Maintenance of canine coronary artery patency following thrombolysis with front loaded plus low dose maintenance conjunctive therapy. A comparison of factor Xa versus thrombin inhibition.

OBJECTIVE: The aim was to examine the abilities of the direct thrombin inhibitor, recombinant hirudin (rHIR), and the coagulation factor Xa inhibitor, recombinant tick anticoagulant peptide (rTAP), given in combination with rt-PA as high dose front loading plus low dose maintenance infusions, to enhance reperfusion and maintain vessel patency in a canine model of left circumflex coronary artery stenosis and electrolytic lesion. METHODS: Occlusive coronary artery thrombosis was induced in anaesthetised dogs by electrical injury (150 microA) of the intimal surface of the vessel. Thirty minutes after occlusive thrombosis, high dose front loading infusions (45 min) of rTAP (200 micrograms.kg-1 x min-1) and rHIR (300 micrograms.kg-1 x min-1) were initiated concomitant with the start of a 90 min infusion of recombinant tissue-type plasminogen activator (rt-PA). Following the termination of front loading infusions, maintenance infusions of rTAP (10 or 20 micrograms.kg-1 x min-1) or rHIR (20 micrograms.kg-1 x min-1) were initiated and continued for the duration of the protocol (180 min after rt-PA termination). RESULTS: Reperfusion was incomplete in the rHIR group (7/9; 78%), whereas all rTAP-treated preparations reperfused (8/8 per group, aggregate 16/16; 100%). Following thrombolysis, the rHIR group had a high incidence of reocclusion, ranging from intermittent to long periods of occlusion, with only 2/7 (29%) of the preparations which initially recanalised remaining patent during the 180 min period following rt-PA termination. In contrast, 5/8 preparations in each of the two rTAP groups [aggregate 10/16; 63%] remained patent during the same period. The greater efficacy of rTAP v rHIR in maintaining vessel patency was also reflected in integrated coronary artery blood flows [91.0(SEM 5.8)% and 84.9(6.1)% of preocclusion flow in rTAP groups v 57.5(12.2)% of preocclusion flow in rHIR group], times to reocclusion [123.3(22.8) and 128.0(6.7) min in rTAP groups v 36.6(23.2) min in rHIR group; p < 0.05], and residual thrombus masses [1.8(0.3) and 2.0(0.3) mg in rTAP groups v 10.4(3.8) mg in rHIR group; p < 0.05]. CONCLUSIONS: With the present front loading plus low dose maintenance infusions designed to limit the duration of "high dose" conjunctive therapy, rTAP was more effective than rHIR at equimolar plasma concentrations in maintaining post-thrombolysis vessel patency, preserving coronary artery blood flow, and reducing residual thrombus mass. These findings further support the therapeutic potential of inhibiting factor Xa in the setting of coronary artery thrombolysis.

Identification and initial structure-activity relationships of a novel class of nonpeptide inhibitors of blood coagulation factor Xa.

The discovery and some of the basic structure-activity relationships of a series of novel nonpeptide inhibitors of blood coagulation Factor Xa is described. These inhibitors are functionalized beta-alanines, exemplified by 2a. Docking experiments placing 2a in the active site of Factor Xa implied that the most expeditious route to enhancing in vitro potency was to modify the group occupying the S3 site of the enzyme. Increasing the hydrophobic contacts between the inhibitor and the enzyme in this region led to 8, which has served as the prototype for this series. In addition, an enantioselective synthesis of these substituted beta-alanines was also developed.

Design of a new class of orally active fibrinogen receptor antagonists.

The integrin receptor recognition sequence Arg-Gly-Asp was successfully used as a template from which to develop a series of potent, selective, orally active, peptide-based fibrinogen receptor antagonists with a long duration of action. Simple modifications centered on the Arg and Gly residues quickly led to a modified peptide (1) with significantly enhanced ability to inhibit in vitro platelet aggregation. Substitution of the guanidino group in 1 by piperidine provided 3, which showed not only a further increase in potency but also a modest degree of oral efficacy. Finally, exploration of the nature of the C-terminal amino acid, with respect to its side-chain functionality and the carboxy terminus, yielded a group of molecules that showed excellent in vitro potency for inhibiting platelet aggregation, excellent integrin selectivity, a high level of oral efficacy, and an extended duration of action.

