Abstract
Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl3 models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.
Footnotes
- Received July 20, 2009.
- Accepted November 30, 2009.
This work was supported by the National Institutes of Health [Grant R01 HL090687] (to V.R.M.); the American Heart Association [Grant SDG 0535258N] (to S.Z.); and Fondo de Investigaciones Sanitarias [Grant PI081795] (to J.-C.M.).
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.159194.
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ABBREVIATIONS:
- tPA
- tissue type plasminogen activator
- PA
- plasminogen activator
- PAI-1
- plasminogen activator inhibitor type 1
- scFv
- single chain antibody variable fragment
- GPA
- glycophorin A
- RBC
- red blood cell
- CNS
- central nervous system
- CR1
- human complement receptor type 1
- ETI
- Erythrina trypsin inhibitor
- SFM
- serum-free cell culture medium
- PAGE
- polyacrylamide gel electrophoresis
- PBS
- phosphate-buffered saline
- BSA
- bovine serum albumin
- PCR
- polymerase chain reaction.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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