A TISSUE-PLASMINOGEN ACTIVATOR P-SELECTIN FUSION PROTEIN IS AN EFFECTIVE THROMBOLYTIC AGENT/

Background P-selectin is expressed on the surface of activated endothe lial cells and platelets. We hypothesized that a tissue plasminogen ac tivator (TPA)/P-selectin fusion protein would have not only thrombolyt ic activity but also might target TPA to the thrombi. In addition, it seemed possible that this chimeric protein would competitively inhibit the binding of native P-selectin on endothelial cells and platelets t o leukocytes and thus further promote thrombolysis. Methods and Result s The full-length, plasminogen activator inhibitor-1-resistant form of TPA (TPA(IR)) together with two TPA(IR)/P-selectin fusion constructs (P280(IR) and P121(IR)) were expressed with the use of baculovirus vec tors. After infection of Sf-21 cells with the recombinant baculovirus, recombinant TPA(IR) and P-selectin/TPA(IR) fusion proteins were purif ied with the use of metal ion chromatography. The intact protease acti vity of TPA(IR), and the ligand binding capability of P-selectin were confirmed through indirect chromogenic and cell binding assays, respec tively. These molecules were assessed both in vitro and in vivo for th rombolytic activity. In vitro clot lysis assays indicated equal effica cy of TPA(IR), P280(IR), and P121(IR) (P>.5). The in vive efficacy was tested in a cyclic flow variation model with the use of the rat mesen teric artery. Compared with saline control treatment, reduction in cyc lic flow variations was significant (P<.05) and similar (P>.5) among T PA(IR), P280(IR), and P121(IR). No significant bleeding was noted amon g treated animals. Conclusions Chimeric proteins P280(IR), and P121(IR ) have clot lysis activities that are similar to TPA(IR) both in vitro and in vivo. These chimeric proteins also bind to P-selectin ligand i n vitro. Thus, these proteins may provide an efficient method of targe ting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determi ne the future value of these proteins as clinical therapeutic agents.