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Master of Science (MS)
Spiegel, P. Clint
Anthony-Cahill, Spencer J.
Hemophilia A is an X-linked blood disorder that results in the inability to form proper blood clots and may also cause spontaneous bleeding in the joints or muscles. This disorder is caused by mutations in the F8 gene that attenuate or abolish the activity of its protein product, factor VIII. Blood coagulation factor VIII (fVIII) is a glycoprotein that serves as a cofactor for the serine protease factor IXa on the surface of activated platelets to form the intrinsic tenase complex, which activates factor X during hemostasis to enable blood clot formation. Previous studies have shown that the C-terminal (C2) domain of fVIII is responsible for binding to phospholipids in platelet membranes. However, there are conflicting models for its structural binding mechanism. Elucidating the mechanism of factor VIII binding to phosphatidylserine-containing membranes in activated platelets is essential in the understanding and progress towards improving current hemophilia A therapeutics. Some mutations that occur in the C2 domain result in nonfunctional factor VIII possibly due to its inability to bind platelet membranes and form the tenase complex. This research helps to further describe this mechanism by analyzing the atomic interactions between the C2 domain of factor VIII and phosphatidylserine membranes by X-ray crystallography and biochemical assays. A 1.4 Å resolution X-ray crystal structure of the porcine C2 domain of factor VIII in complex with O-phospho-L-serine (OPLS), a soluble moiety of the headgroup of phosphatidylserine, revealed a favorable electrostatic interaction between Arg2320 and OPLS, implying the importance of the universally conserved arginine at residue 2320. Within the human hemophilia A population, two mutations at that residue in fVIII, Arg2320Thr/Ser, are of interest due to the ability of these fVIII mutants to stay in circulation as non-functioning proteins. Characterizing these mutants with phosphatidylserine membrane-binding enzyme-linked immunosorbent assays (ELISAs), affinity pull-down assays, and intrinsic fluorescence has allowed for a more comprehensive understanding of the binding mechanism of fVIII. Because the C1 domain of fVIII is structurally homologous to the C2 domain, we attempted to study two naturally occurring mutations (Arg2163His and Arg2159His) in the C1 domain that likely interact with phosphatidylserine-containing membranes. Arg2163His is a mutation located in the same position structurally as Arg2320 in the C2 domain. However, we were unable to purify these C1 mutants to a level suitable for biochemical studies. Furthermore, phosphatidylserine lipid binding studies were conducted on an Arg2215Ala C2 domain mutant due to its proximity to the hydrophobic region of platelet membranes. We were able to express and purify all of the C2 domain mutants, and proper folding of these mutants was verified using affinity pull-down assays. Phosphatidylserine membrane binding ELISAs revealed that all the mutants studied were not able to bind to activated platelet phosphatidylserine-containing membranes in activated platelets. Preliminary intrinsic fluorescence analysis determined that Arg2320Ser was thermodynamically less stable than wild type, but was still predominantly folded. Our data further supported our activated platelet membrane binding mechanism that is centered on Arg2320, with two hydrophobic spikes docked within the hydrophobic lipid bilayer of platelet membranes, and nonspecific interactions between either the phosphate or carboxyl groups of phospholipids and the underlying basic residues of C2 (Arg2209, Arg2215, Lys2183, Arg2220, Arg2222, and Lys2249).
Western Washington University
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Wo, Serena W., "Structural Characterization of Factor VIII C2 Domain Activated Platelet Membrane Binding" (2017). WWU Graduate School Collection. 614.