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.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.

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