Characterization of the inhibition of fibrin assembly by fibrinogen fragment D.
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ABSTRACT:
SUBMITTER: Williams JE
PROVIDER: S-EPMC1163178 | biostudies-other | 1981 Sep
REPOSITORIES: biostudies-other
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Project description:Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the highly pathogenic and highly transmissible human coronavirus that is the causative agent for the worldwide COVID-19 pandemic. COVID-19 manifests predominantly as a respiratory illness with symptoms consistent with viral pneumonia, but other organ systems (e.g., kidney, heart, brain) can also become perturbed in COVID-19 patients. Accumulating data suggest that significant activation of the hemostatic system is a common pathological manifestation of SARS-CoV-2 infection. The clotting protein fibrinogen is one of the most abundant plasma proteins. Following activation of coagulation, the central coagulation protease thrombin converts fibrinogen to fibrin monomers, which selfassemble to form a matrix, the primary structural component of the blood clot. Severe COVID-19 is associated with a profound perturbation of circulating fibrinogen, intra- and extravascular fibrin deposition and persistence, and fibrin degradation. Current findings suggest high levels of fibrinogen and the fibrin degradation product D-dimer are biomarkers of poor prognosis in COVID-19. Moreover, emerging studies with in vitro and animal models indicate fibrin(ogen) as an active player in COVID-19 pathogenesis. Here, we review the current literature regarding fibrin(ogen) and COVID-19, including possible pathogenic mechanisms and treatment strategies centered on clotting and fibrin(ogen) function.
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Project description:Liquid fibrinogen is an injectable platelet concentrate rich in platelets, leukocytes, and fibrinogen obtained by blood centrifugation. The aim of this study was to analyze the release of different growth factors in the liquid fibrinogen at different times and to assess possible correlations between growth factors and cell counts. The concentration of transforming growth factor beta 1 (TGF-?1), platelet-derived growth factor-AB (PDGF-AB), platelet-derived growth factor-BB (PDGF-BB), bone morphogenetic protein 2 (BMP-2), fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF) released by liquid fibrinogen were examined with ELISA at three time points (T0, time of collection; T7, 7 days; T14, 14 days). The cellular content of the liquid fibrinogen and whole blood was also calculated for each volunteer. A mean accumulation of platelets of almost 1.5-fold in liquid fibrinogen compared to whole blood samples was found. An increase of TGF-?1, PDGF-AB, FGF-2, and VEGF levels was detected at T7. At T14, the level of TGF-?1 returned to T0 level; PDGF-AB amount remained high; the levels of FGF-2 and VEGF decreased with respect to T7, but remained higher than the T0 levels; PDGF-BB was high at all time points; BMP-2 level was low and remained constant at all time points. TGF-?1, PDGF-AB, and PDGF-BB showed a correlation with platelet amount, whereas BMP-2, FGF-2, and VEGF showed a mild correlation with platelet amount. Due to the high concentration of platelets, liquid fibrinogen does contain important growth factors for the regeneration of both soft and hard tissue. The centrifugation protocol tested in this study provides a valid solution to stimulate wound healing in oral and periodontal surgery.
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Project description:BackgroundThrombin activates fibrinogen and binds the fibrin E-domain (Kd ~ 2.8 μM) and the splice variant γ'-domain (Kd ~ 0.1 μM). We investigated if the loading of D-Phe-Pro-Arg-chloromethylketone inhibited thrombin (PPACK-thrombin) onto fibrin could enhance fibrin stability.MethodsA 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin.ResultsLarge increases in Tm indicated that calcium led to protein stabilization (0 vs. 2 mM Ca2+) for fibrinogen (54.0 vs. 62.3 °C) and fibrin (62.3 vs. 72.2 °C). Additionally, active site inhibition with PPACK dramatically increased the Tm of thrombin (58.3 vs. 78.3 °C). Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability by ΔTm = 9.3 °C, similar to the ΔTm when fibrinogen was converted to fibrin monomer (ΔTm = 8.8 °C) or to fibrin (ΔTm = 10.4 °C). Addition of PPACK-thrombin at high 5:1 M ratio to fibrin(ogen) had little effect on fibrin(ogen) Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ'-domain interaction.ConclusionsTSA was a sensitive assay of protein stability and detected: (1) the effects of calcium-stabilization, (2) thrombin active site labeling, (3) fibrinogen conversion to fibrin, and (4) GPRP induced changes in fibrinogen stability being essentially equivalent to that of fibrin monomer or polymerized fibrin.SignificanceThe low volume, high throughput assay has potential for use in understanding interactions with rare or mutant fibrin(ogen) variants.
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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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