Document Type

Project

Publication Date

Spring 2017

Keywords

Blood coagulation, Lipid binding, Biochemistry

Abstract

Blood coagulation factor VIII (fVIII) is an essential cofactor in the mammalian blood-clotting cascade. fVIII must bind the phospholipid membrane of activated platelets to function as a cofactor for fIXa. The blood coagulation cascade culminates in the formation of a stable blood clot. In humans, the C1 and C2 domains are implicated in binding phospholipid membranes, however the relative contribution of different residues in the lipid-binding mechanism is unclear. Using site-directed mutagenesis, expression of the isolated C1 and C2 domains in Escherichia coli cells, protein purification with metal affinity chromatography, electrospray ionization mass spectrometry, enzyme-linked immunosorbent assays, liposome sedimentation assays, pull down assays, circular dichroism and intrinsic tryptophan fluorescence, we compare relative binding affinities and stability of five mutations on the C1 and C2 domains. Three of the mutations are on the C2 domain: R2320S, R2320T, and R2215A. The other two mutations are on the C1 domain: R2163H and R2159H. Our results combined with future research will help elucidate the roles of these residues in the function and stability of FVIII with regards to Hemophilia A.

Department

Chemistry

Subjects - Topical (LCSH)

Hemophilia--Treatment--Complications; Bilayer lipid membranes--Therapeutic use; Blood coagulation factor VIII; Binding sites (Biochemistry)

Genre/Form

student projects; term papers

Type

Text

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

Rights Statement

http://rightsstatements.org/vocab/InC/1.0/

Language

English

Format

application/pdf

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