Thrombotic responses of endothelial outgrowth cells to protein-coated surfaces.

There is significant clinical need for viable small-diameter vascular grafts. While there are many graft biomaterials in development, few have been clinically successful. Evaluation of grafts with a clinically relevant model is needed to drive development. This work examined extracellular matrix coatings on the thrombotic phenotype of endothelial outgrowth cells (EOCs). EOCs were tested on flat plates and tubular grafts. Flat plate studies examined collagen I, collagen IV, fibronectin and α-elastin coatings. EOCs attached or proliferated more readily on collagen I and fibronectin surfaces as determined by total DNA. The production of activated protein C (APC) by EOCs was also dependent on the surface coating, with collagen I and fibronectin displaying a higher activity than both collagen IV and α-elastin on flat plate studies. Based on these results, only collagen I and fibronectin coatings were tested on expanded polytetrafluoroethylene (ePTFE) in the ex vivo model. Tubular samples showed significantly greater tissue factor pathway inhibitor gene expression on collagen I than on fibronectin. Platelet adhesion was not significantly different, but EOCs on collagen I produced significantly lower APC than on fibronectin, suggesting that differences exist between the flat plate and tubular cultures. Overall, while the hemostatic phenotype of EOCs displayed some differences, cell responses were largely independent of the matrix coating. EOCs adhered strongly to both fibronectin- and collagen-I-coated ePTFE grafts under ex vivo (100 ml/min) flow conditions suggesting the usefulness of this clinically relevant cell source, testing modality, and shunt model for future work examining biomaterials and cell conditioning before implantation.

Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

OBJECTIVE: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. APPROACH AND RESULTS: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. CONCLUSIONS: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

A biologically active surface enzyme assembly that attenuates thrombus formation.

Activation of hemostatic pathways by blood-contacting materials remains a major hurdle in the development of clinically durable artificial organs and implantable devices. We postulate that surface-induced thrombosis may be attenuated by the reconstitution onto blood contacting surfaces of bioactive enzymes that regulate the production of thrombin, a central mediator of both clotting and platelet activation cascades. Thrombomodulin (TM), a transmembrane protein expressed by endothelial cells, is an established negative regulator of thrombin generation in the circulatory system. Traditional techniques to covalently immobilize enzymes on solid supports may modify residues contained within or near the catalytic site, thus reducing the bioactivity of surface enzyme assemblies. In this report, we present a molecular engineering and bioorthogonal chemistry approach to site-specifically immobilize a biologically active recombinant human TM fragment onto the luminal surface of small diameter prosthetic vascular grafts. Bioactivity and biostability of TM modified grafts is confirmed in vitro and the capacity of modified grafts to reduce platelet activation is demonstrated using a non-human primate model. These studies indicate that molecularly engineered interfaces that display TM actively limit surface-induced thrombus formation.

Prevention of vascular graft occlusion and thrombus-associated thrombin generation by inhibition of factor XI.

The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)-dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti-human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and beta-thromboglobulin (betaTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.

Thrombin formation in vitro in response to shear-induced activation of platelets.

INTRODUCTION: Thromboembolic events caused by implanted vascular devices present serious medical challenges. In particular bileaflet mechanical heart valves (MHVs) are prone to thrombus formation in the hinge region due to a combination of high shear stress and stagnation regions. Most studies of shear-induced platelet activation and aggregation have been performed using viscometers, parallel plate flow, and other non-physiologic in vitro configurations. The present study investigated these events in a physiogically relevant environment in which thrombin formation in response to shear stress activation of platelets plays a more predominant role. MATERIALS AND METHODS: Anticoagulated (citrated) human blood was placed in a steady flow loop containing a 400 microm round orifice or various MHVs in the leakage position. Simultaneous blood recalcification enhanced the thrombus forming potential of the blood. Aggrastat and AN51 were used to block binding to the platelet GPIIb/IIIa and GPIb receptors, respectively, and aspirin was used to block thromboxane production. Thrombin generation was measured indirectly by the thrombin-antithrombin III assay. RESULTS AND CONCLUSIONS: Aggrastat, AN51, and aspirin all suppressed thrombin formation. Furthermore, histological results suggested important roles for vWF and fibrinogen in a two-step model of thrombus formation. Thus, thrombin is reproducibly formed in this in vitro system, a process that can be suppressed by blocking platelet activation. This system has the potential to investigate mechanisms and interventions for medical devices that contact with blood under varying shear stress conditions.

