Evidence that heterodimers exist in the fibrinogen Matsumoto II (gamma308N-->K) proband and participate in fibrin fiber formation.
BACKGROUND
In this report, we established the gamma308K/N fibrinogen-secreting Chinese hamster ovary (CHO) cell line, which is an artificially heterozygous, Matsumoto II (M-II; gamma308K-->N) type of dysfibrinogen, to indirectly demonstrate the existence of heterodimeric molecules in propositus plasma and the participation of these molecules in fibrin fiber formation.


METHODS
We co-transfected the gamma-chain of gamma308K- and gamma308N-coding vectors into CHO cells expressing Aalpha- and Bbeta-chains and selected the clones by utilizing the unique electrophoretic mobility of the variant gamma-chain of gamma308K. Although sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis under reducing conditions showed that the amount of the variant gamma-chain was slightly less than the amount of normal gamma-chain in recombinant gamma308K/N fibrinogen, we judged that our selected clone was still a useful model of the M-II individual.


CONCLUSIONS
Functional analyses demonstrated that thrombin-catalyzed fibrin polymerization decreased in the following order: gamma308N, gamma308K/N, an equimolar mixture of gamma308K with gamma308N. The difference in the polymerization curves between gamma308N and gamma308K/N is highly similar to the difference between plasma fibrinogen from a normal control and the M-II proband. In addition, experimental results using the equimolar mixture indicated that gamma308K is able to polymerize into fibrin fibers and does not inhibit the gamma308N polymerization. In conclusion, our results indirectly demonstrated that gamma308K/N fibrinogen is the mixture of normal homodimers, heterodimers, and variant homodimers, and all of these can participate in the fibrin fiber formation.
Okumura
Nobuo
N
Laboratory of Clinical Chemistry, Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan. nobuoku@gipac.shinshu-u.ac.jp
Terasawa
Fumiko
F
Fujita
Kiyotaka
K
Fujihara
Noriko
N
Tozuka
Minoru
M
Koh
Chang-Sung
CS
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000096:biosynthesis* / Q000378:metabolism / Q000648:ultrastructure; D015241:Fibrinogens, Abnormal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D014162:Transfection
D015241:Fibrinogens, Abnormal; D021122:Protein Subunits; D011994:Recombinant Proteins; C477845:fibrinogen Matsumoto II; D005337:Fibrin; D013917:Thrombin