THE ALPHA-C-E DOMAINS OF HUMAN FIBRINOGEN(420) CONTAIN CALCIUM-BINDING SITES BUT LACK POLYMERIZATION POCKETS

The extended alpha (alpha(E)) isoform unique to Fibrinogen(420) (Fib(4 20)) is distinguished from the conventional alpha chain of Fibrinogen( 340) by the presence of an additional 236-residue carboxyl terminus gl obular domain (alpha(E)C). A recombinant form of alpha(E)C (r alpha(E) C), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography . Purified r alpha(E)C appears to be predominantly intact, as judged b y N-terminal sequence analysis, mass spectral analysis of the C-termin al cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, co upled with analysis of CNBr digestion fragments, confirms hi-linked gl ycosylation at Asn667, the site at which sugar is attached in alpha(E) Analysis of CNBr digestion fragments confirms that two disulfide brid ges exist at cysteine pairs alpha(E)613/644 and alpha(E)780/793. In th e presence of 5 mmol/L EDTA, r alpha(E)C is highly susceptible to plas mic degradation, but Ca2+ (5 mmol/L) renders r alpha(E)C resistant. No protective effect from plasmic degradation was conferred to alpha(E)C by the peptides GPRPamide or GHRP, nor did r alpha(E)C bind to a GPR peptide column. These results suggest that the alpha(E)C domain contai ns a calcium-binding site, but lacks a polymerization pocket. By analo gy with the site elucidated in the gamma C domain, we predict that the alpha(E)C calcium binding site involves residues alpha(E)772-7783 DAD OWEE. (C) 1998 by The American Society of Hematology.