Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3669-3674
The 
EC Domains of Human Fibrinogen420
Contain Calcium Binding Sites But Lack Polymerization Pockets
Dianne Applegate,
Liana Haraga,
Kathe M. Hertzberg,
Lara Stoike Steben,
Jian-Zhong Zhang,
Colvin M. Redman, and
Gerd Grieninger
From the Lindsley F. Kimball Research Institute of the New York Blood
Center, New York, NY.
The extended 
(E) isoform unique to
Fibrinogen420 (Fib420) is distinguished from
the conventional chain of Fibrinogen340 by the presence
of an additional 236-residue carboxyl terminus globular domain
(EC). A recombinant form of EC
(rEC), having a predicted mass of 27,653 Daltons, was
expressed in yeast (Pichia pastoris) and purified by anion
exchange column chromatography. Purified rEC 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 E. Analysis of CNBr digestion fragments confirms that
two disulfide bridges exist at cysteine pairs E613/644
and E780/793. In the presence of 5 mmol/L EDTA,
rEC is highly susceptible to plasmic degradation, but
Ca2+ (5 mmol/L) renders rEC resistant. No
protective effect from plasmic degradation was conferred to
rEC by the peptides GPRPamide or GHRP, nor did
rEC bind to a GPR peptide column. These results suggest
that the EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in
the 
C domain, we predict that the EC calcium binding
site involves residues E772-778: DADQWEE.
