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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Research Article Hematology

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 5

Systemic effects of the uPA-T prodrugs.

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Systemic effects of the uPA-T prodrugs. (A) Representative flow cytometr...
(A) Representative flow cytometric study of a single-dose infusion of PLT/uPA-T (0.25 mg/kg) and uPA-T (5 mg/kg) into hαIIb+ mice, followed by measurements of platelet-bound PLT/uPA-T (left) and uPA-T (right). (B) Data represent the mean ± 1 SEM for 5 studies of infusion of PLT/uPA-T into hαIIb+ mice and were analyzed as in A for PLT/uPA-T. Gray bar indicates a half-life of ~2 hours. (C) Effects of infusions of PBS (white circles), uPA-T (5 mg/kg, gray squares), or PLT/uPA-T (0.25 mg/kg, black triangles) on circulating platelet counts at 0, 1, 4, and 24 hours after iv. infusion of the drug. The drop in platelet count observed with each intervention to ~90% of baseline was due, we believe, to repeated blood draws. Gray dashed line represents the baseline platelet count with no intervention. (D) D-dimer and fibrinogen levels after a single-bolus, 30-minute infusion of uPA, uPA-T, or PLT/uPA-T were determined. The amount infused is shown on the x axis in mg/kg. Values were compared with the levels measured prior to prodrug infusion in each animal. Studies were performed at the end of the infusion. Data represent the mean ± 1 SEM. Dashed lines in D represents settings in which pre– and post–drug infusion levels were identical. n = 5 for all studies. None reached statistical significance when compared with the pre-study levels. Data analysis was done by Student t test for drug-treated mice compared with PBS controls.