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 6

Prodrug efficacy versus nascent thrombi induced by FeCl3-sclerosing versus preexisting thrombi induced by tail-clip injury.

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Prodrug efficacy versus nascent thrombi induced by FeCl3-sclerosing vers...
(A–D) Schematics of the study and results. Results and schematics shown in A and B involve hαIIb+ mice. Prodrugs were given as a 2-minute bolus, followed by a 30-minute infusion dose at the indicated mg/kg dose, except for panel B, which refers to a bolus only. Data represent the mean ± 1 SEM and are shown by n values. ANOVA multiparametric analysis was performed, and P values are relative to the PBS control. (A) AUC of blood flow in the FeCl3 injury model while receiving the indicated prodrug. AUC for blood flow is shown because, while all of the mice exposed to PBS or bolus uPA-T had total occlusions, none of the other mice developed total occlusions, but some developed partial occlusions that were notable by measuring the AUC. In B, tail-clip blood loss that occurred after the prodrug’s infusion was compared with blood loss prior to prodrug infusion. The time to stop bleeding, determined visually during the pre-drug window, was 2.1 ± 0.4 minutes, similar to that seen by others in similar models (32). Dashed lines in A–D indicate equal blood loss before and after drug exposure. Data shown in C and D represent studies analogous to those in A and B, respectively, but were conducted in NSG mice either infused (+) or not infused (–) with human platelets. The uPA and uPA-T dose for both the bolus and infusion was 5 mg/kg and 0.5 mg/kg for PLT/uPA-T. Data shown represent the mean ± 1 SEM. ANOVA multiparametric analysis was performed, and P values are relative to the PBS control.