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. Peptides with an apparent M-r of similar to 94,000 (heavy chain; HC) and similar 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 carboxyl-terminal C2 domain, Using V-a reconstituted with either LC form, we examined the effects of the two LC species on membrane binding and 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 composed 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 changes when bound to a membrane. 3) The activity of bovine V-a varied somewhat with LC species, the difference being greatest at limiting X-a concentration. 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 the 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 both altered membrane packing, with V-a-HC having the largest effect. 2) V-a-HC bound reversibly and in a Ca2+-independent fashion to membranes composed of neutral phospholipid (K-d approximate to 0.3 mu M; stoichiometry approximate to 91), High ionic strength had little effect on binding. 3) The substantial effect of V-a on packing within neutral phospholipid 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 packing, and changes in protein structure.