ROLES OF FACTOR V-A HEAVY AND LIGHT-CHAINS IN PROTEIN AND LIPID REARRANGEMENTS ASSOCIATED WITH THE FORMATION OF A BOVINE FACTOR V-A-MEMBRANE COMPLEX

Factor V-a is an essential protein cofactor of the enzyme factor X-a, which activates prothrombin to thrombin during blood coagulation. Pept ides with an apparent M-r of similar to 94,000 (heavy chain; HC) and s imilar to 74,000 or 72,000 (light chain; LC) interact in the presence of Ca2+ to form active V-a. The two forms of V-a-LC differ in their ca rboxyl-terminal C2 domain, Using V-a reconstituted with either LC form , we examined the effects of the two LC species on membrane binding an d on the activity of membrane-bound V-a. We found that 1) V-a composed of the 72,000 LC bound only slightly more tightly to membranes compos ed of a mixture of neutral and acidic lipids, the K-d being reduced by a factor of similar to 3 at 5 mM and by a factor of 6 at 2 mM Ca2+. 2 ) The two forms of V-a seemed to undergo different conformational chan ges when bound to a membrane. 3) The activity of bovine V-a varied som ewhat with LC species, the difference being greatest at limiting X-a c oncentration. We have also addressed the role of the two V-a peptides in membrane lipid rearrangements and binding: 1) V-a binding increased lateral packing density in mixed neutral/acidic lipid membranes. In t he solid phase, V-a-HC had no effect, whereas V-a-LC and whole V-a had similar but small effects, In the fluid phase, V-a-HC and whole V-a b oth altered membrane packing, with V-a-HC having the largest effect. 2 ) V-a-HC bound reversibly and in a Ca2+-independent fashion to membran es composed of neutral phospholipid (K-d approximate to 0.3 mu M; stoi chiometry approximate to 91), High ionic strength had little effect on binding. 3) The substantial effect of V-a on packing within neutral p hospholipid membranes was mimicked by V-a-HC. 4) Based on measurements of membrane phase behavior, binding of V-a or its peptide components did not induce thermodynamically discernible lateral membrane domains, These results suggest that the membrane association of factor V-a is a complex process involving both chains of V-a, changes in lipid packi ng, and changes in protein structure.