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Inhibited thrombins. Interactions with fibrinogen and fibrin.

ABSTRACT: Fibrin-monomer-Sepharose was used to study thrombin binding to fibrin and the role of the enzyme active centre in this interaction. Binding properties of preformed enzyme-inhibitor complexes, as well as inhibition of thrombin already adsorbed to fibrin monomer, were investigated. No apparent difference was found in binding properties of phenylmethanesulphonyl fluoride-, D-Phe-Pro-Arg-CH2Cl- and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide-inhibited thrombins. Also, the elution profile of phenylmethane-sulphonyl fluoride-inhibited thrombin from fibrinogen-Sepharose was identical with that of active thrombin from fibrin-monomer-Sepharose. Thus far the only low-Mr inhibitor that prevents thrombin from binding to fibrin monomer is pyridoxal 5'-phosphate. Preformed hirudin-thrombin complexes do not interact with fibrin. The extent to which the active centre of thrombin associated with fibrin is still accessible to substrates and inhibitors was also studied. Thrombin bound to fibrin hydrolyses a synthetic substrate at the same rate as the free enzyme. Water-soluble low-Mr inhibitors such as D-Phe-Pro-Arg-CH2Cl and dansylarginine NN-(3-ethylpentane-1,5-diyl)amide can readily modify the active centre of the fibrin-associated enzyme, and the active centre is exposed to the degree that displacement of dansylarginine NN-(3-ethylpentane-1,5-diyl)amide by D-Phe-Pro-Arg-CH2Cl is possible without disturbing the binding. Hirudin disrupts the affinity between thrombin and fibrin. These data indicate that the active centre of thrombin associated with fibrin through extended binding is fully exposed and freely accessible. It is possible that extended binding may play a regulatory role in the activation of Factor XIII by thrombin, as well as inactivation of this enzyme by antithrombin III.

SUBMITTER: Kaminski M

PROVIDER: S-EPMC1147791 | biostudies-other | 1987 Mar

REPOSITORIES: biostudies-other

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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.

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Inhibited thrombins. Interactions with fibrinogen and fibrin.

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