Partial glycosylation at asparagine-2181 of the second C- type domain of human factor V modulates assembly of the prothrombinase complex

Suhng Wook Kim, Thomas L. Ortel, Mary Ann Quinn-Allen, Lina Yoo, Laura Worfolk, Xin Zhai, Barry R. Lentz, William H. Kane

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Abstract

Thrombin-activated factor Va exists as two isoforms, factor Va1 and factor Va2, which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained following incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site at Asn-2181, was partially glycosylated when expressed in COS cells. To confirm the structural basis for factor Va1 and factor Va2, we mutated Asn-2181 to glutamine (N2181Q) and expressed this mutant using a B domain deletion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q released a light chain with mobility identical to that of factor Va2 on SDS-PAGE. The functional properties of purified N2181Q were similar to those of factor Va2 in prothrombinase assays carried out in the presence of limiting concentrations of phosphatidylserine. The binding of human factor Va1 and factor Va2 to 75:25 POPC/POPS vesicles was also investigated in equilibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va2 binding to POPC/POPS vesicles was approximately 3-fold higher than that of factor Va1. These results indicate that partial glycosylation of factor V at asparagine-2181 is the structural basis of the light chain doublet and that the presence of this oligosaccharide reduces the affinity of factor Va for biological membranes.

Original languageEnglish
Pages (from-to)11448-11454
Number of pages7
JournalBiochemistry
Volume38
Issue number35
DOIs
Publication statusPublished - 1999 Aug 31

ASJC Scopus subject areas

  • Biochemistry

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    Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., & Kane, W. H. (1999). Partial glycosylation at asparagine-2181 of the second C- type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry, 38(35), 11448-11454. https://doi.org/10.1021/bi991275y