Hemostatic effect of activated factor VII without promotion of thrombus growth in melagatran-anticoagulated primates.

INTRODUCTION: Pharmacological enhancement of coagulation using activated prothrombin complex concentrate (APCC) or activated factor VII (FVIIa) might be useful hemostatic approaches to bleeding emergencies during anticoagulant therapy. However, any such intervention should not increase thrombotic risk. We therefore investigated their hemostatic and prothrombotic potential during propagation of large arterial-type thrombin in anticoagulated baboons. MATERIALS AND METHODS: High dose melagatran, a competitive inhibitor of thrombin (0.6 mg/kg/h), or inactivated FVIIa (FVIIai), a competitive inhibitor of FVIIa (2 mg/kg) were used for anticoagulation. APCC or FVIIa were administered to melagatran-anticoagulated animals only. Primary hemostasis was assessed as template bleeding time (BT). Thrombus formation was quantified as fibrin deposition (FD) and platelet deposition (PLD) in synthetic vascular grafts that were deployed for 40 min into arteriovenous shunts. RESULTS: Melagatran (n=11) prolonged BT to 279% (95% CI +/-140%; P<0.019), reduced FD to 33% [+/-8%; P<0.001]; and PLD to 39% [+/-11%; P<0.001] of untreated controls. FVIIai (n=3) prolonged BT (222% [+/-51%; P<0.010]) without inhibiting thrombus propagation. APCC (n=10) reduced the antithrombotic effect of melagatran (FD 52% [+/-9%; P<0.002], PLD 61% [+/-17%; P=0.028] versus melagatran alone) at a dose (250 U/kg) that had no effect on the BT (327% [+/-150%; P=0.607]. Meanwhile, FVIIa (n=12) normalized the BT to 115% (+/-32%; P<0.05) at a dose (270 microg/kg) that was not yet prothrombotic (FD 26% [+/-4%; P<0.001], PLD 39% [+/-9%; P=0.970]). CONCLUSION: Administration of FVIIa during antithrombotic treatment with direct thrombin inhibitors might support hemostasis before promoting the intraluminal expansion of thrombi.

In vivo platelet redistribution and acute transient thrombocytopenia after eptifibatide injection in baboons.

BACKGROUND: The occurrence of thrombocytopenia has been reported during clinical eptifibatide (Integrilin) therapy, but the exact mechanism is not yet established to explain the varied duration and severity of thrombocytopenia associated with glycoprotein (GP) IIb/IIIa inhibitors. We assessed the redistribution of platelets in juvenile baboons during acute transient thrombocytopenia that was observed after eptifibatide injection. METHODS: Eptifibatide was administered intravenously to eight baboons by infusion at 20 microg/kg/min or a bolus injection of 10 mg. Platelet distribution was measured with a gamma scintillation camera using 111In-labeled autologous platelets. Platelet function and GP IIb/IIIa receptor inhibition were evaluated using the Plateletworks system. The effects of pretreatment with abciximab (0.4 mg/kg) or human immunoglobulin concentrate (0.75 g/kg) were also investigated. RESULTS: Eptifibatide, administered as an infusion or a bolus, caused transient thrombocytopenia with uptake of platelets predominantly by the liver. The recovery of platelet aggregation was associated with the re-entry of platelets from the liver into the systemic circulation. Pretreatment with either abciximab (0.4 mg/kg) or human intravenous immunoglobulin (IVIG, 0.75 g/kg) attenuated eptifibatide-induced thrombocytopenia and the hepatic uptake of radiolabeled platelets. CONCLUSION: Acute thrombocytopenia after eptifibatide injection was caused by the transient redistribution of platelets to the liver. Attenuation of the decrease in platelet count and hepatic sequestration by abciximab and IVIG suggests that thrombocytopenia may have been caused by ligand-induced binding site antigen induction and recognition by the reticuloendothelial system.

