Platelet-Targeted Pro-Urokinase as a Novel Thromboprophylaxis Fibrinolytic Strategy

Abstract 3339

Use of plasminogen activators (PAs) is restricted to life-threatening thrombotic conditions because high concentrations are required to diffuse into clots, overcome PA inhibitors and compensate for rapid clearance, thereby predisposing to bleeding. We hypothesized that targeting pro-PAs to platelets would circumvent these obstacles and preferentially lyse nascent, pathological clots that are actively recruiting platelets, while sparing preformed hemostatic clots. To test this concept, we expressed a chimeric protein (anti-PLT scFv/uPA-T) composed of an N-terminal scFv generated from a monoclonal antibody MoAb 312.8 directed to human αIIb integrin chain linked via a serine-rich linker peptide to human low molecular weight thrombin activatable pro-urokinase (uPA-T) wherein the plasmin cleavage site was replaced with a thrombin cleavage site, which we reasoned would be activated preferentially at sites of active clot propagation. Anti-PLT scFv/uPA-T expressed in Drosophila S2 insect cells bound specifically to human platelets and to transgenic mouse platelets that contain only human αIIb/mouse β3 on their cell surface (HαIIb+ Tg), but not to wild type mouse platelets. The anti-PLT scFv/uPA-T bound specifically to immobilized human αIIbβ3 with a Kd of ∼80 nM. The anti-PLT scFv/uPA-T did not interfere with either ADP-induced platelet aggregation or activation as demonstrated by expression of P-selectin by flow cytometry, and retained its zymogenic properties until activated specifically by thrombin. The fibrinolytic activity of anti-PLT scFv/uPA-T and uPA-T were compared using the FeCl₃ carotid artery injury model in HαIIb+ Tg mice. The mice were protected from forming occlusive thrombi for at least 10 hrs post injection of anti-PLT scFv/uPA-T whereas even five-fold higher concentrations of uPA-T were effective for only 2–5 min. These studies support a novel approach for prophylactic targeting drug delivery by combining a pro-drug that requires activation by thrombin with platelet delivery to sites of incipient thrombotic vascular occlusion.