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Anticoagulant efficacy and immunogenicity of the selective factor Xa inhibitor antistasin following subcutaneous administration in the rhesus monkey.

The antithrombotic efficacy and duration of action of a single subcutaneous administration of the selective factor Xa inhibitor recombinant antistasin (rATS) was evaluated in a rhesus monkey model of mild disseminated intravascular coagulation. rATS (1 mg/kg) was shown to be fully effective and comparable to standard heparin (1,000 U/kg) in the suppression of thromboplastin-induced fibrinopeptide A generation for at least 5 h following a single subcutaneous administration. The absorption rate of rATS, as measured by ex vivo activated partial thromboplastin times (aPTT), mirrored that of standard heparin exhibiting peak anticoagulant activity between 1 and 2 h post administration. The anticoagulant effects of a single rATS dose lasted for longer than 30 h maintaining an aPTT value at least 2-fold higher than baseline. Repeated subcutaneous administrations of rATS resulted in the generation of fully neutralizing antibodies. These results suggest that specific factor Xa inhibition may be as effective as standard heparin in the treatment of venous thrombosis. Due to its antigenicity however, rATS is probably not suitable for chronic subcutaneous anticoagulant therapy.

Comparison of the in vivo anticoagulant properties of standard heparin and the highly selective factor Xa inhibitors antistasin and tick anticoagulant peptide (TAP) in a rabbit model of venous thrombosis.

An in vivo thromboplastin (TP)-induced venous stasis thrombosis model in rabbits was used to compare the efficacy of standard heparin with the selective factor Xa inhibitors, recombinant tick anticoagulant peptide (rTAP) and recombinant antistasin (rATS), in prophylactic prevention of thrombus formation. Heparin significantly reduced TP-induced clot formation at doses of 55 and 100 U kg-1h-1 yielding clot weights of 9 +/- 4 and 6 +/- 2%, respectively. Clot formation was significantly decreased by i.v. infusions of rTAP at doses of 21, 37 and 64 micrograms kg-1 min-1 resulting in normalized clot weights of 13 +/- 3, 8 +/- 2 and 2 +/- 1%, respectively. rATS was approximately 10-fold more potent than rTAP, reducing normalized clot weights to 16 +/- 5, 2 +/- 1 and 1 +/- 0.8% at rATS doses of 1.25, 2.5 and 5.0 micrograms kg-1 min-1, respectively. These data suggest that factor Xa-mediated inhibition of coagulation with rTAP and rATS is as effective as conventional anticoagulant treatment with heparin in preventing venous thrombosis.

Comparison of enoxaparin, hirulog, and heparin as adjunctive antithrombotic therapy during thrombolysis with rtPA in the stenosed canine coronary artery.

A canine model of electrolytic injury-induced coronary artery thrombosis and rtPA-induced thrombolysis was used to evaluate the relative antithrombotic efficacy of enoxaparin (a low molecular weight heparin), conventional therapy (heparin or heparin plus aspirin), and hirulog (a direct thrombin inhibitor), when used as adjunctive therapy during thrombolysis. After 60 min of clot aging, adjunctive therapy was begun at doses which elevated APTT approximately 2-fold over baseline. Fifteen minutes after the start of adjunctive therapy, recombinant tissue plasminogen activator (rtPA) was administered (100 microg/kg i.v. bolus + 20 microg/kg/min for 60 min). Adjunctive therapy continued for 1 h after termination of rtPA and blood flow was monitored for two additional hours. Enoxaparin (1 mg/kg i.v. bolus + 30 microg/kg/min, n = 10 for each treatment group) was the only adjunctive treatment that significantly increased the total minutes of flow (143 +/- 25 min out of a possible 240 min, vs 54 +/- 25 min for vehicle, p <0.05) and decreased thrombus mass (6.0 +/- 1.3 mg vs 11.8 +/- 3.2 mg for vehicle). Although hirulog (2 mg/kg i.v. bolus + 40 microg/kg/min) did not significantly increase the minutes of flow (120 +/- 27 min, p <0.06) or decrease thrombus mass (8.7 +/- 1.7 mg) compared to vehicle, these values were not significantly different than those measured in the enoxaparin group. However, the results with hirulog were achieved at the expense of a significantly greater increase in template bleeding time than that measured during enoxaparin treatment. Minutes of flow for heparin (50 U/kg i.v. bolus + 0.6 U/kg/min) and heparin plus aspirin (5 mg/kg i.v. bolus) were 69 +/- 20 and 60 +/- 23 min, respectively; thrombus masses were 8.2 +/- 1.3 and 7.3 +/- 1.0 mg, respectively. In summary, enoxaparin was more effective than conventional therapy in this model in terms of vessel patency and thrombus mass, and was as effective as hirulog, at least at a dose of hirulog that only modestly impaired hemostasis. Therefore, enoxaparin may prove to be a safe and effective alternative agent for adjunctive therapy during thrombolysis with rtPA.

