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

Summary

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 α1 (dsPAα1) to HuSZ51 and compared the fibrinolytic activity of P-selectin-targeted dsPAα1 (HuSZ51-dsPAα1) to unmodified dsPAα1 in vitro and in vivo. HuSZ51-dsPAα1 and dsPAα1 were expressed in CHO cells and purified to homogeneity by affinity chromatography. HuSZ51dsPAα1 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 dsPAα1 in chromogenic and clot lysis assays, indicating that dsPAα1 is fully functional when fused to HuSZ51. Compared to dsPAα1, HuSZ51-dsPAα1 had similar thrombolytic efficacy in a rat pulmonary embolism model and anti-thrombotic potency in a dog model of femoral artery thrombosis. However, HuSZ51dsPAα1 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-dsPAα1 because antigen level and amidolytic activity were higher in plasma from HuSZ51-dsPAα1-treated groups than corresponding dsPAα1-treated groups. These data indicate that the thrombolytic efficacy of HuSZ51-dsPAα1 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-dsPAα1 in the dog model.

Keywords

P-selectin - dsPAα1 - fusion protein - clot targeting - thrombolysis

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