The activation of bovine protein C by factor Xa.
A complex composed of factor Xa and phospholipid vesicles assembled in the presence of calcium ions catalyzes a discrete cleavage of the heavy chain of bovine protein C that is indistinguishable from that produced by thrombin as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This cleavage generates an active site capable of hydrolyzing small substrates and inactivating factor Va function in the prothrombinase complex. Activation of protein C by factor Xa requires both calcium ions and phospholipid vesicles and proceeds at a rate an order of magnitude greater than that observed for alpha-thrombin in solution. gamma-Carboxyglutamic acid-domainless protein C is not activated by factor Xa, consistent with the requirement for phospholipid and distinguishing this reaction from protein C activation by thrombin. Thrombomodulin serves as a cofactor for the factor Xa-catalyzed reaction, forming a 1:1 complex with factor Xa (apparent Kd = 5.7 X 10(-10) M) and stimulating the saturated rate of protein C activation by factor Xa (kcat = 149 min-1) to levels comparable with the thrombin-thrombomodulin complex. Protein C activation by factor Xa is not inhibited by the specific thrombin inhibitor dansyl-N-(3-ethyl-1,5-pentanediyl)amide but is inhibited by antithrombin III, tripeptide-chloromethyl ketones, and the monoclonal antibody alpha-BFX-2b that is highly specific for factor Xa. These data indicate that thrombomodulin is promiscuous in its role as a cofactor and suggest the existence of an alternative pathway for protein C activation in vivo.
Haley
P E
PE
Department of Biochemistry, University of Vermont College of Medicine, 05405-0068.
Doyle
M F
MF
Mann
K G
KG
D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D005165:Factor V / Q000378:metabolism; D005170:Factor X / Q000378:metabolism; D015951:Factor Xa; D006801:Humans; D007700:Kinetics; D011486:Protein C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism; D018179:Receptors, Thrombin; D012697:Serine Endopeptidases / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism
D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C022475:prothrombinase complex; D005165:Factor V; D005170:Factor X; D012697:Serine Endopeptidases; D013917:Thrombin; D015951:Factor Xa