RPR120844, a novel, specific inhibitor of coagulation factor Xa inhibits venous thrombosis in the rabbit.

The in vivo antithrombotic activity of RPR 20844, a novel synthetic coagulation factor Xa (fXa) inhibitor (Ki = 7 nM), was assessed by its ability to inhibit thrombus formation in a damaged segment of the rabbit jugular vein. Intravenous dose-response studies were performed and thrombus mass (TM), activated partial thromboplastin time (APTT), prothrombin time (PT), inhibition of ex vivo fXa activity and plasma drug levels (PDL) were determined. TM, measured at the end of a 50 min infusion, was significantly reduced (p<0.05 vs. saline-treated animals) by RPR120844 at 30 and 100 microg/kg/min. At doses of 10, 30 and 100 microg/kg/min, APTT was prolonged by 2.1, 4.2 and 6.1-fold, and PT was prolonged by 1.4, 2.2 and 3.5-fold, respectively. PDL were determined by measuring anti-fXa activity using an amidolytic assay. Peak PDL were 0.8+/-0.3, 1.5+/-0.9 and 2.4+/-0.6 microM, respectively. The drug effect was reversible with APTT, PT and PDL returning toward pretreatment values 30 min after termination of treatment. The results suggest that RPR 120844, or similar compounds, may provide an efficacious, yet easily reversible, means of inhibiting thrombus formation.

Anti-thrombotic activity of RG13965, a novel platelet fibrinogen receptor antagonist.

RG13965, a pseudotetrapeptide analogue of Arg-Gly-Asp (RGD), inhibited collagen-induced dog, monkey, human, hamster, mouse, and pig platelet aggregation in vitro with IC50 values of 3.7, 4.6, 6.3, 126, 136 and 1600 microM, respectively. RG13965 (3, 10, and 30 mg/kg, i.v.) decreased the incidence of collagen/epinephrine-induced thrombosis in mice from 90% in untreated animals to 63, 37, and 0%, respectively. In hamsters, RG13965 (10 and 30 mg/kg, i.v.) prolonged the time required for formation of a hemostatic plug in severed mesenteric arteries by 1.6- and 3.6-fold, respectively. In a canine model of repetitive platelet thrombus formation in the coronary artery, RG13965 (0.1, 0.3, and 1 mg/kg, i.v.) reversibly inhibited cyclic flow reductions (CFRs) and inhibited ADP-induced ex vivo platelet aggregation by 29, 57, and 77%, respectively. RG13965 (1 mg/kg) completely inhibited CFRs for at least 40 min. Platelet count was not altered at any dose and template bleeding time was prolonged modestly (1.8-fold) at only the highest dose. RG13965 dose-dependently and reversibly inhibited thrombus formation at doses which did not completely inhibit ex vivo platelet aggregation and only modestly prolonged template bleeding time.

In vitro characterization of a novel factor Xa inhibitor, RPR 130737.

