Abstract TP260: Synergistic Inhibitors Halt Brain and Systemic Hemorrhage After Tissue Plasminogen Activator Treatment for Ischemic Stroke

Tissue plasminogen activator (TPA) treatment for ischemic stroke is complicated by brain hemorrhage and major systemic bleeding for which there is no specific antidote. These bleeding complications reduce the therapeutic value of TPA as well as undermine its therapeutic use in eligible patients. Although the primary causes of TPA-induced hemorrhage are unknown, it may be related to the clot dissolving or fibrinolytic effects of TPA-catalyzed plasminogen activation. We examined whether specific inhibitors of TPA’s fibrin-dependent activation of plasminogen could reduce bleeding after the initiation of TPA therapy for ischemic stroke. METHODS: Hybridomas were screened for the production of monoclonal antibodies (MAbs) that inhibited TPA-induced fibrinolysis. After molecular characterization and purification, these MAbs were tested for their ability to stop brain hemorrhage and systemic bleeding triggered by TPA therapy for ischemic stroke. RESULTS: Two MAbs were selected that bound with high affinity (sub-nanomolar dissociation constants) to different epitopes on the TPA molecule. These MAbs preferentially inhibited TPA’s fibrin-dependent plasminogen activation but not other catalytic functions; they did not affect plasminogen activation by other agents. When added to human clots, these MAbs synergistically blocked TPA fibrinolysis with greater specificity and potency than a stabilized form of plasminogen activator inhibitor 1. When administered after the onset of TPA therapy for ischemic stroke, these MAbs significantly reduced brain hemorrhage (p<0.05) but did not worsen infarction. The MAbs also markedly diminished TPA-triggered tail bleeding and hemoglobin loss in anesthetized mice. CONCLUSION: TPA-induced brain and systemic bleeding in vivo is blocked by potent synergistic inhibitors of TPA’s fibrin-dependent plasminogen activation. This implies that hemorrhage is related to TPA’s fibrin-dependent mechanism of plasminogen activation and that targeted inhibitors of this process may serve as specific antidotes for TPA-associated hemorrhage.