A Human-Specific, Platelet-Targeted, Thrombin-Activatable Urokinase (PLT/uPA-T) Demonstrates Selective Thromboprophylactic Properties in Two in Vivo Models

Two major clinical limitations to the use of thrombolytic agents are their short half-life and lack of targeting specificity. We aimed to circumvent these limitations by targeting a platelet-bound, thrombin-activatable, low molecular weight urokinase, termed PLT/uPA-T, to nascent thrombi by a combination of two strategies: (1) Attaching the drug through its N-terminal scFv portion to human αIIb/β3 on the surface of platelets with nM affinity. (2) Protecting the uPA from rapid activation/inactivation in the circulation while simultaneously requiring its activation by thrombin through a two amino acid deletion at the plasmin cleavage site that concurrently creates a thrombin cleavage/activation site. These two properties constrain the activity of PLT/uPA-T on mature clots, which express low levels of thrombin and transiently recruit only a few new platelets to the shell rather than the core of the thrombus. These two properties of PLT/uPA-T also enhance the drug’s lifespan by attaching it to the platelet cell surface and preventing its inactivation before reaching its intended target. PLT/uPA-T binds specifically to human platelets and to hαIIb mouse platelets that transgenically expressed human (h) αIIb on its surface. In hαIIb mice, the half-life of retro-orbitally infused PLT/uPA-T was ~2 hours, ~100-fold longer than similarly infused uPA-T and did not cause any spontaneous bleeding or lower platelet counts 4 to 24 hours later. We now report two in vivo models to test the efficacy of the PLT/uPA-T as a thromboprophylactic agent versus uPA-T, taking into account the much shorter circulating half-life of the latter. In the hαIIb mice model, a tail-vein clipping model was done to represent the “mature clot” as follows: following clipping, blood was collected into 37°C water for 10 minutes. The tail was then removed from the water and a bolus of PLT/uPA-T (0.5 µg/g mouse) injected retro-orbitally followed by a continuous infusion of the same dose over the next 30 minutes. uPA-T was similarly infused but both bolus and infusion were given at 10-fold higher doses. A no-drug treatment control was also included. After the drug infusion was started, the tail was placed into fresh 37°C water and bleeding was documented from these “mature clots” over the ensuing 30 minutes. To study “nascent thrombi”, mice were bolused/infused with same drug regimens, and a FeCl3 carotid artery injury study was performed contemporaneously. PLT/uPA-T was as effective at 1/10th the dose as uPA-T at preventing these “nascent thrombi” (FeCl3 injuries), but did not cause bleeding from “mature clots” (tail clippings) relative to the no-treatment control, while the uPA-T treatment lead to ~5-fold greater rebleeding compared to the no-treatment control (p<0.001) with N > 10 animals per arm. The second in vivo model targeted human platelets infused into immunocompromized NOD-SCID γ-interferon-deficient (NSG) male mice to generate a calculated 10% of all the circulating platelets being human at the initiation of the studies. The pre-drug “mature clots” and the post-drug “nascent thrombi” were both arteriolar laser cremaster injuries. We enumerated mouse platelets incorporated into these thrombi over time. There was an ~50% decrease in the size of laser-induced post-drug “nascent thrombi” after PLT/uPA-T or a 10-fold higher dose of uPA-T relative to no drug treatment. However, PLT/uPA-T did not affect the size of the laser-induced pre-drug “mature clots” relative to no drug-treatment, while there was a decrease in size of the mature clots after treatment with uPA-T. These studies describe two preclinical models of comparative thromboprophylactic efficacy and safety that are independent of drug half-life. Our studies demonstrate that a combination of platelet-targeting and need for thrombin-activation makes PLT/uPA-T a very potent and targeted thrombolytic agent to prevent new thrombus formation, while leaving older, hemostatic clots intact.