Conjugation of Plasminogen Activators to Carrier Erythrocytes Imparts New Biological Properties to Their Soluble Counterparts.

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.