Recombinant γ′ fibrinogen and its interaction with factor XIII Public Deposited

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Alternate title
  • Recombinant [gamma prime] Fibrinogen and its Interaction with Factor XIII
Last Modified
  • March 21, 2019
Creator
  • Gersh, Kathryn Connell
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Because the γ′ chain of fibrinogen has been hypothesized as a binding site for factor XIII zymogen, I prepared two recombinant fibrinogen variants, a γ′/γ′ fibrinogen homodimer and a γ/γ′ fibrinogen heterodimer. Characterization of their expression in CHO cells revealed the following: 1) γ′ chain protein is produced in about the same proportion as γ′ mRNA in any given cell line. 2) γ and γ′ chains assemble into homodimeric fibrinogen molecules as readily as they do heterodimers. 3) Mass spectrometry studies indicate that one of two tyrosine residues in the recombinant γ′ chain is sulfated. I next characterized the polymerization of purified recombinant γ′/γ′ and γ/γ′ fibrinogen. As measured by turbidity, the presence of the γ′ chain leads to decreased lateral aggregation at all calcium concentrations studied. I monitored fibrinopeptide release by HPLC and found that release of fibrinopeptide A occurs at the same rate regardless of the presence of a γ′ chain, but fibrinopeptide B release is faster from γ′/γ′ than γ/γ or γ/γ′ fibrinogen. Factor XIIIa-catalyzed cross-linking of the variants starts at a slower rate for fibrinogens with a γ′ chain than it does for those without. I then used the γ′/γ′ and γ/γ′ fibrinogen variants in an ELISA assay to measure factor XIII binding to fibrinogen and fibrin. I studied binding of factor XIII zymogen, recombinant factor XIII composed of just A subunits, and active-site-inhibited, activated recombinant factor XIIIa. No matter what form the factor XIII is in, or whether fibrinogen or fibrin is used, these experiments clearly showed that the γ′ chain has no role in factor XIII binding to fibrinogen.
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  • In Copyright
Advisor
  • Lord, Susan T.
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