Carbohydrate moieties on the procofactor factor V, but not the derived cofactor factor Va, regulate its inactivation by activated protein C.
Factor V (FV) is a single-chain plasma protein containing 13-25% carbohydrate by mass. Studies were done to determine if these carbohydrate moieties altered the activated protein C (APC)-catalyzed cleavage and inactivation of both FV and the cofactor which results from its activation by alpha-thrombin, factor Va(IIa) (FVa(IIa)). Treatment of purified FV with N-glycanase and neuraminidase under nonprotein-denaturing conditions removed approximately 20-30% of the carbohydrate from the heavy chain region of the molecule. When glycosidase-treated FV was analyzed in an aPTT (activated partial thromboplastin time)-based APC sensitivity assay, the APC sensitivity ratio (APC-SR) increased from 2.34 to 3.33. In contrast, when glycosidase-treated FV was activated with alpha-thrombin, the addition of the resulting FVa(IIa) to the plasma-based APC sensitivity assay produced no substantial increase in the APC-SR. Additional functional analyses of the APC-catalyzed inactivation of FVa(IIa) in an assay consisting of purified components indicated that both glycosidase-treated and untreated FVa(IIa) expressed identical cofactor activities and were inactivated at identical rates. Analyses of the APC-catalyzed cleavage of glycosidase-treated FV at Arg(306), the initial cleavage site, revealed a 10-fold rate increase when compared to untreated FV. In contrast, and consistent with functional assays, similar analyses of FVa(IIa), derived from those FV species, revealed near-identical rates of APC-catalyzed cleavage at both the Arg(506) and Arg(306)sites. These combined results indicate that N-linked carbohydrate moieties play a substantial role in the APC-catalyzed cleavage and inactivation of FV but not FVa(IIa) at position Arg(306) and that the Arg(306) cleavage sites of FV and FVa(IIa) are distinct substrates for APC.
Silveira
Jay R
JR
Department of Biochemistry, University of Vermont, College of Medicine, Burlington, Vermont 05405, USA.
Kalafatis
Michael
M
Tracy
Paula B
PB
D001120:Arginine / Q000378:metabolism; D002236:Carbohydrate Conformation; D002241:Carbohydrates / Q000737:chemistry*; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005165:Factor V / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000378:metabolism; D015943:Factor Va / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D010314:Partial Thromboplastin Time; D011486:Protein C / Q000737:chemistry*; D011498:Protein Precursors / Q000737:chemistry* / Q000378:metabolism
D002241:Carbohydrates; D011486:Protein C; D011498:Protein Precursors; D015943:Factor Va; D005165:Factor V; D001120:Arginine; D006026:Glycoside Hydrolases