Immobilization of actively thromboresistant assemblies on sterile blood-contacting surfaces.

Rapid one-step modification of thrombomodulin with alkylamine derivatives such as azide, biotin, and PEG is achieved using an evolved sortase (eSrtA) mutant. The feasibility of a point-of-care scheme is demonstrated herein to site-specifically immobilize azido-thrombomodulin on sterilized commercial ePTFE vascular grafts, which exhibit superior thromboresistance compared with commercial heparin-coated grafts in a primate model of acute graft thrombosis.

Reversal of rivaroxaban anticoagulation by haemostatic agents in rats and primates.

Rivaroxaban is an oral, direct factor Xa inhibitor for the management of thromboembolic disorders. Despite its short half-life, the ability to reverse rivaroxaban anticoagulation could be beneficial in life-threatening emergencies. The potential of prothrombin complex concentrate (PCC; Beriplex®), activated PCC (aPCC; FEIBA®) or recombinant activated factor VII (rFVIIa; NovoSeven®) to reverse rivaroxaban in rats and baboons was investigated. Anaesthetised rats pre-treated with intravenous rivaroxaban (2 mg/kg) received intravenous rFVIIa (100/400 µg/kg), PCC (25/50 U/kg) or aPCC (50/100 U/kg) after initiation of bleeding. Clotting times and bleeding times (BTs) were recorded. Rivaroxaban was administered as an intravenous 0.6 mg/kg bolus followed by continuous 0.6 mg/kg/hour infusion in baboons. Animals received intravenous aPCC 50 U/kg (2 U/kg/minute) or rFVIIa 210 µg/kg. BT and clotting parameters were measured. In rats pretreated with high-dose rivaroxaban, PCC 50 U/kg, aPCC 100 U/kg and rFVIIa 400 µg/kg significantly reduced BT vs rivaroxaban alone (5.4 ± 1.4-fold to 1.5 ± 0.4-fold [p<0.05]; 3.0 ± 0.4-fold to 1.4 ± 0.1-fold [p<0.001]; and 3.5 ± 0.7-fold to 1.7 ± 0.2-fold [p<0.01] vs baseline, respectively). In baboons pre-infused with rivaroxaban and then given aPCC, BT increased by 2.0 ± 0.2-fold and aPCC returned BT to baseline for the duration of its infusion. rFVIIa reduced BT from 2.5 ± 0.3-fold over baseline to 1.7 ± 0.3-fold over baseline. Prolongation of prothrombin time was reduced by PCC, aPCC and rFVIIa in both species. Rivaroxaban reduced thrombin-antithrombin levels; application of PCC and aPCC, but not rFVIIa, increased these levels. In conclusion, PCC, aPCC or rFVIIa have the potential to reverse the anticoagulant and anti-haemostatic effects of rivaroxaban.

Transport-reaction model of mural thrombogenesis: comparisons of mathematical model predictions and results from baboon models.

Thrombogenesis depends on biochemical reactions affected by blood flow dynamics. While mathematical models of mural thrombogenesis provide a means of understanding how blood flow affects thrombus growth, comparisons to experimental data are needed to validate the models and enable prediction of thrombus growth under diverse conditions. In this paper, we present mathematical models of mural thrombogenesis under flow and validation of the models with experimental data collected from a thrombogenic vascular graft segment. The grafts were placed in exteriorized high-flow arteriovenous (AV) shunts in baboons. Radiolabeled platelet deposition onto the thrombogenic segment, a marker of thrombus size, and plasma thrombin-antithrombin (TAT) concentration downstream of the graft, a marker of local thrombin generation, were monitored over time. The mathematical model of mural thrombogenesis consisted of transport-reaction equations in which platelets and thrombin were explicitly considered. We found that the transport-reaction model captured the order of magnitude of TAT sampled levels, while calculated rates of platelet deposition agreed well with radioimaging results. Analysis of experimental and modeling data indicates that, at least during part of thrombus growth progression, thrombin generation is in excess and platelet adhesion rates would be sustained even at lower local thrombin concentrations.

