Protein C activation by an activator purified from the venom of <i>Agkistrodon halys halys</i>

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 K(i) 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 K(m) for protein C of 0.52 muM and a k(cat) of 0.17 s-1 at 1 = 0.05 (k(cat)/K(m) = 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 K(m) 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 K(i)s for calcium ion and NaCl inhibition of bovine protein C activation were &gt; 10 mM and 220 mM, respectively. At I = 0.1 and in the absence of Ca2+ ions bovine protein C was activated with a K(m) of 0.056 muM and a k(cat) of 0.24 s-1 (k(cat)/K(m) = 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

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels\ud

Objectives: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. Methods and Results: We genotyped the control populations of two population-based studies for four AVPR2 variations: a-245c, G12E, L309L, and S331S. Rare alleles of a-245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R–green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (KD) was 4.5 nm [95% confidence interval (CI) 3.6–5.4] for 12E-V2R–GFP and 16.5 nm (95% CI 10.1–22.9) for 12G-V2R–GFP. AVP-induced cAMP generation was enhanced in 12E-V2R–GFP cells. Conclusions: The 12E-V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVII