Thrombin-activatable fibrinolysis inhibitor (TAFI) activation during thrombolysis may limit the effectiveness of tissue plasminogen activator (tPA) treatment. Our data suggest that an antithrombotic human thrombin analog, E-WE thrombin (ProCase), which is a selective protein C activator under development for treating acute thrombotic emergencies, also promotes tPA-induced fibrinolysis. We found that E-WE thrombin is a poor activator of TAFI compared to wild-type (WT) thrombin. In the presence of thrombomodulin (TM), WT thrombin induced TAFI activation within 5 min. In contrast, detectable amounts of activated TAFI were not generated by E-WE thrombin in the presence of TM until 60 min. Further studies using a quantitative TAFI activity assay showed that E-WE thrombin inhibited thrombin/thrombomodulin-mediated TAFI activation in a concentration-dependent manner. The ability of E-WE to enhance tPA induced clot lysis was also evaluated in vitro. When both E-WE thrombin and tPA were incorporated within plasma clots, lysis was accelerated by up to 74% compared with the addition of tPA alone. We then tested the ability of E-WE thrombin to interrupt arterial-type experimental thrombus formation in baboons when combined with a standard interventional dose of tPA (1mg/kg). Thrombosis was initiated in the baboons by interposing 4mm internal diameter collagen coated ePTFE vascular grafts within an arterio/venous shunt. Thrombus formation was monitored by real-time gamma camera imaging of autologous 111In-labelled platelet accumulation in the grafts for a total of 90 min. Fibrin deposition was determined by direct endpoint measurement of incorporated 125I-labelled fibrinogen. Antithrombotic interventions were injected intravenously at 30 min after graft deployment into the shunt. Treatment with tPA (1mg/kg, iv) reduced fibrin deposition by 57%, but did not reduce graft-associated platelet accumulation compared with controls (n=8 and 9, respectively). E-WE thrombin, at doses ranging from 2-10µg/kg (n=8), interrupted thrombus growth within 10 min of treatment, and reduced platelet and fibrin deposition by 53-70% and 45-58% at 90 min, respectively, compared with controls. When E-WE thrombin (2µg/kg) was co-administered with tPA (1mg/kg), a profound 91% reduction in thrombus fibrin content was observed (n=1), with platelet deposition being reduced by 34%. Bleeding time and volume were also assessed during the studies, with E-WE treated animals showing no increased bleeding compared with controls. However, tPA caused ecchymoses that were not observed with E-WE thrombin treatment. The combination therapy showed no overt anti-hemostatic effects beyond tPA administration alone. These data suggest that E-WE thrombin can inhibit TAFI activation, and co-administration of E-WE thrombin with tPA may improve the efficacy of thrombolysis without additional hemostasis impairment.
Tucker:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Markway:Aronora, Inc: Employment, Equity Ownership. Wallisch:Aronora, Inc: Employment, Equity Ownership. Verbout:Aronora, Inc: Employment, Equity Ownership. Lorentz:Aronora, Inc: Employment, Equity Ownership. Carris:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
Asterisk with author names denotes non-ASH members.