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

Abstract

Background: 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 pre‐existent 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. Objectives: Fibrinogen Detroit is a clinically observed mutation (AαR19→S) with non‐engaging GPS A‐knobs. By analogy, a similar Bβ‐chain mutation, BβR17→S, should produce non‐engaging 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. Methods: DD‐FG, wild‐type recombinant (WT‐FG), and human plasma (hp‐FG) fibrinogen self‐association was studied by turbidimetry coupled with fibrinopeptides release 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 ~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‐associations findings. Conclusions: (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