A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation
Rudy E. Fuentes, … , Vladimir R. Muzykantov, Mortimer Poncz
Rudy E. Fuentes, … , Vladimir R. Muzykantov, Mortimer Poncz
Published December 21, 2015
Citation Information: J Clin Invest. 2016;126(2):483-494. https://doi.org/10.1172/JCI81470.
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A chimeric platelet-targeted urokinase prodrug selectively blocks new thrombus formation

Abstract

The use of fibrinolytic agents to prevent new thrombus formation is limited by an increased risk of bleeding due to lysis of hemostatic clots that prevent hemorrhage in damaged blood vessels. We sought to develop an agent that provides thromboprophylaxis without carrying a significant risk of causing systemic fibrinolysis or disrupting hemostatic clots. We previously showed that platelet (PLT) α granule–delivered urokinase plasminogen activator (uPA) is highly effective in preventing thrombosis, while being associated with little systemic fibrinolysis or bleeding. Here, we generated a chimeric prodrug composed of a single-chain version of the variable region of an anti-αIIbβ3 mAb fused to a thrombin-activatable, low-molecular-weight pro-uPA (PLT/uPA-T). PLT/uPA-T recognizes human αIIbβ3 on both quiescent and activated platelets and is enzymatically activated specifically by thrombin. We found that this prodrug binds tightly to human platelets even after gel filtration, has a prolonged half-life in mice transgenic for human αIIb compared with that of uPA-T, and prevents clot formation in a microfluidic system. Importantly, in two murine injury models, PLT/uPA-T did not lyse preexisting clots, even when administration was delayed by as little as 10 minutes, while it concurrently prevented the development of nascent thrombi. Thus, PLT/uPA-T represents the prototype of a platelet-targeted thromboprophylactic agent that selectively targets nascent over preexisting thrombi.

Authors

Rudy E. Fuentes, Sergei Zaitsev, Hyun Sook Ahn, Vincent Hayes, M. Anna Kowalska, Michele P. Lambert, Yuhuan Wang, Donald L. Siegel, Daniel W. Bougie, Richard H. Aster, Daniel D. Myers, Victoria Stepanova, Douglas B. Cines, Vladimir R. Muzykantov, Mortimer Poncz

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Figure 7

Prodrug efficacy in nascent versus preexisting thrombi in the cremaster laser injury model.

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Prodrug efficacy in nascent versus preexisting thrombi in the cremaster ...
Xenotransfused NSG mice were used for these studies, as for Figure 6, C and D. (A) Schematic of studies. The degree of prevention of growth of nascent thrombi under the bolus infusion of a prodrug is an indication of the thromboprophylactic effectiveness of the tested prodrug. Laser injuries performed prior to drug bolus infusions generated the preexisting thrombi. The degree by which they decreased in size after a bolus infusion of a prodrug was an indication of the effect of the tested prodrug on lysing preexisting thrombi. (B) Platelet and fibrin accumulation in nascent injuries during bolus infusion of PBS, 5 mg/5 mg uPA or uPA-T, or 0.125 mg/0.125 mg PLT/uPA-T. n = 5 animals per arm, with up to 5 pre- and post-drug injuries per animal. (C) Relative size of the thrombus as indicated by measuring accumulated platelets and fibrin in preexisting injuries that had then been exposed to a bolus infusion of the same drugs as used in B. Size is shown relative to the preexisting size, i.e., prior to drug exposure. ANOVA multiparametric analysis was performed, and P values are relative to the PBS control. P values shown in black are relative to the PBS control, and values in red are relative to uPA.