title: Hydrodynamic characterization of recombinant human fibrinogen species.
abstract: BACKGROUND
  Fibrinogen is a key component of the blood coagulation system and plays important, diverse roles in several relevant pathologies such as thrombosis, hemorrhage, and cancer. It is a large glycoprotein whose three-dimensional molecular structure is not fully known. Furthermore, circulating fibrinogen is highly heterogeneous, mainly due to proteolytic degradation and alternative mRNA processing. Recombinant production of human fibrinogen allows investigating the impact on the three-dimensional structure of specific changes in the primary structure.
  
  
  METHODS
  We performed analytical ultracentrifugation analyses of a full-length recombinant human fibrinogen, its counterpart purified from human plasma, and a recombinant human fibrinogen with both Aα chains truncated at amino acid 251, thus missing their last 359 amino acid residues.
  
  
  RESULTS
  We have accurately determined the translational diffusion and sedimentation coefficients (Dt(20,w)(0), s(20,w)(0)) of all three species. This was confirmed by derived molecular weights within 1% for the full length species, and 5% for the truncated species, as assessed by comparison with SDS-PAGE/Western blot analyses and primary structure data. No significant differences in the values of Dt(20,w)(0) and s(20,w)(0) were found between the recombinant and purified full length human fibrinogens, while slightly lower and higher values, respectively, resulted for the recombinant truncated human fibrinogen compared to a previously characterized purified human fibrinogen fragment X obtained by plasmin digestion.
  
  
  CONCLUSIONS
  Full-length recombinant fibrinogen is less polydisperse but hydrodynamically indistinguishable from its counterpart purified from human plasma. Recombinant Aα251-truncated human fibrinogen instead behaves differently from fragment X, suggesting a role for the Bβ residues 1-52 in inter-molecular interactions. Overall, these new hydrodynamic data will constitute a reliable benchmark against which models of fibrinogen species could be compared.
authors: Institut Pasteur, Protéopole, Plate-Forme de Biophysique des Macromolécules et de leurs Interactions, CNRS UMR 3528, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France. bertrand.raynal@pasteur.fr
authors: Bertrand
authors: B
authors: Raynal
authors: Barbara
authors: B
authors: Cardinali
authors: Jos
authors: J
authors: Grimbergen
authors: Aldo
authors: A
authors: Profumo
authors: Susan T
authors: ST
authors: Lord
authors: Patrick
authors: P
authors: England
authors: Mattia
authors: M
authors: Rocco
mesh_terms: D015153:Blotting, Western; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry / Q000235:genetics; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D057446:Hydrodynamics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014461:Ultracentrifugation
chemical_list: D005338:Fibrin Fibrinogen Degradation Products; D011994:Recombinant Proteins; C030568:fibrinogen fragment X; D005340:Fibrinogen
keywords: AUC-SV; Analytical Ultracentrifugation; Blood Coagulation; FG; FpA; FpB; Hydrodynamics; Recombinant Fibrinogen; analytical ultracentrifugation sedimentation velocity; chicken plasma FG; cpFG; fibrinogen; fibrinopeptide A; fibrinopeptide B; hpFrX-FG; hpHMW-FG; hrHMW-FG; hrα251-FG; human plasma FG fragment X; human plasma high molecular weight FG; human recombinant FG with Aα chains truncated after residue 251; human recombinant high molecular weight FG
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