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

Thrombin-activated factor Va exists as two isoforms, factor Va(1) and facto r Va(2), which differ in the size of their light chains and their affinity for biological membranes. The heterogeneity of the light chain remained fol lowing incubation of factor Va with N-glycanase. However, we found that the factor V C2 domain, which contains a single potential glycosylation site a t Asn-2181, was partially glycosylated when expressed in COS cells. To conf irm the structural basis for factor Va(1) and factor Va(2), we mutated Asn- 2181 to glutamine (N2181Q) and expressed this mutant using a B domain delet ion construct (rHFV des B) in COS cells. Thrombin activation of N2181Q rele ased a light chain with mobility identical to that of factor Va(2) on SDS-P AGE. The functional properties of purified N2181Q were similar to those of factor Va(2) in prothrombinase assays carried out in the presence of limiti ng concentrations of phosphatidylserine. The binding of human factor Va(1) and factor Va(2) to 75:25 POPC/POPS vesicles was also investigated in equil ibrium binding assays using proteins containing a fluorescein-labeled heavy chain. The affinity of human factor Va(2) binding to POPC/POPS vesicles wa s approximately 3-fold higher than that of factor Va(1). These results indi cate that partial glycosylation of factor V at asparagine-2181 is the struc tural basis of the light chain doubler and that the presence of this oligos accharide reduces the affinity of factor Va for biological membranes.