Biophysical Characterization of the Thermodynamic Basis and Binding Orientation for Factor VIII Membrane Association

After vesicular damage occurs, factor VIII (fVIII) is proteolytically activated by thrombin and binds to activated factor IX (fIX) on the surface of activated platelets during the blood coagulation cascade to form the intrinsic “tenase” complex. Once activated, fVIII dissociates from von Willebrand factor and C1 and C2 domains bind to activated platelet surfaces. Previous studies have identified that the C1 and C2 domains bind to negatively charged membranes through both surface exposed hydrophobic residues located in the beta hairpin loops and adjacent positively charged residues. The thermodynamics of fVIII lipid binding are currently uncharacterized and our current understanding of the binding orientation of the C1 and C2 domains of fVIII on lipid membranes is incomplete. In this study, we investigate the thermodynamics and binding orientation of the C domains via mutagenesis to both surface exposed hydrophobic residues and adjacent charged residues. Binding measurements between phosphatidylserine (PS) containing lipid nanodiscs and fVIII C domains have revealed that the C1 domain lipid binding is driven by enthalpic interactions, whereas the C2 domain binding is driven by entropic interactions. Binding experiments on fVIII C2 domain mutants for both surface exposed hydrophobic and positively charged residues have revealed differential disruption to lipid binding. Lastly, we have utilized molecular dynamics to perform simulations on the C1 and C2 domains binding to lipid nanodiscs to elucidate the binding orientation of the C domains and identify novel residues that interact with the membrane. Our results from these simulations agree with previous work, showing that C domain lipid binding centers on R2163 (C1) and R2320 (C2).