Hydrodynamic characterization of recombinant human fibrinogen species

Abstract

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 (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.

Fibrin Formation, Structure and Properties

“…The αC regions fold back from the distal ends of the triple coiled-coils to form a fourth strand and then extend outward via a flexible connector to relatively compact C-terminal domains that interact with the central globule of fibrinogen (Veklich et al 1993; Litvinov et al 2007b). Truncation of the αC regions affects substantially the hydrodynamic behavior of fibrinogen (Raynal et al 2013). …”

Fibrinogen species as resolved by HPLC-SAXS data processing within theUltraScan Solution Modeler(US-SOMO) enhanced SAS module

“…This suggests that the new, lower band represents a degradation product forming in-column, either by the action of a contaminating protease or by autolysis, perhaps favored by conformational changes resulting from the gel filtration procedure (the starting material had undergone extensive dialysis in the elution buffer without any noticeable change in composition). From western blots of reduced samples of the same fractions stained with an antibody recognizing the A-chain N-terminal end (data not shown), and by comparison with our previous hpHMW-FG analyses (see Figs 1-2 and Table 1 of Raynal et al, 2013), this band originates from the degradation of the C-terminal part of the long, mostly unstructured A chains. Combining all the densitometric analyses, we could reasonably assign the top band to homo-and hetero-dimers of FG species having the A610 and A601 chains, and heterodimers of either A610 or A601 with A583 chains (total molecular weights $339 000-335 000; average E 280 = 1.53 ml mg À1 cm À1 and v 2 = 0.715 ml g À1 ), and the lower band to heterodimers containing A601-A461 and A583-A461 (plus traces of A583-A424 and A461-A424), and to A583 and A461 homodimers (total molecular weights $333 000-307 000; average E 280 = 1.58 ml mg À1 cm À1 and v 2 = 0.715 ml g À1 ).…”

“…For BSA, E 280 = 0.65 ml mg À1 cm À1 and v 2 = 0.733 ml g À1 . For the injected hpHMW-FG samples, the values were computed taking into account the inherent polydispersity (Raynal et al, 2013), and were E 280 = 1.55 ml mg À1 cm À1 and v 2 = 0.715 ml g À1 . Sample analyses by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) without or with urea, and western blotting, all followed by densitometry, were performed as previously reported (Cardinali et al, 2010).…”

“…Yang et al, 2001;Litvinov et al, 2007;Tsurupa et al, 2009). As a part of an ongoing study aimed at characterizing nearly intact FG and C domain-less species (see Cardinali et al, 2010;Raynal et al, 2013), and their covalent and noncovalent adducts, we have performed size-exclusion (SE) HPLC-SAXS studies on a human plasma high-molecular-weight FG fraction (hpHMW-FG). This material presents severe aggregation problems, and the SE-HPLC-SAXS analysis showed non-baseline-resolved oligomers peaks and a split non-symmetrical main peak.…”

Caracterização do fator de elongação Tu (EF-Tu) de Leptospira: aspectos relacionados à colonização e evasão ao sistema complemento do hospedeiro

“…Fibrinogênio também está associado a diversas patologias, como a trombose, hemorragia, e cancro (RAYNAL et al, 2013).…”