Safety and antithrombotic efficacy of moderate platelet count reduction by thrombopoietin inhibition in primates.

Most heart attacks and strokes are caused by blood clots (thrombi) that block the vasculature. Because disease-causing arterial thrombosis depends on blood platelets, platelet inhibitors such as aspirin and clopidogrel effectively decrease the risk of thrombosis; however, they also impair platelet-dependent hemostasis that staunches bleeding from wounds and can therefore produce excessive bleeding. Experimental studies show that a reduction in the number of platelets also inhibits thrombosis, but these treatments also interfere with platelet function. Because normal hemostasis requires that the platelet concentration remain within a physiological range in the circulation, we evaluated whether lowering the number of circulating platelets--but only to a value still within the normal range--by inhibiting platelet formation in the bone marrow inhibits acute thrombogenesis in baboons. We reduced the platelet count with an inhibitor against the megakaryocyte-promoting hormone thrombopoietin and then showed that experimental occlusive thrombogenesis on collagen-coated vascular grafts was reduced, without impairment of primary hemostasis. These results suggest that suppressing platelet production without interfering with the hemostatic function of platelets may offer a safe alternative to current therapies for prevention of stroke and heart attack triggered by blood clotting.

Inhibition of acute vascular thrombosis in chimpanzees by an anti-human tissue factor antibody targeting the factor X binding site.

Tissue factor (TF) antagonists targeting the factor VII (FVII) binding domain have been shown to interrupt acute vascular thrombus formation without impairing haemostasis in non-human primates. In this study, we evaluate whether a human/mouse chimeric monoclonal antibody (ALT-836, formerly known as Sunol-cH36) blocking the factor X/factor IX (FX/FIX) binding site of tissue factor could achieve similar clinical benefits in an arterial thrombosis model induced by surgical endarterectomy in chimpanzees. In this model, sequential surgical endarterectomies on right and left superficial femoral arteries were performed 30 days apart in five chimpanzees. A bolus (1 mg/kg) of ALT-836 was injected intravenously immediately preceding the restoration of flow in the endarterectomised femoral artery. Pre-surgical labelling of autologous platelets using (111)In-Oxine and post-surgical gamma camera imaging of (111)In-platelet deposition at endarterectomy sites was performed. The manipulated arterial segments were harvested for patency analysis 30 days following surgery. The results indicate that ALT-836 was highly effective at reducing acute vascular thrombosis, with no significant variations in surgical blood loss and template-bleeding time in the treated group compared to the control animals. These data suggest that ALT-836 is an effective and safe antithrombotic agent in preventing TF-initiated vascular thrombogenesis without compromising haemostasis.

Procoagulant properties of flow fields in stenotic and expansive orifices.

In the United States, over 125,000 mechanical heart valves (MHVs) are implanted each year. Flow through the MHV hinge can cause thromboemboli formation. The purpose of this study was to examine various orifice geometries representing the MHV hinge region and how these geometries may contribute to platelet activation and thrombin generation. We also characterized these flow fields with digital particle image velocimetry (DPIV). Citrated human blood at room temperature was forced through the orifices (400 and 800 microm ID) with a centrifugal bypass pump, continuously infusing calcium chloride to partially reverse the citrate anticoagulant. Blood samples were tested for the presence of thrombin-antithrombin complex (TAT) and platelet factor 4 (PF4). Velocity and shear stress were measured with DPIV using a blood analog fluid seeded with fluorescent microbeads. The results indicate that small changes in geometry, although they do not affect the bulk flow, change the coagulation propensity as blood flows through the orifices. A more abrupt geometry allows more stagnation to occur resulting in more thrombin generation. PF4 measurements indicated similar levels of platelet activation for all orifices. DPIV showed differences in the jets with respect to entrainment of stagnant fluid. These results help to pinpoint the important parameters that lead to flow stasis and subsequent thrombus formation.

