Fibrinogen Alpha C-Regions Are Not Directly Involved In Fibrin Polymerization As Evidenced By A "Double-Detroit" Recombinant Fibrinogen Mutant And Knobs-Mimic Peptides

Background Fibrin polymerization, following fibrinopeptides A and B (FpA, FpB) cleavage, relies on newly exposed alpha- and beta-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' gamma- and (B)beta-chains C-terminal regions. A role for mostly disordered (A)alpha-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed. Objectives Fibrinogen Detroit is a clinically observed mutation (A alpha R19 -> S) with nonengaging GPS A-knobs. By analogy, a similar B beta-chain mutation, B beta R17 -> S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (A alpha R19 -> S, B beta R17 -> S; DD-FG) was developed: with A-a and B-b engagements endogenously blocked, other interactions would become apparent. Methods 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). Results 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 similar to 50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial B beta-chain N-terminal and A alpha-chain C-terminal degradation. Nevertheless, similar to 70% DD-FG molecules bearing (A)alpha C-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. Conclusions (A)alpha C-regions interactions appear too weak to assist native fibrin polymerization, at least without knobs engagement. Their role in all stages should be carefully reconsidered.