous injection of lipopolysaccharide from E. coli (25 mg/kg; serotype 0127:B8) caused decreases in blood pressure which were significantly reduced (about 60%) in animals pretreated with Hirulog. Rat survival to endotoxin was significantly increased in Hirulog pretreated group (5 and 10 mg/kg) up to 24 hours. Hirulog at the dose of 10 mg/kg inhibited both endotoxin-induced leukopenia at 30 and 60 minute points and thrombocytopenia at 30 minute point but not at 90 and 120 minute points. Fibrinogen levels were significantly reduced after 2 hours following endotoxin administration. Pretreatment with Hirulog (5-10 mg/kg i.v.) 30 min prior to administration of endotoxin prevented changes in fibrinogen plasma levels. These results demonstrate that Hirulog-induced inhibition of thrombin is effective in reducing toxic and lethal effects of endotoxin.

Kinetic mechanism for the interaction of Hirulog with thrombin.

Hirulog (D-FPRPGGGGDGDFEEIPEEYL) is a bivalent inhibitor of thrombin consisting of a moiety (D-FPRP) that binds to the active-site cleft and a hirudin-like C-terminal region (DGDFEEIPEEYL) that binds to the positively charged surface groove of thrombin known as the anion-binding exosite. The formation of the thrombin-Hirulog complex was studied using steady-state and rapid kinetics at 37 degrees C. The inhibition constant for Hirulog was found to be 1.9 nM. Hirulog was slowly degraded by thrombin with a kcat value of 0.01 s-1. The formation of the complex resulted in an enhancement of 44% in the intrinsic fluorescence of thrombin. The kinetics of the increase in thrombin fluorescence were described by a double-exponential decay. The dependence of the rate constant for the fast phase on the concentration of Hirulog could be described by the Michaelis-Menten equation with Km and kmax values of 0.75 +/- 0.12 microM and 325 +/- 17 s-1. The data were consistent with a mechanism in which the C-terminal region of Hirulog binds to the anion-binding exosite with a dissociation constant of 0.75 microM in the first step, followed by two intramolecular steps with rate constants of about 300 and 30 s-1. A C-terminal fragment of hirudin was found to compete in the first step confirming that this process corresponded to the binding of the hirudin-like C-terminus of Hirulog to the anion-binding exosite.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The arterial thrombotic process and emerging drugs for its control.

Unabated, the arterial thrombotic process continues to be a major challenge in the management of acute coronary artery disease. Pharmacologic and mechanical revascularization therapies, which have proliferated over the last decade, remain impeded by arterial thrombosis and its clinical sequelae. New antithrombotic drugs aimed at specific points in the arterial thrombotic process offer the potential for substantial improvements in the management of coronary artery disease. The use of these agents as a mainstay of patient care is becoming a reality as controlled clinical studies test their safety and potential benefits.

Structure of the hirulog 3-thrombin complex and nature of the S' subsites of substrates and inhibitors.

The X-ray crystallographic structure of the human alpha-thrombin complex with hirulog 3 (a potent, noncleavable hirudin-based peptide of the "hirulog" class containing a beta-homoarginine at the scissile bond), which is isomorphous with that of the hirugen-thrombin crystal structure, was solved at 2.3-A resolution by starting with a model for thrombin derived from the hirugen-thrombin complex and was refined by restrained least squares methods (R = 0.132). Residues of hirulog 3 were well-defined in the electron density, which included most of the pentaglycine linker and the C-terminal helical turn that was disordered in a related structure of thrombin with hirulog 1. The interactions of D-Phe1'-Pro2'-beta-homoArg3' with the active site of thrombin were essentially identical to those of related structures of PPACK- (D-Phe-Pro-Arg chloromethyl ketone) and hirulog 1-thrombin, with the guanidinium function of the arginyl P1 residue forming a hydrogen-bonding ion pair with Asp189 of the S1 site. A noticeable shift in the CA atom of beta-homoArg3' due to the methylene insertion displaces the scissile bond from attack by Ser195, thus imparting proteolytic stability to the beta-homoArg hirulog derivative. Resolution of the pentaglycine spacer, linking N- and C-terminal functional domains into a single oligopeptide bivalent inhibitor, permitted delineation of corresponding S' subsites of thrombin. The position of Gly4' (P1') is stabilized by three hydrogen bonds with His57, Lys60F, and Ser195, while the conformational angles maintained in a strained, nonallowed configuration for non-glycyl amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin.

