Summary
Limited proteolysis of human α-thrombin by various proteases has been efficiently used to demonstrate the importance of two insertion loops 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 α-thrombin giving rise to a transient derivative, consisting of two non-covalently linked subunits. Although the thrombin derivative conserves its activity towards the synthetic substrate S-2238 (Km = 8.4 μM and kcat = 145 s-1 versus respectively 4.5 μM and 149 s-1 for α-thrombin), most of its coagulant activity is lost (140 NIH u/mg versus 3000 NIH u/mg) and its ability to activate platelets is considerably reduced (threshold for full platelet aggregation 2.5 nM versus 0.25 nM). The thrombin fragments were separated by HPLC and after reduction and S-carboxyamidemethylation were digested with a lysylendopeptidase; the resulting peptides were separated by HPLC and sequenced. One fragment corresponded to B chain fragment 1-73 and the second to B chain fragment 74-259 covalently linked to the A chain, indicating that tcu-PA cleaves selectively the peptide bond Arg 73-Asn 74 in the B chain. The proteolytic derivative obtained, designated βu-thrombin, is therefore identical to the transient proteolytic derivative, αt-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to γt-thrombin.
These results show that the interactions of α-thrombin and u-PA are not limited to the inactivation of scu-PA by α-thrombin previously reported by other groups, but that reciprocally tcu-PA, the fully active form of scu-PA, may induce the loss of α-thrombin clotting and platelet stimulating activities. Furthermore, these data underscore the functional importance of the integrity of the peptide bond Arg 73-Asn 74 of the B chain on the recognition of macromolecular substrates by α-thrombin.
