Limited proteolysis of human alpha-thrombin by urokinase yields a non-clotting enzyme.
Limited proteolysis of human alpha-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 alpha-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 microM and kcat = 145 s-1 versus respectively 4.5 microM 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 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 beta u-thrombin, is therefore identical to the transient proteolytic derivative, beta 1-thrombin, produced by trypsin. Prolonged incubation with tcu-PA resulted in further conversion in a derivative analogous to gamma t-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
Bezeaud
A
A
Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
de Raucourt
E
E
Miyata
T
T
Bouton
M C
MC
Angles-Cano
E
E
Guillin
M C
MC
D000595:Amino Acid Sequence; D001120:Arginine; D001216:Asparagine; D001777:Blood Coagulation / Q000187:drug effects*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D013917:Thrombin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D001216:Asparagine; D001120:Arginine; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator