Amplified anticoagulant activity of tissue factor-targeted thrombomodulin In-vivo validation of a tissue factor-neutralizing antibody fused to soluble thrombomodulin

Tissue factor (TF) exposure is a potent pro-thrombotic trigger that initiates activation of the coagulation cascade, while thrombomodulin (TM) is a potent anticoagulant protein that limits the extent of activation. Both TF neutralizing antibodies and soluble TM (sTM) are effective anticoagulants. We have developed a novel anticoagulant fusion protein, Ab(TF)-TM, by fusing a TFneutralizing single-chain antibody,Ab(TF), to an active fragment ofTM. Ab(TF)- TM is a novel anticoagulant targeting to sites ofTF exposure with a dual mechanism of action.The Ab(TF) portion of the molecule inhibitsTF/factorVIIa mediated activation of FIX and FX, and the TM portion of the molecule acts as a cofactor for activation of protein C. In-vitro coagulation assays show that Ab(TF)- TM more potently inhibitsTF-initiated coagulation (prothrombin time) than can its individual components, Ab(TF) (20-fold) and sTM (80-fold) alone, or in combination (10-fold).In contrast, the potency of Ab(TF)-TM in the activated partial thromboplastin and thrombin clotting time assays was similar to sTM alone. In a rat model of disseminated intravascular coagulation (DIC), intravenous injection of a human TF-containing thromboplastin reagent (0.5 ml/kg) resulted in an immediate death in ~60% of the animals and a clinical score of ~2.5. Pre-injection of Ab(TF)-TM or Ab(TF) and sTM, given alone or in combination, showed dose-dependent efficacy. At a dose of 0.7 nmol/kg, Ab(TF)-TM completely prevented death and reduced clinical scores by 79%,while neitherAb(TF) nor sTM,given alone or in combination, showed significant therapeutic effects. Calculated effective doses that reduced mortality by 50% relative to that in the control group (ED50 , nmol/kg) were 0.21 for Ab(TF)- TM, 3.2 for an equimolar mixture of Ab(TF) and sTM, 4.3 for sTM and 20 for Ab(TF). Thus, Ab(TF)-TM presented 10– to 100-fold enhancement of the anticoagulant potency, relative to the ED50 in Ab(TF) and sTM given either alone or in combination, in a rat DIC model.