Capture of flowing endothelial cells using surface-immobilized anti-kinase insert domain receptor antibody.

In humans, self-endothelialization of synthetic grafts is severely limited, but a recent interesting idea is to attract endothelial progenitor cells (EPCs) from peripheral blood onto grafts via antibodies directed at proposed EPC markers. Results with anti-CD34 antibodies have shown some promise, but it is unclear whether CD34 is the best marker for cells with re-endothelializing potential. Much evidence points to kinase insert domain receptor (KDR) as an important indicator of endothelial potential if not a definitive marker. Because KDR is not an adhesion molecule (like CD34), we first demonstrated the ability to use adsorbed and protein G-oriented antibody to this receptor to capture flowing cells onto a solid surface. Using endothelial cells and smooth muscle cells, we show in a model system under low shear rates the ability to selectively capture cells by this receptor. Furthermore, our results indicate that concomitant flow of cells lacking the receptor does not affect the efficiency of capture of KDR(+) cells but that orienting the antibody significantly increases the efficiency of capture.

Fibrinogen gamma' chain carboxy terminal peptide selectively inhibits the intrinsic coagulation pathway.

The minor gammaA/gamma' isoform of fibrinogen contains a high affinity binding site for thrombin exosite II that is lacking in the major fibrinogen isoform, gammaA/gammaA fibrinogen. The biological consequences of gamma' chain binding to thrombin were therefore investigated. Coagulation assays, thrombin activity assays, and a primate thrombosis model were used to characterize the biological effects of the gamma' 410-427 peptide. The gamma' peptide had little effect on thrombin cleavage of the small peptidyl substrate tosyl-glycyl-prolyl-arginine-4-nitranilide acetate. However, in vitro assays demonstrated that the gamma' peptide inhibited thrombin cleavage of larger proteinaceous substrates, including fibrinogen and factor VIII. The gamma' peptide inhibited the activated partial thromboplastin time in plasma and showed greater inhibition of activated partial thromboplastin time assays than prothrombin time assays, consistent with the inhibition of factor VIII cleavage. Studies in a baboon thrombosis model showed that the gamma' 410-427 peptide inhibited fibrin-rich thrombus formation (typical of venous thrombi) and, to a lesser extent, platelet-rich thrombus formation (typical of arterial thrombi). These results indicate that binding of thrombin exosite II by the gamma' peptide has selective effects on the intrinsic pathway.

Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates.

The anticoagulant and anti-inflammatory enzyme, activated protein C (APC), naturally controls thrombosis without affecting hemostasis. We therefore evaluated whether the integrity of primary hemostasis was preserved during limited pharmacological antithrombotic protein C activator (PCA) treatment in baboons. The double-mutant thrombin (Trp215Ala/Glu217Ala) with less than 1% procoagulant activity was used as a relatively selective PCA and compared with systemic anticoagulation by APC and low-molecular-weight heparin (LMWH) at doses that inhibited fibrin deposition on thrombogenic segments of arteriovenous shunts. As expected, both systemic anticoagulants, APC (0.028 or 0.222 mg/kg for 70 minutes) and LMWH (0.325 to 2.6 mg/kg for 70 minutes), were antithrombotic and prolonged the template bleeding time. In contrast, PCA at doses (0.0021 to 0.0083 mg/kg for 70 minutes) that had antithrombotic effects comparable with LMWH did not demonstrably impair primary hemostasis. PCA bound to platelets and leukocytes, and accumulated in thrombi. APC infusion at higher circulating APC levels was less antithrombotic than PCA infusion at lower circulating APC levels. The observed dissociation of antithrombotic and antihemostatic effects during PCA infusion thus appeared to emulate the physiological regulation of intravascular blood coagulation (thrombosis) by the endogenous protein C system. Our data suggest that limited pharmacological protein C activation might exhibit considerable thrombosis specificity.

