In vitro assembly of the component chains of fibrinogen requires endoplasmic reticulum factors.
Human fibrinogen (340 kDa) is a dimer, with each identical half-molecule composed of three different polypeptides (Aalpha, 66 kDa; Bbeta, 55 kDa; and gamma, 48 kDa). To understand the mechanisms of chain assembly, a coupled in vitro transcription translation system capable of assembling fibrinogen chains was developed. Fibrinogen chain assembly was assayed in an expression system coupled to rabbit reticulocyte lysate in the presence or absence of dog pancreas microsomal membranes. Fibrinogen chain assembly required microsomal membranes and oxidized glutathione. Co-expression of two of the chains, Bbeta and gamma or Aalpha and gamma, yielded free chains and two-chain complexes. Unlike combinations of Aalpha with gamma and Bbeta with gamma, co-expression of Aalpha and Bbeta did not form a single two-chain complex but produced a mixture of two-chain complexes. Co-expression of all three chains yielded free chains, two-chain complexes, and higher molecular weight complexes that corresponded to a half-molecule and to fully formed fibrinogen. Upon treatment of this mixture with thrombin and factor XIIIa, a gamma.gamma dimer, similar to that obtained from cross-linked human fibrin, was produced, indicating that properly folded fibrinogen was formed in vitro. Molecular chaperones may participate in fibrinogen assembly, since antibodies to resident proteins of the endoplasmic reticulum (BiP, Hsp90, protein disulfide isomerase, and calnexin) co-precipitated the chaperones together with nascent fibrinogen chains and complexes.
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Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, USA.
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Redman
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D000818:Animals; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D018832:Molecular Chaperones; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011503:Transglutaminases / Q000378:metabolism
D018832:Molecular Chaperones; D005340:Fibrinogen; D011503:Transglutaminases