Design and evaluation of a thrombin-activable plasminogen activator.

Abstract

A new chimeric plasminogen activator with high fibrin affinity was designed to bind fibrin and to initiate clot destruction, following activation by thrombin. The chimeric activator, 59D8-scuPA-T, was made from the Fab fragment of an anti-fibrin antibody (59D8) and a C-terminal portion of a thrombin-activable low molecular weight single-chain urokinase plasminogen activator, scuPA-T, obtained by deletion of Phe-157 and Lys-158 from low molecular weight single-chain urokinase-type plasminogen activator (scuPA) by site-directed mutagenesis. The chimeric molecule had a molecular mass of 91,000, a value consistent with one 59D8 light chain (M(r) = 27,000) and one 59D8 heavy-chain Fd fragment fused to low molecular weight scuPA (M(r) = 64,000). According to its design, 59D8-scuPA-T was activated by thrombin but not by plasmin, whereas the control chimeric molecule, 59D8-scuPA, was activated by plasmin but not by thrombin. When activated by thrombin, 59D8-scuPA-T converted plasminogen to plasmin. In vitro plasma clot lysis assays showed that 59D8-scuPA-T lysed clots performed by thrombin and that heparin and hirudin could prevent clot lysis. When incorporated as part of a thrombin-induced clot, only 59D8-scuPA-T was able to lyse the clot while 59D8-scuPA and high molecular weight scuPA were ineffective. Together these results demonstrate that 59D8-scuPA-T is a thrombin-activable plasminogen activator that offers selective thrombolysis of thrombin-rich clots over more established, aged clots, and may also act as an antithrombotic agent.