Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.
OBJECTIVE
We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg.


METHODS
A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles.


RESULTS
By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone).


CONCLUSIONS
These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.
Department of Pathology, Shinshu University School of Medicine, Shinshu University Hospital, Japan.
M
M
Hirota-Kawadobora
F
F
Terasawa
T
T
Suzuki
M
M
Tozuka
K
K
Sano
N
N
Okumura
D000409:Alanine / Q000737:chemistry; D000818:Animals; D016466:CHO Cells; D002384:Catalysis; D006224:Cricetinae; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D026122:Factor XIIIa / Q000737:chemistry*; D005337:Fibrin / Q000737:chemistry* / Q000648:ultrastructure; D005340:Fibrinogen / Q000737:chemistry / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008855:Microscopy, Electron, Scanning; D010957:Plasmids / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D013053:Spectrophotometry; D013917:Thrombin / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D011108:Polymers; D011994:Recombinant Proteins; D006639:Histidine; D005337:Fibrin; D005340:Fibrinogen; D026122:Factor XIIIa; D013917:Thrombin; D000409:Alanine