A Tissue Plasminogen Activator/P-Selectin Fusion Protein Is an Effective Thrombolytic Agent

Abstract

Background P-selectin is expressed on the surface of activated endothelial cells and platelets. We hypothesized that a tissue plasminogen activator (TPA)/P-selectin fusion protein would have not only thrombolytic 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 to leukocytes and thus further promote thrombolysis. Methods and Results 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 vectors. After infection of Sf-21 cells with the recombinant baculovirus, recombinant TPA IR and P-selectin/TPA IR fusion proteins were purified with the use of metal ion chromatography. The intact protease activity of TPA IR and the ligand binding capability of P-selectin were confirmed through indirect chromogenic and cell binding assays, respectively. These molecules were assessed both in vitro and in vivo for thrombolytic activity. In vitro clot lysis assays indicated equal efficacy of TPA IR , P280 IR , and P121 IR ( P >.5). The in vivo efficacy was tested in a cyclic flow variation model with the use of the rat mesenteric artery. Compared with saline control treatment, reduction in cyclic flow variations was significant ( P <.05) and similar ( P >.5) among TPA IR , P280 IR , and P121 IR . No significant bleeding was noted among 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 in vitro. Thus, these proteins may provide an efficient method of targeting TPA to the thrombotic region. Further experimental analysis with the use of larger animal coronary occlusion models should help determine the future value of these proteins as clinical therapeutic agents.