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 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. Objectives 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. 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 ~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. 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.

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region

“… 20 – 22 , 25 In agreement with this, we observed no differences in GPVI-fibrinogen and GPVI-fibrin binding profiles by ELISA. Furthermore, we obtained similar binding profiles with our new fibrin mutant that is unable to polymerize 26 when compared with polymerized fibrin preparations, confirming that polymerization of fibrinogen into fibrin is not required to elicit GPVI binding. Although GPVI is able to bind both fibrinogen and fibrin, we hypothesize that platelet activation only occurs when GPVI is clustered, which should happen if fibrin is polymerized into fibers or film, 40 but not with fibrinogen in solution.…”

“…We next compared the ability of fibrinogen variants to bind GPVI, including plasma-purified, recombinant wild-type, nonpolymerizing-fibrinogen, and fibrin. 26 Similar binding profiles to GPVI monomer and dimer were observed for all fibrinogen variants, suggesting that fibrin polymerization is not required for GPVI binding (Figure 1 C dimer and 1D monomer). Indeed, when these fibrinogen variants were converted to fibrin through thrombin treatment, their binding profiles to GPVI remained similar (Figure 1 C dimer and 1D monomer).…”

“…Here, we investigate the molecular interaction mechanisms of GPVI and fibrin(ogen), using a combination of 4 different protein-protein interaction methods, that is, ELISA, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM), to gain detailed understanding of the binding affinity and kinetics while also identifying the GPVI binding regions on the fibrinogen molecule. We also investigate the effects of fibrin polymerization on GPVI binding using a novel recombinant fibrinogen mutant that is unable to polymerize on conversion to fibrin by thrombin, called nonpolymerizing (previously also called double-Detroit in Duval et al 26 ) fibrinogen. Our data show that both monomeric and dimeric GPVI bind the αC-region of fibrinogen and that their affinities and kinetic profiles are distinct from each other based on avidity.…”

Principles of Fibrinogen Fiber Assembly In Vitro

“…[ 37,38 ] However, a recent study has cast doubts to the effective role of the αC regions in basic fibrin polymerization. [ 39 ] In any case, these flexible and polar αC arms participate in crosslinking of fibrin during blood coagulation [ 29 ] and could lead to different arrangements of the fibrinogen molecule.…”

Application of fibrin-based hydrogels for nerve protection and regeneration after spinal cord injury

“…The incomplete knowledge of the B–b interaction function reaches an agreement that closely relates to the lateral aggregation of fibrin and the stability after polymerization [ 56 ]. The mutual binding of αC domains of adjacent molecules is thought to promote the lateral aggregation of the protofibrils, and even the weak binding force does not play a decisive role [ 57 , 58 ]. Protofibrils finally form a branched 3D network gel structure through longitudinal extension and associate laterally.…”