Thrombosis is a common complication of surgery that increases perioperative morbidity and mortality. For thromboprophylaxis to be practical in the perioperative setting, an approach must sustain physiologic fibrin formation to avoid perioperative bleeding and permit wound repair, while at the same time prevent pathological thrombi from forming. The investigators postulate that coupling plasminogen activators (PA's) to carrier red blood cells (RBC) will deny the enzymes ready access to mature clots or to extravascular fibrin, but will prolong their life-span within the circulation and permit them to be incorporated within newly formed clots. The preliminary results support this hypothesis and show that PAs can be coupled to RBC without loss of biocompatibility, circulate for a prolonged time and display selective fibrinolytic activity against newly formed clots in vitro and likely in vivo. The investigators have also observed that: (1) single chain urokinase (scuPA) bound to its soluble receptor (suPAR) is enzymatically active, fibrin specific, resistant to plasminogen activator inhibitor-1 (PAI-1), but its activity is regulated by the fourth kringle of plasminogen (K4); (2) a scuPA variant lacking the kringle (DK-scuPA) displays enhanced enzymatic activity, is relatively resistant to PAI-1. Retains high affinity binding to suPAR, and acquires anti-vasoconstrictor activity; (3) suPAR can be coupled to circulating RBC via complement receptor-1 (CR-1) allowing scuPA to bind to circulating RBC without ex vivo manipulation. The investigators hypothesize that coupling scuPA or DK-scuPA to RBC via suPAR will generate a latent pro-drug which has little, if any, access to mature clots, but which enjoys a prolonged life-span and ready access to nascent clots which induce its activity. Based on these findings, the investigators propose to study the behavior of RBC-coupled scuPA and DK-scuPA and efficacy and safety in animal models that recapitulate common post surgical thrombotic situations. The following specific aims will be pursued: (1) Biologic activity of uPA coupled to RBC (uPA/RBC). The stability, enzymatic and vasoregulatory activity, resistance to PAI-1, adhesivity and regulation by plasminogen of uPA/RBC will be investigated in vitro. (2) Bioavailability of UPA/RBC in vivo. The investigators will characterize the stability, blood clearance, biodistribution, and activities of uPA/RBC injected into intact animals. (3) Efficacy of uPA/RBC in animal mdels of thromboembolism. Animal models of pulmonary and cerebral embolism that have been developed in the investigators' laboratory will be employed to study uPA/RBC-mediated thromboprophylaxis in intact rodents and in genetically altered mice. The results of this study may provide new means to achieve thromboprophylaxis in surgical settings such as angioplasty and endarterectomy, as well as be a platform for the development of novel forms of antithrombotic therapy.
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