Bakker H M, Tans G, Yukelson L Y, Janssen-Claessen T W, Bertina R M, Hemker H C, Rosing J
Cardiovascular Research Institute Maastricht, University of Limburg, The Netherlands.
Blood Coagul Fibrinolysis. 1993 Aug;4(4):605-14. doi: 10.1097/00001721-199308000-00012.
The protein C activator from Agkistrodon halys halys venom was purified 533-fold by ion-exchange chromatography on QAE-Sephadex A-50, affinity chromatography on aprotinin-Sepharose and Mono-Q fast protein liquid chromatography. The purified enzyme is a single chain protein with an apparent molecular weight of 36,000 that activates protein C by proteolytic removal of a small fragment from the heavy chain. The protein C activator exhibited a high amidolytic activity towards the tripeptide substrates D-Pro-Phe-Arg-pNA (S2302) and D-Phe-(pipecolyl)-Arg-pNA (S2238). The activity of the activator was not affected by thiolprotease or metalloprotease inhibitors. The activator was inhibited, however, by benzamidine, Phe-Pro-Arg chloromethyl ketone, p-nitrophenyl p-guanidinobenzoate and soy bean trypsin inhibitor, which classifies the enzyme as a serine protease. The purified protease was capable of activating both human and bovine protein C. Activation of human protein C only occurred at an appreciable rate in a calcium-free reaction medium at low ionic strength. Ca2+ ions inhibited the activation of human protein C with an apparent Ki of 0.8 mM. Addition of NaCl to the reaction medium also strongly inhibited human protein C activation (50% inhibition at 20 mM NaCl). Kinetic analysis of human protein C activation by the venom activator (in a calcium-free medium) revealed an apparent Km for protein C of 0.52 microM and a kcat of 0.17 s-1 at I = 0.05 (kcat/Km = 3.3 x 10(5) M-1 s-1). At I = 0.15 rates of human protein C activation became linear with protein C indicating a strong increase in Km with increasing ionic strength. Activation of bovine protein C was hardly affected by variation of Ca2+ and NaCl concentrations in the reaction medium. The apparent Kis for calcium ion and NaCl inhibition of bovine protein C activation were > 10 mM and 220 mM, respectively. At I = 0.1 and in the absence of Ca2+ ions bovine protein C was activated with a Km of 0.056 microM and a kcat of 0.24 s-1 (kcat/Km = 4.3 x 10(6) M-1 s-1). Our data are indicative for a rather large conformational and/or structural difference between human and bovine protein C at physiological ionic strength.
