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Fibrinogen αC‐regions are not directly involved in fibrin polymerization as evidenced by a “Double‐Detroit” recombinant fibrinogen mutant and knobs‐mimic peptides

2019; Elsevier BV; Volume: 18; Issue: 4 Linguagem: Inglês

10.1111/jth.14725

1538-7933

Cédric Duval, Aldo Profumo, Anna Aprile, Annalisa Salis, Enrico Millo, Gianluca Damonte, Julia S. Gauer, Robert A. S. Ariëns, Mattia Rocco,

Blood properties and coagulation

Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed α- and β-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' γ- and (B)β-chains C-terminal regions. A role for mostly disordered (A)α-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed.Fibrinogen Detroit is a clinically observed mutation (AαR19 → S) with nonengaging GPS A-knobs. By analogy, a similar Bβ-chain mutation, BβR17 → S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (AαR19 → S, BβR17 → S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent.DD-FG, wild-type recombinant (WT-FG), and human plasma (hp-FG) fibrinogen self-association was studied by turbidimetry coupled with fibrinopeptides release high-performance liquid chromatography (HPLC)/mass spectrometry analyses, and by light-scattering following size-exclusion chromatography (SE-HPLC).In contrast to WT-FG and hp-FG, DD-FG produced no turbidity increase, irrespective of thrombin concentration. The SE-HPLC profile of concentrated DD-FG was unaffected by thrombin treatment, and light-scattering, at lower concentration, showed no intensity and hydrodynamic radius changes. Compared with hp-FG, both WT-FG and DD-FG showed no FpA cleavage difference, while ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial Bβ-chain N-terminal and Aα-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)αC-regions potentially able to associate were available. Higher-concentration, nearly intact hp-FG with 500-fold molar excess GPRP-NH2 /GHRP-NH2 knobs-mimics experiments confirmed these no-association findings.(A)αC-regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.