His2315/Gln2316 of the Factor VIII C2 Domain Interact with Phospholipid Membranes and Influence Activity of the Factor Xase Complex.

A major phospholipid-binding motif of factor VIII is localized in the C2 domain. Two pairs of hydrophobic amino acids in the factor VIII C2 domain (Met2199/Phe2200, Leu2251/Leu2252) have previously been shown to interact with phospholipid membranes. However mutations of hydrophilic amino acids (Gln2213, Arg2215, Asn2217, Arg2220, Lys2249) that were predicted to interact with the membrane surface, based upon the crystallographic structure, have not altered phospholipid binding activity. The established hydrophobic interactions do not account for the demonstrated hydrophilic interactions of factor VIII with the phospholipid bilayer. We hypothesized that His2315 and/or Gln2316, interact with phospholipid membranes, based upon their contribution to the epitope for phospholipid-blocking mAb B02C11 and based upon phosphatidylserine binding by C2 domain peptides that include these residues. Factor VIII His2315Ala/Gln2316Ala (fVIII 2315/16) was prepared by PCR mutagenesis and expressed from COS-1 cells. Secreted factor VIII was purified by immunoaffinity chromatography and evaluated in ELISA assays, aPTT assays with factor VIII deficient plasma, and in defined assays with varying phospholipids. The specific activity of fVIII 2315/16 was 67 ± 9% of wild type factor in a commercial aPTT assay with a large excess of phospholipid. The apparent affinity for extruded phospholipid vesicles (0.1 μm diameter) of composition phosphatidylserine:phosphatidylethanolamine: phosphatidylcholine 4:20:76 was 32 ± 10% of wild type factor VIII in a factor Xase assay with varying phospholipid. When the phospholipid concentration was saturating, the Vmax was 55% of factor Xase with wild type factor VIII. We conclude that His2315 and/or Gln2316 constitute the first identified hydrophilic amino acids of factor VIII that interact with a phospholipid membrane. His2315/Gln2316, combined with the two pairs of phospholipid-interactive hydrophobic amino acids constitute three spatially separated phospholipid-interactive points that define a phospholipid-interactive plane on the factor VIII C2 domain. This is consistent with the hypothesis that the phospholipid-binding plane of the C2 domain overlaps substantially with the epitope for BO2C11 and is nearly coincident with the phospholipid-binding surface predicted from the crystallographic structure.