RPR 130737 inhibited factor Xa (FXa) with a Ki of 2.4 nM and also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C, plasmin, tissue-plasminogen activator and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose-dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 microM (human), 0.61 microM (monkey), 0.44 microM (dog), 0.15 microM (rabbit), and 0.82 microM (rat). The concentrations required to double prothrombin time were 0.86 microM (human), and 1.26 microM (monkey), 1.15 microM (dog), 0.39 microM (rabbit) and 7.31 microM (rat). Kinetic studies revealed that RPR 130737 was a fast binding, reversible and competitive inhibitor for FXa when Spectrozyme FXa, a chromogenic substrate, was used. A coupled-enzyme assay measuring thrombin activity following prothrombinase conversion of prothrombin to thrombin indicated that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In this assay, RPR 130737 showed IC50s of 17 nM and 35.9 nM, respectively when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or gel-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet aggregation in vitro. These results indicate that RPR 130737 has the potential to be developed as an antithrombotic agent based on its potent and selective inhibitory effect against FXa.

Pharmacological characterization of a novel factor Xa inhibitor, FXV673.

FXV673 is a novel, potent, and selective factor Xa (FXa) inhibitor. FXV673 inhibited human, dog, and rabbit FXa with a K(i) of 0.52, 1.41, and 0.27 nM, respectively. FXV673 also displayed excellent specificity toward FXa relative to other serine proteases. It showed selectivity of more than 1000-fold over thrombin, activated protein C (aPC), plasmin, and tissue-plasminogen activator (t-PA). FXV673 prolonged plasma activated partial thromboplastin time (APTT) and prothrombin time (PT) in a dose-dependent fashion. In the APTT assays, the concentrations (microM) required for doubling coagulation time were 0.41 (human), 0.65 (monkey), 1.12 (dog), 0.25 (rabbit), and 0.80 (rat). The concentrations (microM) required in the PT assays were 1.1 (human), 1.32 (monkey), 2.31 (dog), 0.92 (rabbit), and 1.69 (rat). A coupled-enzyme assay was performed to measure thrombin activity following prothrombinase conversion of prothrombin to thrombin. FXV673 showed IC(50)s of 1.38 and 2.55 nM, respectively, when artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or fresh platelets were used as the phospholipid source for prothrombinase complex formation. It was demonstrated that FXV673 could inhibit further thrombin generation in the prothrombinase complex using PS/PC liposomes. FXV673 dose-dependently prolonged the time to vessel occlusion and inhibited thrombus formation in well-characterized canine models of thrombosis. Interspecies extrapolation (approximately 2.5-fold higher sensitivity for FXa inhibition in human than in dog) suggested that 100 ng/ml of FXV673 would be an effective plasma concentration for clinical studies. Currently FXV673 is undergoing clinical studies to be developed as an antithrombotic agent.

Site-directed mutagenesis of the leech-derived factor Xa inhibitor antistasin. Probing of the reactive site.

Antistasin (ATS) is a leech-derived 119-amino-acid protein which exhibits potent and highly selective inhibition of coagulation Factor Xa. It inhibits Factor Xa according to a common mechanism of serine-proteinase inhibitors in which a conformationally rigid substrate-like reactive site is presented to the enzyme. In this study a recombinant version of ATS was expressed and purified utilizing a yeast expression system in order to probe the reactive site P1 (Arg-34) and P1' (Val-35) residues by site-directed mutagenesis. The results demonstrate the requirement for a positively charged residue in the P1 position of ATS, with an arginine residue preferred over a lysine, yielding K1 values of 61 pM and 1.28 nM respectively. Mutation of the P1 arginine residue to the non-polar amino acid leucine abolished its inhibitory potency toward Factor Xa. The role of the C-terminal domain of ATS, which shares significant amino acid sequence identity with the N-terminal domain, was investigated by creating a second reactive site in the corresponding position of the C-terminal domain. The inhibitory activity of this mutant demonstrated that the C-terminal domain of ATS is not folded into the proper conformation necessary to create a functional inhibitory domain.

The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa.

Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.

Molecular cloning and characterization of gag-, pol-, and env-related gene sequences in the ev- chicken.

Using less stringent hybridization conditions and cloned viral DNA probes representing the avian sarcoma virus gag, pol, env, and long terminal repeat (LTR) gene sequences, we detected related sequences in two avian species purportedly lacking all endogenous avian leukosis viruses, the ev- chicken and the Japanese quail. The blot hybridization patterns obtained with the various probes suggest the presence of between 40 and 100 copies of retrovirus-related sequences in the genomes of these two species. An ev- chicken genomic DNA library was prepared and screened with gag-specific and pol-specific DNA probes. Several different clones were obtained from this library and characterized. Analysis of these clones revealed that the retrovirus-related gene sequences are linked in the order LTR-gag-pol-env-LTR, a structure indicative of a complete provirus. These data indicate the presence of previously unidentified endogenous retrovirus species in avian cells, suggesting that under the appropriate conditions of hybridization additional, more distantly evolved families of endogenous retrovirus genes may be identified in vertebrate species.

