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Fibrinogen ?C-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 ?- and ?-chains N-termini (GPR, GHR; A-, B-knobs, respectively) engaging preexistent a and b pockets in other fibrin(ogen) molecules' ?- and (B)?-chains C-terminal regions. A role for mostly disordered (A)?-chains C-terminal regions "bridging" between fibrin molecules/fibrils has been proposed. OBJECTIVES:Fibrinogen Detroit is a clinically observed mutation (A?R19 ? S) with nonengaging GPS A-knobs. By analogy, a similar B?-chain mutation, B?R17 ? S, should produce nonengaging GHS B-knobs. A homozygous "Double-Detroit" mutant (A?R19 ? S, B?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 ~50% FpB was not recovered. Correspondingly, SDS-PAGE/Western-blots revealed partial B?-chain N-terminal and A?-chain C-terminal degradation. Nevertheless, ~70% DD-FG molecules bearing (A)?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)?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.

SUBMITTER: Duval C

PROVIDER: S-EPMC7186824 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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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.

Duval Cédric C Profumo Aldo A Aprile Anna A Salis Annalisa A Millo Enrico E Damonte Gianluca G Gauer Julia S JS Ariëns Robert A S RAS Rocco Mattia M

Journal of thrombosis and haemostasis : JTH 20200129 4

<h4>Background</h4>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.<h4>Objectives</h4>Fibrinogen Detroit is a clinically observed mutation (AαR19 → S) ...[more]

PMID: 31889430

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Project description:The fibrin polymers formed in solution during the earliest phase of the fibrinogen-fibrin conversion are shown to be stable soluble molecules at pH7.4 and 0.15m- or 0.3m-NaCl. The various sequential soluble fibrin polymers produced from the fibrinogen-thrombin reaction can be observed by gel chromatography and can be isolated for characterization. The mechanism of fibrin polymerization proposed from the present studies suggests that the initial event is the thrombin activation at only one of the Aalpha-chains in fibrinogen. The resulting highly reactive intermediate is the true fibrin monomer and it rapidly, and irreversibly, self-associates to form the stable fibrin dimer (s(20.w)=12S). Fibrin dimer possesses the N-terminal pattern alanine/glycine/tyrosine (1:1:2) per 340000 molecular weight, and possesses the chain structure [(alpha)Aalpha)(Bbeta)(2)(gamma)(2)](2). The fibrin dimer is a soluble inert molecule, but additional thrombin activation of its remaining intact Aalpha-chains leads to new associations into larger inert soluble fibrin polymers. In this manner progressively larger fibrin oligomers are constructed with thrombin continually in control of the process because of the necessity to repeatedly re-activate the various fibrin polymers in solution. The inert character of the soluble fibrin polymers can be explained by the reciprocal alignment of the associating molecules, which mutually consumes their active surfaces and leaves an intact Aalpha-chain at either end of each fibrin oligomer. The soluble fibrin polymers will proceed to further association only if thrombin activates these remaining Aalpha-chains, otherwise the fibrin molecules are stable indefinitely. The intermolecular associations within the soluble fibrin polymers are essentially irreversible under these nearly physiological conditions. However, the bonding is not covalent. This mechanism accounts for the clinical observations of stable fibrinogen-derived polymers in the plasma from patients undergoing thrombotic processes. Since it is shown that the intermediate fibrin polymers, themselves, are stable soluble molecules, it is no longer necessary, nor warranted, to invoke hypothetical ;fibrinogen-fibrin complexes' to explain observations of fibrin solubility.

Project description:Fibrin is a major component of the provisional extracellular matrix formed during tissue repair following injury, and enables cell infiltration and anchoring at the wound site. Macrophages are dynamic regulators of this process, advancing and resolving inflammation in response to cues in their microenvironment. Although much is known about how soluble factors such as cytokines and chemokines regulate macrophage polarization, less is understood about how insoluble and adhesive cues, specifically the blood coagulation matrix fibrin, influence macrophage behavior. In this study, we observed that fibrin and its precursor fibrinogen elicit distinct macrophage functions. Culturing macrophages on fibrin gels fabricated by combining fibrinogen with thrombin stimulated secretion of the anti-inflammatory cytokine, interleukin-10 (IL-10). In contrast, exposure of macrophages to soluble fibrinogen stimulated high levels of inflammatory cytokine tumor necrosis factor alpha (TNF-α). Macrophages maintained their anti-inflammatory behavior when cultured on fibrin gels in the presence of soluble fibrinogen. In addition, adhesion to fibrin matrices inhibited TNF-α production in response to stimulation with LPS and IFN-γ, cytokines known to promote inflammatory macrophage polarization. Our data demonstrate that fibrin exerts a protective effect on macrophages, preventing inflammatory activation by stimuli including fibrinogen, LPS, and IFN-γ. Together, our study suggests that the presentation of fibrin(ogen) may be a key switch in regulating macrophage phenotype behavior, and this feature may provide a valuable immunomodulatory strategy for tissue healing and regeneration.Statement of significanceFibrin is a fibrous protein resulting from blood clotting and provides a provisional matrix into which cells migrate and to which they adhere during wound healing. Macrophages play an important role in this process, and are needed for both advancing and resolving inflammation. We demonstrate that culture of macrophages on fibrin matrices exerts an anti-inflammatory effect, whereas the soluble precursor fibrinogen stimulates inflammatory activation. Moreover, culture on fibrin completely abrogates inflammatory signaling caused by fibrinogen or known inflammatory stimuli including LPS and IFN-γ. Together, these studies show that the presentation of fibrin(ogen) is important for regulating a switch between macrophage pro- and anti-inflammatory behavior.

Dataset Information

Fibrinogen ?C-regions are not directly involved in fibrin polymerization as evidenced by a "Double-Detroit" recombinant fibrinogen mutant and knobs-mimic peptides.

Publications

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

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