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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4122-4131
Hypofibrinogenemia Associated With a Heterozygous Missense Mutation
 153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates
Defective Secretion of the Variant Fibrinogen
Fumiko Terasawa,
Nobuo Okumura,
Kiyoshi Kitano,
Nobuaki Hayashida,
Makoto Shimosaka,
Mitsuo Okazaki, and
Susan T. Lord
From the Gene Research Center and Department of Applied Biology,
Faculty of Textile Science and Technology, Shinshu University, Ueda,
Japan; the Department of Medical Technology, School of Allied Medical
Sciences Shinshu University and Second Department of Internal Medicine,
Shinshu University School of Medicine, Matsumoto, Japan; and the
Department of Pathology and Laboratory Medicine, University of North
Carolina, Chapel Hill, NC.
We genetically analyzed a case of hypofibrinogenemia that showed no
bleeding or thrombotic tendency. Direct sequencing of a polymerase
chain reaction-amplified  -chain gene segment showed a novel
nucleotide substitution. This heterozygous mutation encodes both Cys
(TGT) and Arg (CGT) at residue 153. To examine the basis for the
fibrinogen deficiency, we prepared expression vectors containing mutant
-chain DNAs encoding 153R and 153A for in vitro expression in
Chinese hamster ovary (CHO) cells. Enzyme-linked immunosorbent assay
and immunoblot analysis of the culture media and cell lysates showed
that CHO cells transfected with 153R or 153A synthesized the
variant -chain, but did not secrete variant fibrinogen into the
culture medium. Metabolic pulse-chase experiments showed that
fibrinogen assembly was impaired when either variant -chain was
expressed. In cells expressing normal fibrinogen,
assem- bly intermediates and intact fibrinogen were seen in cell
lysates prepared after short (3 minutes) or long (1 hour) incubation
with 35S-methionine. Neither intermediates nor intact
fibrinogen was seen with the variant -chains. These data suggest
that -chains have an important early role in fibrinogen assembly.
Thus, our results support the model for fibrinogen assembly proposed by Huang et al (J Biol Chem 268:8919, 1993), in which the first
step in assembly is the formation of  or  dimers, or both.
This model implies that Cys153 has a critical role in the formation of these early assembly intermediates. We concluded that the
153Cys Arg substitution does not allow fibrinogen assembly
and secretion, and this is manifest in vivo as a fibrinogen deficiency.
We designated this variant as fibrinogen Matsumoto IV.
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