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Article
Carbohydrate Moieties on the Procofactor Factor V, but Not the Derived Cofactor Factor VA, Regulate Its Inactivation by Activated Protein C
Biochemistry
  • Jay R Silveira
  • Michael Kalafatis, Cleveland State University
  • Paula B Tracy
Document Type
Article
Publication Date
2-5-2002
Disciplines
Abstract

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.

DOI
10.1021/bi011304g
Citation Information
Jay R Silveira, Michael Kalafatis and Paula B Tracy. "Carbohydrate Moieties on the Procofactor Factor V, but Not the Derived Cofactor Factor VA, Regulate Its Inactivation by Activated Protein C" Biochemistry Vol. 41 Iss. 5 (2002) p. 1672 - 80 ISSN: 0006-2960
Available at: http://works.bepress.com/michael_kalafatis/22/