DESIGN AND EVALUATION OF A THROMBIN-ACTIVABLE PLASMINOGEN-ACTIVATOR

A new chimeric plasminogen activator with high fibrin affinity was des igned to bind fibrin and to initiate clot destruction, following activ ation by thrombin. The chimeric activator, 59D8-scuPA-T, was made from the Fab fragment of an anti-fibrin antibody (59D8) and a C-terminal p ortion of a thromb-inactivable low molecular weight single-chain uroki nase plasminogen activator, scuPA-T, obtained by deletion of Phe-157 a nd Lys-158 from low molecular weight single-chain urokinase-type plasm inogen activator (scuPA) by site-directed mutagenesis. The chimeric mo lecule had a molecular mass of 91000, a value consistent with one 59D8 light chain (M(r) 27000) and one 59D8 heavy-chain Fd fragment fused t o low molecular weight scuPA (M(r) = 64000). 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 n ot by thrombin. When activated by thrombin, 59D8-scuPA-T converted pla sminogen to plasmin. In vitro plasma clot lysis assays showed that 59D 8-scuPA-T lysed clots preformed by thrombin and that heparin and hirud in could prevent clot lysis. When incorporated as part of a thrombin-i nduced clot, only 59D8-scuPA-T was able to lyse the clot while 59D8-sc uPA and high molecular weight scuPA were ineffective. Together these r esults demonstrate that 59D8-scuPA-T is a thrombin-activable plasminog en activator that offers selective thrombolysis of thrombin-rich clots over more established, aged clots, and may also act as an antithrombo tic agent.