Hypofibrinogenemia Associated With a Heterozygous Missense Mutation γ153Cys to Arg (Matsumoto IV): In Vitro Expression Demonstrates Defective Secretion of the Variant Fibrinogen

Abstract

We genetically analyzed a case of hypofibrinogenemia that showed no bleeding or thrombotic tendency. Direct sequencing of a polymerase chain reaction-amplified γ-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 γ-chain DNAs encoding γ153R and γ153A 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 γ153R or γ153A synthesized the variant γ-chain, but did not secrete variant fibrinogen into the culture medium. Metabolic pulse-chase experiments showed that fibrinogen assembly was impaired when either variant γ-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 γ-chains. These data suggest that γ-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 γ or βγ dimers, or both. This model implies that γCys153 has a critical role in the formation of these early assembly intermediates. We concluded that the γ153Cys→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.

Molecular Mechanisms of Hypo‐ and Afibrinogenemia

“…The Matsumoto IV (γ153 Cys→Arg) mutation affects the proximal A region of the γD domain. 17 Transient expression of 153 Arg γ chains with Aα and Bβ chains in Chinese hamster ovary cells demonstrated that, although the variant chain was synthesized, no variant fibrinogen molecules were secreted into the culture medium.…”

“…Since γ153 Cys forms an inter chain S-S bond to γ182 Cys it appears that either, γ153 Cys has a direct role in complex formation or, that the freed thiol at γ182 impairs the formation of S-S bonds in the αγ or βγ complexes. 17 The Bβ353 Leu→Arg and Bβ400 Gly→Asp mutations 18 are located in the distal P region of the βD domain. 11 Heterozygosity for either lowers fibrinogen levels by approximately 50%, whereas homozygosity produces afibrinogenemia.…”

Identification and Characterization of Five New Fibrinogen Gene Polymorphisms

“…One of these differences, γ 796 T → A, has been identified previously 10 and has recently been redescribed. 11 In that study, a total of five variations were noted, all of which were identified in our study. In agreement with our interpretation, these variations were classified by the authors as errors in the Genbank entry.…”

Heterozygous variant fibrinogen γA289V (Kanazawa III) was confirmed as hypodysfibrinogenemia by plasma and recombinant fibrinogens

“…Culture media and cell lysates were prepared, and fibrinogen concentrations were measured by ELISA. 15…”