The a EC Domains of Human Fibrinogen 420 Contain Calcium Binding Sites But Lack Polymerization

The extended a (aE) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional a chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (aEC). A recombinant form of aEC (raEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified raEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in aE. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs aE613/644 and aE780/793. In the presence of 5 mmol/L EDTA, raEC is highly susceptible to plasmic degradation, but Ca21 (5 mmol/L) renders raEC resistant. No protective effect from plasmic degradation was conferred to raEC by the peptides GPRPamide or GHRP, nor did raEC bind to a GPR peptide column. These results suggest that the aEC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the gC domain, we predict that the aEC calcium binding site involves residues aE772-778: DADQWEE. r 1998 by The American Society of Hematology.