CHARACTERIZATION OF 2 FORMS OF HUMAN FACTOR-VA WITH DIFFERENT COFACTOR ACTIVITIES

Factor Va is an essential cofactor in factor Xa-catalyzed prothrombin activation. Purified human factor Va appears to consist of a heavy cha in (M(r) almost-equal-to 105,000) and a light chain doublet with M(r) almost-equal-to 74,000 and almost-equal-to 71,000. We separated factor Va by chromatography on a Mono-S column into two fractions, designate d factors Va1 and Va2. Factor Va1 contains the light chain with M(r) a lmost-equal-to 74,000, and factor Va2 exclusively contains the light c hain with M(r) almost-equal-to 71,000. The two forms of factor Va expr ess different cofactor activities when prothrombin is activated at low phospholipid concentrations or on membranes containing low amounts of phosphatidylserine in phosphatidylcholine. Compared with factor Va2, much higher amounts of factor Va1 are required for factor Xa.Va comple x formation at the membrane surface. Once incorporated into the prothr ombinase complex, factors Va1 and Va2 are equally active in prothrombi n activation. This indicates that the two forms of factor Va do not di ffer in their ability to promote the catalytic activity of factor Xa o r to interact with prothrombin. Direct binding experiments show that t he different cofactor activities are explained by a greatly impaired a bility of factor Va1 to bind to negatively charged membranes. Factor V is also separated into two protein peaks after chromatography on a Mo no-S column. Upon incubation with thrombin, the first peak yields fact or Va1 and the second peak factor Va2. The same two forms of factor Va were generated when freshly prepared plasma samples or platelet suspe nsions were treated with thrombin. This shows that the heterogeneity o f the light chain domain is an intrinsic property of both plasma and p latelet factor V. It is hypothesized that the heterogeneity is caused by small differences in the carboxyl-terminal C2 domain of factor V th at are introduced as the result of post-ribosomal processing.