LIMITED PROTEOLYSIS OF HUMAN ALPHA-THROMBIN BY UROKINASE YIELDS A NON-CLOTTING ENZYME

Limited proteolysis of human alpha-thrombin by various proteases has b een efficiently used to demonstrate the importance of two insertion lo ops located on the surface of this molecule. In the present study, we demonstrate that two-chain urokinase (tcu-PA) specifically cleaves the B chain of alpha-thrombin giving rise to a transient derivative, cons isting of two non-covalently linked subunits. Although the thrombin de rivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 mu M and kcat = 145 s(-1) versus respectively 4.5 mu M and 149 s(-1) for alpha-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platel ets is considerably reduced (threshold for full platelet aggregation 2 .5 nM versus 0.25 nm). The thrombin fragments were separated by HPLC a nd after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded bo B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, in dicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 7 4 in the B chain. The proteolytic derivative obtained, designated beta (u)-thrombin, is therefore identical to the transient proteolytic deri vative, beta(t)-thrombin, produced by trypsin. Prolonged incubation wi th tcu-PA resulted in further conversion in a derivative analogous to gamma(t)-thrombin. These results show that the interactions of alpha-t hrombin and u-PA are not limited to the inactivation of scu-PA by alph a-thrombin previously reported by other groups, but that reciprocally tcu-PA, the fully active form of scu-PA, may induce the loss of alpha- thrombin clotting and platelet stimulating activities. Furthermore, th ese data underscore the functional importance of the integrity of the peptide bond Arg 73-Asu 74 of the B chain on the recognition of macrom olecular substrates by alpha-thrombin.