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 4

Thrombolytic effects of PLT/uPA-T in human blood.

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Thrombolytic effects of PLT/uPA-T in human blood. (A) Schematic beginnin...
(A) Schematic beginning with isolated human platelets incubated with prodrugs or control PBS and then gel filtered. These platelets are then used to reconstitute whole blood. Clotting in whole blood is activated by adding either FVIIa or PF4 and the HIT-like mAb KKO to induce a prothrombotic state. The blood is then flowed through a vWF-coated channel, and adherence of platelets and fibrinogen to the channel’s wall is monitored. (B) The prodrugs uPA-T (10 μg/ml) and PLT/uPA-T (10 μg/ml) were incubated with human platelets and then gel filtered. Representative flow cytometric studies of platelet surface–bound prodrug after gel filtration are shown. (C) Relative fibrin accumulation and (D) platelet deposition in the microfluidic channel of reconstituted “whole” blood activated by FVIIa with platelet-bound prodrug is shown compared with preincubation with PBS (dashed line). Data represent the mean ± 1 SEM. n = 5 independent experiments, each performed in duplicate. *P ≤ 0.001 compared with PBS. Data analysis was done by Student’s t test for drug-treated samples compared with PBS controls.