FACTOR-VA MEMBRANE INTERACTION IS MEDIATED BY 2 REGIONS LOCATED ON THE LIGHT-CHAIN OF THE COFACTOR

Factor Va was incubated with 1-azidopyrene, a fluorescent lipophilic probe, in the presence of phospholipid vesicles composed of various proportions of phosphatidylcholine (PC) and phosphatidylserine (PS). The majority of the label was associated with the light chain of factor Va. The light chain was found to be labeled in the presence of phospholipid vesicles containing either 100% PC or 100%PS. After cleavage by factor Xa and incubation with PC/PS vesicles composed of 75% PC and 25% PS, label was found both on the M(r) = 30 000 fragment, derived-from the NH2-terminal portion of the bovine factor Va light chain (residues 1537-1752), and on the M(r) = 46 000/48 000 carboxyl-terminal fragment of the factor Va light chain (residues 1753-2183). The M(r) = 46 000/48 000 fragment incorporated I-azidopyrene independent of the phospholipid composition, while label incorporation into the M(r) = 30 000 fragment required phospholipid vesicles containing PC. No labeling of the M(r) = 30 000 fragment was observed with phospholipid vesicles composed of 100% PS. The label incorporation into the two portions of the molecule was found to be independent of the ionic strength in the presence of phospholipid vesicles containing 75% PC and 25% PS. In contrast, the labeling of the M(r) = 46 000/48 000 fragment with phospholipid vesicles composed of 1 00% PS was ionic strength dependent. These data suggest that two regions of factor Va light chain interact with the lipid bilayer and have different requirements for interaction: the binding site located on the M(r) = 30 000 fragment of the cofactor (A3 domain) interacts with phospholipid vesicles containing neutral phospholipid and is most likely hydrophobic in nature whereas the binding site located on the M(r) = 46 000/48 000 carboxyl-terminal fragment (C1-C2 domains ) interacts with membranes composed of anionic and neutral phospholipid and displays partly ionic binding characteristics.