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.

Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor.

Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin-specific inhibition by and slow cleavage of hirulog-1.

Hirulog-1 [D-Phe-Pro-Arg-Pro-[Gly]4-desulphohirudin-(53-64) (HV1)] was designed to bind by its first four and last 12 residues to the alpha-thrombin catalytic site and anion-binding exosite for fibrin(ogen) recognition respectively, with a [Gly]4 bridge and an Arg-Pro bond at the scissional position. Human alpha-, gamma- and zeta-thrombins, as well as bovine trypsin, readily hydrolyse Spectrozyme-TH (D-hexahydrotyrosyl-Ala-Arg p-nitroanilide) at pH 7.4 and approx. 23 degrees C. Both alpha- and zeta-thrombins, which have high fibrinogen-clotting activities (greater than 3000 kunits/g), were inhibited with this substrate by hirulog-1 [Ki = 2.56 +/- 0.35 nM (n = 3) and 1.84 +/- 0.15 nM (n = 3) respectively] and slowly cleaved the inhibitor [k = 0.326 +/- 0.082 min-1 (n = 12) and 0.362 +/- 0.056 min-1 (n = 18) respectively], whereas gamma-thrombin, which has essentially no clotting activity (approx. 4 kunits/g), and trypsin were not inhibited with greater than 1000-fold molar excess of hirulog-1. Similar inhibition parameters were also obtained for hirulog-1 incubated with alpha-thrombin or zeta-thrombin at approx. 23 degrees C and by measuring thrombin activity with fibrinogen in the clotting assay at 37 degrees C. Cleavage of the Arg-3-Pro-4 bond in hirulog-1 by either alpha- or zeta-thrombin was shown by identical cleavage products of either thrombin on h.p.l.c. and by sequence analysis of the alpha-thrombin products. These data demonstrate that hirulog-1 is a specific inhibitor of thrombin forms with high fibrinogen-procoagulant activities and that its Arg-3-Pro-4 bond is slowly cleaved by these thrombin forms.

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 thrombin's 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 Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.

Structure of the hirugen and hirulog 1 complexes of alpha-thrombin.

The isomorphous structures of the hirugen (N-acetylhirudin 53'-64' with sulfato-Tyr63') and hirulog 1 (D-Phe-Pro-Arg-Pro-(Gly)4 desulfato-Tyr63'-hirugen) complexes of human alpha-thrombin have been determined and refined at 2.2 A resolution to crystallographic R-factors of 0.167 and 0.163, respectively. The binding of hirugen to thrombin is similar to that of the binding of the C-terminal dodecapeptide of hirudin, including that of the terminal 3(10) helical turn. The sulfato Tyr63', which, as a result of sulfation, increases the binding affinity by an order of magnitude, is involved in an extended hydrogen bonding network utilizing all three sulfato oxygen atoms. The hirugen-thrombin complex is the first thrombin structure determined to have an unobstructed active site; this site is practically identical in positioning of catalytic residues and in its hydrogen bonding pattern with that of other serine proteinases. Hirulog 1, which is a poor thrombin substrate, is cleaved at the Arg3'-Pro4' bond in the crystal structure. The Arg3' of hirulog 1 occupies the specificity site, the D-Phe-Pro-Arg tripeptide is positioned like that of D-Phe-Pro-Arg chloromethylketone in the active site and the Pro4'(Gly)4 spacer to hirugen is disordered in the structure, as is the 3(10) turn of hirugen. The latter must be related to the simultaneous absence both of sulfation and of the last residue of hirudin (Gln65'). In addition, the autolysis loop of thrombin (Lys145-Gly150) is disordered in both structures. Changes in circular dichroism upon hirugen binding are therefore most likely the result of the flexibility associated with this loop.