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 1

Schematics of the construction and biology of PLT/uPA-T.

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Schematics of the construction and biology of PLT/uPA-T. (A) Schematic c...
(A) Schematic construct of the scFv N-terminal and LMW uPA-T C-terminal portions of PLT/uPA-T. Right: C-terminal zymogen of human single-chain uPA (scuPA) lacking the full molecule domains and modified by deleting Phe157 and Lys158 (in red), which convert a plasmin activation site into the thrombin-activatable uPA-T. tcuPA, two-chain urokinase; P, proline; R, argine; F, phenylalanine; K, lysine; I, isoleucine; s-s, cysteine disulfide bonds. (B) Intended biology of a chimeric PLT/uPA-T that would bind to the surface of resting or activated platelets without activating these cells and be attracted to a nascent thrombus, whereby available thrombin would activate the urokinase moiety uPA-T, leading to plasminogen activation and subsequent fibrinolysis. Preexisting clots, which do not bind new platelets except transiently within the shell and have little or no active thrombin, or at least no accessible thrombin, are protected from lysis by PLT/uPA-T. (C) Schematic of the final expression vector for PLT/uPA-T in insect cells with removal of the rbc/scFv. Bip SS, Ig-binding protein; V5, simian virus 5 epitope; 6XHis, polyhistidine peptide; PMT, metallothionein promoter; Amp, ampicillin-resistant gene; PUC ori, plasmid origin of replication. (D) DNA sequence of the PLT/uPA-T insert in C, with key components boxed in red.