Flow and thrombosis at orifices simulating mechanical heart valve leakage regions.

BACKGROUND: While it is established that mechanical heart valves (MHVs) damage blood elements during leakage and forward flow, the role in thrombus formation of platelet activation by high shear flow geometries remains unclear. In this study, continuously recalcified blood was used to measure the effects of blood flow through orifices, which model MHVs, on the generation of procoagulant thrombin and the resulting formation of thrombus. The contribution of platelets to this process was also assessed. METHOD OF APPROACH: 200, 400, 800, and 1200 microm orifices simulated the hinge region of bileaflet MHVs, and 200, 400, and 800 microm wide slits modeled the centerline where the two leaflets meet when the MHV is closed. To assess activation of coagulation during blood recirculation, samples were withdrawn over 0-47 min and the plasmas assayed for thrombin-antithrombin-llI (TAT) levels. Model geometries were also inspected visually. RESULTS: The 200 and 400 microm round orifices induced significant TAT generation and thrombosis over the study interval. In contrast, thrombin generation by the slit orifices, and by the 800 and 1200 microm round orifices, was negligible. In additional experiments with nonrecalcified or platelet-depleted blood, TAT levels were markedly reduced versus the studies with fully anticoagulated whole blood (p < 0.05). CONCLUSIONS: Using the present method, a significant increase in TAT concentration was found for 200 and 400 microm orifices, but not 800 and 1200 microm orifices, indicating that these flow geometries exhibit a critical threshold for activation of coagulation and resulting formation of thrombus. Markedly lower TAT levels were produced in studies with platelet-depleted blood, documenting a key role for platelets in the thrombotic process.

Soluble thrombomodulin is antithrombotic in the presence of neutralising antibodies to protein C and reduces circulating activated protein C levels in primates.

We studied whether there was a relationship between the anticoagulant effects of recombinant human soluble thrombomodulin (rhsTM) and activation of protein C in a primate model of acute vascular graft thrombosis in 11 baboons (Papio species). Baboons were pretreated with 0.1, 1 and 5 mg/kg of rhsTM, with or without co-injection of a neutralising monoclonal antibody to protein C (HPC4) in the 1 mg/kg rhsTM group. Subsequently, thrombogenic polyester grafts were deployed for 3 h into chronic exteriorised arteriovenous shunts. Thrombus growth in the graft, plasma-activated protein C (APC) levels, coagulation and thrombosis markers were determined. In untreated baboons, baseline circulating APC levels more than doubled and graft thrombi propagated until reaching equilibrium in about 1 h. Treatment with rhsTM reduced thrombus propagation rates, prolonged the clotting and bleeding times, decreased thrombin-antithrombin complex, beta-thromboglobulin and fibrinopeptide A levels, and, surprisingly, also decreased systemic APC levels, in a dose-dependent manner. In the presence of HPC4 antibody to inhibit APC generation, the acute antithrombotic activity of rhsTM on graft thromboses was not attenuated for up to 80 min, but sustained thrombus accumulation was observed over a 180-min period. These findings suggest that, in contrast to the prevailing hypotheses, the primary antithrombotic activity of rhsTM is independent of protein C, at least in this primate model. Direct inhibition of thrombin's prothrombotic activity upon complex formation with rhsTM might explain the molecular mechanism of the observed antithrombotic effect.

Factor VIIa inhibitors: chemical optimization, preclinical pharmacokinetics, pharmacodynamics, and efficacy in an arterial baboon thrombosis model.

Highly selective and potent factor VIIa-tissue factor (fVIIa.TF) complex inhibitors were generated through structure-based design. The pharmacokinetic properties of an optimized analog (9) were characterized in several preclinical species, demonstrating pharmacokinetic characteristics suitable for once-a-day dosing in humans. Analog 9 inhibited platelet and fibrin deposition in a dose-dependent manner after intravenous administration in a baboon thrombosis model, and a pharmacodynamic concentration-response model was developed to describe the platelet deposition data. Results for heparin and enoxaparin (Lovenox) in the baboon model are also presented.

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo.

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.