Abstract

Conjugation of plasminogen activators (PA) to carrier red blood cells (RBC) generates a new agent (RBC/PA), which selectively lyses nascent blood clots. In this work, we studied two types of RBC/PA conjugates, carrying Alteplase (tissue type plasminogen activator, tPA) or Reteplase (an engineered form of tPA, Ret). Compared to non-conjugated 125I-PA counterparts, both 125I-tPA and 125I-Ret coupled to 51Cr-RBC via biocompatible biotin-streptavidin cross-linker showed markedly prolonged circulation in rats and mice. Despite slightly faster blood clearance, RBC/tPA retained significantly higher fibrinolytic activity in circulation than RBC/Ret. In part, the higher fibrinolytic activity of RBC/tPA vs RBC/Ret in the circulation was due to its lessened susceptibility to plasma inhibitors. Analysis of amidolytic activity of RBC-coupled vs free tPA and Ret using chromogenic substrates in vitro revealed that coupling to RBC rendered Ret, but not tPA, insensitive to stimulation of fibrinolytic activity by fibrin. In vitro binding assay in cultural wells showed that RBC/tPA specifically binds to fibrin clot (6±0.2x104 RBC/tPA bound per well vs. 1±0.4x104 RBC/Ret or 7.3±0.6x103 naive RBC). RBC/tPA also bind specifically to immobilized fibrinogen and plasminogen (8±2x104 and 2±0.4x104 RBC/well), but not to non-cleavable plasminogen, collagen, fibronectin or thrombospondin (<1.1±0.3x103RBC/well). Neither RBC/Ret nor naïve RBC bind to these proteins (<1.1±0.2x103 RBC/well). Therefore, RBC/PA complexes display a considerable functional diversity, a result of interplay between the pharmacokinetic features offered by carrier RBC, individual features of a given PA and alterations of the latter caused by RBC coupling. In theory, this diversity may enhance flexibility and utility of potential applications of RBC/PA for thromboprophylaxis.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.