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Blood, 15 May 2002, Vol. 99, No. 10, pp. 3654-3660
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Analysis of fibrinogen  -chain truncations shows the
C-terminus, particularly Ile387, is essential for assembly and
secretion of this multichain protein
Nobuo Okumura,
Fumiko Terasawa,
Hitoshi Tanaka,
Masako Hirota,
Hiroyoshi Ota,
Kiyoshi Kitano,
Kendo Kiyosawa, and
Susan T. Lord
From the Laboratory of Clinical Chemistry, Department
of Medical Technology, School of Allied Medical Sciences, Shinshu
University, the Second Department of Internal Medicine, Shinshu
University School of Medicine, and the Central Clinical Laboratory,
Shinshu University Hospital, Matsumoto, Japan; and the Department of
Pathology and Laboratory Medicine, University of North Carolina, Chapel
Hill.
To examine the role of the fibrinogen  chain in the assembly and
secretion of this multichain protein, we synthesized a series of
fibrinogen variants with truncated chains, terminating between residues 379 and the C-terminus, 411. The variant fibrinogens were synthesized from altered -chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the chain, from 1.4 µg/mL for normal fibrinogen to 0.39 µg/mL for 387 fibrinogen. Immunoassays of cell
lysates showed that all variant chains were synthesized, although
the levels varied significantly. For variants longer than 386 residues,
levels decreased with length but remained near normal. In contrast,
expression of the 4 variants with 386 residues or less was about
20-fold reduced. Quantitative reverse transcription-polymerase chain
reaction demonstrated that the -chain messenger RNA level was
independent from chain length. Western blot analyses showed that
lysates expressing variants with 387 residues or more contained species
comparable to the known intermediates in fibrinogen assembly, including
half-molecules. For shorter variants, these intermediates were not
evident. We conclude that residues near the C-terminus of the chain
are essential for fibrinogen assembly, and more specifically, that
387 is critical. We propose that the loss of residue 387
destabilized the structure of chain, preventing assembly of 
and  dimers, essential intermediates in the assembly of normal fibrinogen.
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