Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

Hanson, Stephen R.; Griffin, John H.; Harker, Laurence A.; Kelly, Andrew B.; Esmon, Charles T.; Gruber, András

doi:10.1172/jci116795

Cited by 176 publications

(146 citation statements)

References 59 publications

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“…Thrombosis experiments were conducted on non-anticoagulated, awake animals that had chronic exteriorized femoral arteriovenous shunts, as described elsewhere. 24 Baseline shunt blood flow exceeded 250 mL/min in all study animals. Anxiety was managed with low-dose ketamine (Ͻ 2 mg/ kg per hour).…”

Section: Experimental Animalsmentioning

confidence: 84%

“…24 The hypothrombogenic graft (expanded-polytetrafluoroethylene; W. L. Gore & Associates, Flagstaff, AZ) 20 was coated with collagen, which consistently triggers plateletdependent thrombus formation. Graft segments 20 mm in length (with internal diameters [id] of either 2 or 4 mm) were filled with equine type I collagen (1 mg/mL; Nycomed Arzeneimittel, Munich, Germany) for 15 minutes, and then dried overnight under sterile airflow.…”

Section: Thrombosis Modelmentioning

confidence: 99%

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

Tucker¹,

Marzec²,

White³

et al. 2009

Blood

194

197

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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 ␤-thromboglobulin (␤TG) 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 collagencoated 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. (Blood. 2009;113:936-944) IntroductionBlood coagulation during hemostasis is initiated by the tissue factor (TF)/factor VIIa complex (the extrinsic pathway) that activates factors IX and X, and ultimately produces thrombin at sites of vascular injury. 1 In thrombosis, intravascular blood coagulation may also be initiated by the extrinsic pathway. 2,3 However, impairment of the TF/factor VIIa pathway does not provide full protection from thrombosis, since symptomatic factor VII deficient subjects can develop concurrent thrombosis and severe bleeding. 4 The functions of the contact proteins (factor XI, factor XII, prekallikrein, and high-molecular-weight kininogen) in hemostasis are less clear. The physiologic role of factor XI (FXI) has been difficult to determine because of the variable bleeding disorder associated with FXI deficiency, 5 and because monospecific FXI inhibitors have not been widely available for experimental investigation. FXI activation is thought to proceed through thrombin-and/or factor XIIdependent mechanisms, and activated FXI (FXIa) contributes to sustained thrombin generation after initiation of blood clotting by activating factor IX. These activities ultimately promote coagulation, platelet activation, and preservation of fibrin clot integrity. 6,7 Thrombin also increases the density of fibrin networks 8 and indirectly inhibits fibrinolysis through activation of carboxypeptidase B (thrombin-activatable fibrinolysis inhibitor, TAFI). 9 Thus, FXI may support thrombus propagation and clot stability by increasing thrombin generation. 10,11 Compelling circumstantial evidence suggests a contributory role for FXI in the p...

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Section: Experimental Animalsmentioning

confidence: 84%

Section: Thrombosis Modelmentioning

confidence: 99%

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

Tucker¹,

Marzec²,

White³

et al. 2009

Blood

194

197

View full text Add to dashboard Cite

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“…Noting the high affinity of thrombin for TM (Ͻ1 nM) (29), physiological concentrations of thrombin may primarily interact with TM in lipid rafts, thereby blocking its interaction with PAR-1 and activating EPCR-bound protein C that is located next to PAR-1 in the same lipid raft and/or PAR-1 recruited from another nearby raft through a regulated mechanism. In support of a primarily antiinflammatory role for thrombin under normal physiological conditions, it is known that the infusion of a low concentration of thrombin to primates exhibits a protective effect by increasing the concentration of APC in circulation without increasing the markers of platelet activation (30). Similarly, a low concentration of thrombin has been demonstrated to block the lethal inflammatory effect of endotoxin (31).…”

Section: Discussionmentioning

confidence: 99%

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

Bae

Yang

Rezaie

2007

Proc. Natl. Acad. Sci. U.S.A.

