Kinetic analyses of the enhancement of the activities of t-pa induced by the presence of monoclonal antibody (C9-5)

Abstract

Nine monoclonal antibodies (Mab) against two chain tissue plasminogen activator (t-PA(W) ) were obtained. The effects of these Mabs on the enzymatic activities of one chain t-PA (t-PA(TD)) and two chain t-PA (t-PA(W)) were examined by incubating t-PA and Mabs with S-2288, or with plasminogen (plg) and S-2251 in the presence or absence of fibrin. One of Mabs called C9-5 significantly enhanced the activities of t-PA, which were kinetically analyzed. The kinetic analyses of the hydrolysis of S-2288 by t-PA showed increase in kcat from 5.17/sec to 7.75/sec in the presence of 75 pg/ml of C9-5 without change in Km. When Glu-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 2 μM to 0.17 μM in the presence of C9-5 without change in Vmax. The addition of fibrin resulted in further decrease in Km from 0.133 μM to 0.077 μM. When Lys-plg was activated by t-PA(TD) in the absence of fibrin, Km decreased from 0.408 μM to 0.185gmM in the presence of C9-5, and kcat increased from 0.131 sec −1 to 0.465 sec −1. The presence of fibrin further increased kcat of the activation of Lys-plg. Similar results were obtained when plasminogen was activated by t-PA(W). Mab, C9-5, was shown to bind to B-chain of t-PA by immunoblotting. These results suggest that a dimolecular complex of t-PA and C9-5 has a higher affinity to plasminogen in the presence or absence of fibrin.