Discipline: Chemistry and Chemical Sciences
Subcategory: Biochemistry (not Cell and Molecular Biology and Genetics)
Stephanie Thompson - Georgia State University
Co-Author(s): Michelle Wuerth and Marcus Carlson, Western Washington University, Bellingham, WA
Hemophilia A is a genetic disorder in which the blood is unable to clot normally and is caused by the lack or absence of coagulation Factor VIII. Factor VIII (fVIII) is a glycoprotein functioning as a cofactor. The most effective treatment for hemophilia A patients include infusions of plasma-derived or recombinant fVIII. A major impediment of fVIII replacement therapy is the development of inhibitory antibodies. The C2 domain of fVIII is a highly immunogenic domain and is essential to the binding of activated platelet surfaces. Once inhibitory antibodies are produced, the C2 domain of fVIII binds to the inhibitory antibodies rather than activated platelets. Mechanistic investigations of the inhibitory antibodies suggest that it prevents the binding of fVIII to phospholipids which aid in the coagulation process. Several residues of the C2 domain are thought to be responsible for binding to these antibodies including Arg2320. Mutations of this residue and binding affinity studies potentially aid in developing new treatments designed to improve blood clotting in hemophilia A patients. Site-directed mutagenesis was used to generate three different point mutants of Arg2320 (R2320S, R2320T, and R2320M). Each point mutant was over expressed and purified from E. coli. Preliminary membrane binding studies (ELISAs) were performed to further understand the role of Arg2320 in binding to platelet surfaces. The results of this study concluded that residue R2320, when mutated, did bind less to blood platelet like membranes. Future work include supplementary binding assays and stability test to further gather data on this residue.
Funder Acknowledgement(s): National Science Foundation
Faculty Advisor: P. Clint Spiegel,