174

212

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Ever-increasing evidence in the literature suggests that the antiinflammatory and cytoprotective properties of activated protein C (APC) are mediated through its endothelial protein C receptor (EPCR)-dependent cleavage of protease-activated receptor 1 (PAR-1) on endothelial cells. However, recent results monitoring the cleavage rate of PAR-1 on human umbilical vein endothelial cells, transfected with an alkaline phosphatase-PAR-1 fusion reporter construct, have indicated that the catalytic activity of thrombin toward PAR-1 is several orders of magnitude higher than that of APC. Because thrombin is required for generation of APC, and because it also functions in the proinflammatory pathways through the activation of PAR-1, it has been difficult to understand how APC can elicit protective cellular responses through the activation of PAR-1 when thrombin is present. In this study we provide a plausible answer to this question by demonstrating that the critical receptors required for both protein C activation (thrombomodulin and EPCR) and APC cellular signaling (EPCR and PAR-1) pathways are colocalized in the membrane lipid rafts in endothelial cells. We further show that the APC cleavage of PAR-1 on cells transfected with a PAR-1 cleavage reporter construct is not sensitive to the cofactor function of EPCR. Thus, the colocalization of EPCR and PAR-1 in lipid rafts is a key requirement for the cellular signaling activity of APC. Thrombomodulin colocalization with these receptors on the same membrane microdomain can also recruit thrombin to activate the EPCR-bound protein C, thereby eliciting PAR-1 signaling events that are involved in the APC protective pathways. endothelial protein C receptor ͉ protease-activated receptor 1 ͉ thrombin ͉ thrombomodulin

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“…1 The abbreviations used are: WT, wild-type human thrombin; APC, activated protein C; WE, W215A/E217A; APTT, activated partial thromboplastin time; AV, arteriovenous. determination of fibrin deposition by 125 iodine-fibrinogen/fibrin counting after allowing at least 30 days for 111 indium decay as described previously (4). After each thrombosis study, blood flow was restored by reconnecting the segments of the chronic shunt using 2-cm-long Teflon tubing connectors (3.0-mm inner diameter).…”

Section: Methodsmentioning

confidence: 99%

The Thrombin Mutant W215A/E217A Shows Safe and Potent Anticoagulant and Antithrombotic Effects in Vivo

Gruber¹,

Cantwell²,

Di³

et al. 2002

Journal of Biological Chemistry

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Administration of the thrombin mutant W215A/E217A (WE), rationally designed for selective activation of the anticoagulant protein C, elicits safe and potent anticoagulant and antithrombotic effects in a baboon model of platelet-dependent thrombosis. The lowest dose of WE tested (0.011 mg/kg bolus) reduced platelet thrombus accumulation by 80% and was at least as effective as the direct administration of 40-fold more (0.45 mg/kg bolus) activated protein C. WE-treated animals showed no detectable hemorrhage or organ failure. No procoagulant activity could be detected for up to 1 week in baboon plasma obtained following WE administration. These results show that engineered thrombin derivatives that selectively activate protein C may represent useful therapeutic agents for the treatment of thrombotic disorders.Thrombosis involves the localized accumulation of fibrin, platelets, and other blood elements, which may restrict blood flow to and from organs and tissues (1, 2). Thrombo-occlusive events with significant morbidity and mortality include pulmonary and peripheral thromboembolism, myocardial infarction, ischemic stroke, sepsis, and heparin-induced thrombocytopenia (1). Thrombin possesses intrinsic procoagulant (fibrinogen clotting and platelet aggregation) and anticoagulant (activation of protein C) activities (3). Continuous infusion of low dose wildtype human thrombin (WT) 1 has previously been shown to be a relatively safe antithrombotic agent in baboons (4) capable of binding to thrombomodulin and generating endogenous activated protein C (APC), a naturally circulating anticoagulant enzyme (5). However, infusion of WT produces some fibrin formation and platelet activation, effects that would be enhanced at higher doses or in regions where WT might be concentrated locally. To fully exploit thrombin as an anticoagulant, its ability to cleave fibrinogen and to activate platelets must be selectively compromised (6). Toward this end, several thrombin mutants have been engineered to significantly tip the balance between procoagulant and anticoagulant activities in favor of protein C activation (6 -9). Although thrombin mutants have produced encouraging anticoagulant effects in vivo (7), no information regarding their antithrombotic potential has been available. Accordingly we tested in a baboon model the effectiveness and safety of the most potent anticoagulant thrombin produced to date, the mutant WE, which was rationally engineered to be practically devoid of activity toward fibrinogen and the platelet receptor PAR1 but to retain the ability of thrombin to activate protein C in the presence of thrombomodulin (9). MATERIALS AND METHODSWT and WE were expressed, purified, and characterized in detail as described previously (9). WT and WE were stored in frozen aliquots until use in thrombosis or coagulation experiments. To confirm that injection of WE would be at least as safe as injection of WT in baboons, we compared the procoagulant activities of the two thrombins in baboon plasma prepared by pooling citrated p...

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Antithrombotic effects of thrombin-induced activation of endogenous protein C in primates.

Cited by 176 publications

References 59 publications

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

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

Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells

The Thrombin Mutant W215A/E217A Shows Safe and Potent Anticoagulant and Antithrombotic Effects in Vivo

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