Purification and characterization of recombinant antistasin: a leech-derived inhibitor of coagulation factor Xa.

Antistasin (ATS) is a selective, tight-binding inhibitor of blood coagulation Factor Xa originally isolated from the salivary glands of the Mexican leech Haementeria officinalis. In order to provide sufficient quantities of ATS to further investigate the role of Factor Xa in blood coagulation, a recombinant version of ATS has been produced in an insect baculovirus host-vector system. In this study, we describe the purification and in vitro and in vivo characterization of a single recombinant antistasin (rATS) isoform. The purified protein constitutes a minor isoform relative to the more abundant ATS isoforms present in leech salivary gland extracts. In vitro, rATS inhibits purified human Factor Xa stoichiometrically, prolongs plasma-based clotting assays at nanomolar concentrations, and like native ATS, is cleaved at a single position by Factor Xa during the course of inhibition. An initial evaluation of the in vivo efficacy of rATS was addressed utilizing a rhesus monkey model of mild disseminated intravascular coagulation. rATS was shown to fully suppress thromboplastin-induced fibrinopeptide A generation in a dose-dependent fashion. The availability of rATS should provide a valuable tool for the critical evaluation of the specific role played by Factor Xa in coagulation.

Inhibition of repetitive thrombus formation in the stenosed canine coronary artery by enoxaparin, but not by unfractionated heparin.

Experiments were designed to compare the antithrombotic efficacy of enoxaparin and unfractionated heparin (UH) in a model of platelet-dependent cyclic flow reductions (CFRs) in the stenosed canine circumflex coronary artery. Low-molecular-weight heparins (LMWHs) are safe and effective in the prevention and treatment of venous thromboembolism. The present experiments were designed to evaluate the potential use of LMWHs in arterial thrombotic indications by comparing the antithrombotic effect of an LMWH with that of UH in an animal model of unstable angina. After establishment of consistent CFRs by experimentally induced vascular stenosis and damage, vehicle (saline), enoxaparin, or UH was administered intravenously as a loading dose plus a continuous infusion for 1 hour. The inhibition of CFRs was taken as an indicator of antithrombotic efficacy. Enoxaparin inhibited repetitive platelet thrombus formation in a dose-dependent manner, with significant inhibition of CFRs achieved at 0.5 mg/kg + 5 microg/kg per minute. This dose of enoxaparin resulted in anti-Xa levels of 0.9 to 1.0 IU/mL, anti-IIa levels of 0.2 to 0.3 IU/mL, activated partial thromboplastin time (APTT) of 1.3-fold over baseline, and a 1.4-fold increase (NS) in template bleeding time. Near-complete abolishment of CFRs was achieved with enoxaparin at 1.0 mg/kg + 10 microg/kg per minute. This dose of enoxaparin produced anti-Xa levels of 2 to 2.2 IU/mL, anti-IIa levels of 0.5 to 0.6 IU/mL, an increase in APTT of 1.4- to 1.5-fold over baseline, and a 1.9-fold increase (P<0.05) in template bleeding time. In contrast, UH had no significant effect on CFRs at a dose (100 U/kg + 10 U/kg per minute) that resulted in anti-Xa levels of 1.2 to 1.6 IU/mL, anti-IIa levels of 1.8 to 2.4 IU/mL, an increase in APTT greater than 10-fold over baseline, and a 2.5-fold increase (P<0.05) in template bleeding time. Compared with the vehicle group, circulating platelet count and hematocrit were not changed significantly by any dose of enoxaparin or UH tested. Enoxaparin, unlike UH, prevented repetitive platelet-dependent thrombus formation in the dog, thereby supporting the potential use of enoxaparin as a replacement for heparin in the treatment of arterial thrombotic disorders such as unstable angina.