Hypofibrinogenemia associated with a heterozygous missense mutation gamma153Cys to arg (Matsumoto IV): in vitro expression demonstrates defective secretion of the variant fibrinogen.
We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified gamma-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 gamma-chain DNAs encoding gamma153R and gamma153A 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 gamma153R or gamma153A synthesized the variant gamma-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant gamma-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 (35)S-methionine. Neither intermediates nor intact fibrinogen was seen with the variant gamma-chains. These data suggest that gamma-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 alphagamma or betagamma dimers, or both. This model implies that gammaCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the gamma153Cys-->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.[1]References
- Hypofibrinogenemia associated with a heterozygous missense mutation gamma153Cys to arg (Matsumoto IV): in vitro expression demonstrates defective secretion of the variant fibrinogen. Terasawa, F., Okumura, N., Kitano, K., Hayashida, N., Shimosaka, M., Okazaki, M., Lord, S.T. Blood (1999) [Pubmed]
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