Hydrodynamic characterization of recombinant human fibrinogen species
Résumé
Introduction: 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 (D t(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 D t(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.
Mots clés
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