P-selectin-targeting of the fibrin selective thrombolytic Desmodus rotundus salivary plasminogen activator at α1

During thrombosis, P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that targeting a plasminogen activator (PA) to P-selectin would enhance local thrombolysis and reduce bleeding risk. Previously, a urokinase (uPA)/anti-P-selectin antibody (HuSZ51) fusion protein was shown to increase fibrinolysis in a hamster pulmonary embolism model. To explore the therapeutic potential of this targeting strategy, we fused the fibrin-selective Desmodus rotundus salivary PA alpha1 (dsPAalpha1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPAalpha1 (HuSZ51-dsPAalpha1) to unmodified dsPAalpha1 in vitro and in vivo. HuSZ51-dsPAalpha1 and dsPAalpha1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51-dsPAalpha1 bound to thrombin-activated human and dog platelets with comparable affinities to that of parental antibody SZ51. The fusion protein retained the catalytic activities of dsPAalpha1 in chromogenic and clot lysis assays, indicating that dsPAalpha1 is fully functional when fused to HuSZ51. Compared to dsPAalpha1, HuSZ51-dsPAalpha1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51-dsPAalpha1 was less effective in lysis of preexisting arterial thrombi in the dog model. The reduced arterial thrombolysis was not due to the pharmacokinetic properties of HuSZ51-dsPAalpha1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPAalpha1-treated groups than corresponding dsPAalpha1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPAalpha1 varied dependent on the physical composition of thrombi. The lack of stimulation by fibrin in arterial thrombi may contribute to the attenuated thrombolytic efficacy of HuSZ51-dsPAalpha1 in the dog model.