Characterization of two 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 chain (M(r) approximately 105,000) and a light chain doublet with M(r) approximately 74,000 and approximately 71,000. We separated factor Va by chromatography on a Mono-S column into two fractions, designated factors Va1 and Va2. Factor Va1 contains the light chain with M(r) approximately 74,000, and factor Va2 exclusively contains the light chain with M(r) approximately 71,000. The two forms of factor Va express 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 complex formation at the membrane surface. Once incorporated into the prothrombinase complex, factors Va1 and Va2 are equally active in prothrombin activation. This indicates that the two forms of factor Va do not differ in their ability to promote the catalytic activity of factor Xa or to interact with prothrombin. Direct binding experiments show that the different cofactor activities are explained by a greatly impaired ability of factor Va1 to bind to negatively charged membranes. Factor V is also separated into two protein peaks after chromatography on a Mono-S column. Upon incubation with thrombin, the first peak yields factor Va1 and the second peak factor Va2. The same two forms of factor Va were generated when freshly prepared plasma samples or platelet suspensions were treated with thrombin. This shows that the heterogeneity of the light chain domain is an intrinsic property of both plasma and platelet factor V. It is hypothesized that the heterogeneity is caused by small differences in the carboxylterminal C2 domain of factor V that are introduced as the result of post-ribosomal processing.
Rosing
J
J
Department of Biochemistry, University of Limburg, Maastricht, The Netherlands.
Bakker
H M
HM
Thomassen
M C
MC
Hemker
H C
HC
Tans
G
G
D001665:Binding Sites; D001792:Blood Platelets / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D015943:Factor Va / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D011516:Prothrombin / Q000378:metabolism
D015943:Factor Va; D011516:Prothrombin