Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Abstract

Human coagulation factor V is a protein cofactor that is an essential component of the thrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 .+-. 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 .+-. 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 .+-. 0.012 to 0.124 .+-. 0.044 unit/mL following activation by the factor V activating enzyme from Russell''s viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 .+-. 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 .+-. 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (.apprx.330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441revealed a single-chain polypeptide with Mr .apprx.230 kDa. This mutant constitutively expressed 38 .+-. 7% of the activity of the RVV-V-activated protein. These results suggest that one of the functions of the large connecting region in factor V is to inhibit constitutive procoagulant activity.