The overall objective of this research is to understand the structure- function relationships of the vitamin K-dependent proteins that are involved in hemostasis, and the genes that encode these proteins. Some of the focus in this application will be placed on murine proteins and genes, since most targeted gene alterations, which have considerably advanced this field, are performed in mouse models. Proteins of specific interest that are contained in this general class, viz., factors (f) VII, IX, X, and protein C (PC), are assembled through similar domains, which include the gamma-carboxyglutamic acid (Gla) domain, a hydrophobic stretch, two consecutive epidermal growth factor-like (EGF) motifs, an activation peptide module, and a serine protease domain. These regions incorporate different properties of these proteins, and appear to function independently. It is hypothesized that by closely defining the critical regions of these protein domains, and their genes, that are essential to their various functions, it will be possible to regulate their specific features, and, likely, to transfer specific functions of one protein into another.
Five specific aims are proposed to achieve these goals: (1) to identify and assess the functional roles of transcriptional regulatory of the murine genes, fVII, fX, PC, and the endothelial cell PC receptor (EPCR), through in vitro and in vivo methodology; (2) to employ a synthetic peptide library to determine sequences that are most effectively cleaved by fVIIa and the fVIIa/TF complex, by thrombin and the thrombin/TM complex, and by aPC and the aPC/EPCR complex. To exchange various optimized peptide sequences identified for each enzyme with comparable residues in another protein (fIX) to determine whether cleavages by these enzymes occur as predicted from the peptide study; (3) to express wild-type and variants of the first growth factor-like domain (EGF1) of human PC and to characterize their divalent cation binding proteins. TO determine, by NMR, the solution structures and backbone dynamics of EGF1-PC in the presence and absence of Ca2+, along with a variant of this module lacking its extra disulfide loop sequence; (4) to chemically synthesize variants of the gamma-carboxyglutamic acid (Gla) domain of human PC and to characterize their Ca2+-dependent properties. Peptides that are selectively-labeled with 13C-Gla will be emphasized to evaluate divalent cation binding to peptides mutated in the same manners as variant proteins that show defective Ca2+-related properties; and (5) to investigate amino acid determinants of the binding of human PC to EPCR.
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