scholarly journals Impact of fibrinogen carbamylation on fibrin clot formation and stability

2017 ◽  
Vol 117 (05) ◽  
pp. 899-910 ◽  
Author(s):  
Stéphane Jaisson ◽  
Philippe Gillery ◽  
Carsten Scavenius ◽  
Endy Spriet ◽  
Anne Nyhaug ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Mechanical Stability ◽  
Inflammatory Diseases ◽  
Plasma Concentrations ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Cross Linking ◽  
Post Translational Modification ◽  
The Impact

SummaryCarbamylation is a non-enzymatic post-translational modification induced upon exposure of free amino groups to urea-derived cyanate leading to irreversible changes of protein charge, structure and function. Levels of carbamylated proteins increase significantly in chronic kidney disease and carbamylated albumin is considered as an important biomarker indicating mortality risk. High plasma concentrations and long half-life make fibrinogen a prime target for carbamylation. As aggregation and cross-linking of fibrin monomers rely on lysine residues, it is likely that carbamylation impacts fibrinogen processing. In this study we investigated carbamylation levels of fibrinogen from kidney disease patients as well as the impact of carbamylation on fibrinogen cleavage by thrombin, fibrin polymerisation and cross-linking in vitro. In conjunction, all these factors determine clot structure and stability and thus control biochemical and mechanical properties. LC-MS/MS analyses revealed significantly higher homocitrulline levels in patient fibrinogen than in fibrinogen isolated from control plasma. In our in vitro studies we found that although carbamylation does not affect thrombin cleavage per se, it alters fibrin polymerisation kinetics and impairs cross-linking and clot degradation. In addition, carbamylated fibrin clots had reduced fiber size and porosity associated with decreased mechanical stability. Using mass spectroscopy, we discovered that N-terminally carbamylated fibrinopeptide A was generated in this process and acted as a strong neutrophil chemoattractant potentially mediating recruitment of inflammatory cells to sites of fibrin(ogen) turnover. Taken together, carbamylation of fibrinogen seems to play a role in aberrant fibrin clot formation and might be involved in haemostatic disorders associated with chronic inflammatory diseases.

scholarly journals Staphylococcus epidermidis Has Growth Phase Dependent Affinity for Fibrinogen and Resulting Fibrin Clot Elasticity

2021 ◽  
Vol 12 ◽  
Author(s):  
Carolyn Vitale ◽  
Tianhui Maria Ma ◽  
Janice Sim ◽  
Christopher Altheim ◽  
Erika Martinez-Nieves ◽  
...  
Keyword(s):  
Bacterial Infection ◽  
Growth Phase ◽  
Staphylococcus Epidermidis ◽  
Fibrin Clot ◽  
Exponential Phase ◽  
Clot Formation ◽  
Coagulase Negative Staphylococci ◽  
Phase Dependence ◽  
The Impact

Bacterial infection and thrombosis are highly correlated, especially in patients with indwelling medical devices. Coagulase-negative staphylococci, typified by Staphylococcus epidermidis, are a common cause of medical device infections owing to their biofilm forming capacity which provides protection from antibiotics and host immune response. Attention has been drawn to the interaction between S. epidermidis and host proteins, specifically fibrinogen. However, little is known regarding the impact of the transition from planktonic to biofilm forming phenotype on this interaction. Here we investigate the growth phase dependence of bacteria-fibrinogen interaction and the resulting effect on fibrin clot formation, structure, and mechanics. Flow cytometry demonstrated growth phase dependent affinity for fibrinogen. To mimic intravascular device seeding, we quantified the adhesion of S. epidermidis to a fibrinogen coated surface under continuous flow conditions in vitro. The bacterial deposition rate onto fibrinogen was significantly greater for stationary (5,360 ± 1,776 cells/cm2s) versus exponential phase (2,212 ± 264, cells/cm2 s). Furthermore, the expression of sdrG–a cell surface adhesion protein with specificity for fibrinogen–was upregulated ∼twofold in the stationary versus the exponential phase. Rheometry and confocal microscopy demonstrated that stationary phase S. epidermidis slows clot formation and generates a more heterogeneous fibrin network structure with greater elasticity (G′ = 5.7 ± 1.0 Pa) compared to sterile fibrinogen (G′ = l.5 ± 0.2 Pa), while exponential phase cells had little effect. This work contributes to the current understanding of the growth phase dependent regulation of bacterial virulence factors and the correlation between bacterial infection and thrombosis.

scholarly journals An In Vivo Predictive Dissolution Methodology (iPD Methodology) with a BCS Class IIb Drug Can Predict the In Vivo Bioequivalence Results: Etoricoxib Products

Pharmaceutics ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 507
Author(s):  
Isabel Gonzalez-Alvarez ◽  
Marival Bermejo ◽  
Yasuhiro Tsume ◽  
Alejandro Ruiz-Picazo ◽  
Marta Gonzalez-Alvarez ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
In Vitro Dissolution ◽  
Case Examples ◽  
Dissolution Studies ◽  
Weak Bases ◽  
Transit Times ◽  
Class Iib ◽  
The Impact

The purpose of this study was to predict in vivo performance of three oral products of Etoricoxib (Arcoxia® as reference and two generic formulations in development) by conducting in vivo predictive dissolution with GIS (Gastro Intestinal Simulator) and computational analysis. Those predictions were compared with the results from previous bioequivalence (BE) human studies. Product dissolution studies were performed using a computer-controlled multicompartmental dissolution device (GIS) equipped with three dissolution chambers, representing stomach, duodenum, and jejunum, with integrated transit times and secretion rates. The measured dissolved amounts were modelled in each compartment with a set of differential equations representing transit, dissolution, and precipitation processes. The observed drug concentration by in vitro dissolution studies were directly convoluted with permeability and disposition parameters from literature to generate the predicted plasma concentrations. The GIS was able to detect the dissolution differences among reference and generic formulations in the gastric chamber where the drug solubility is high (pH 2) while the USP 2 standard dissolution test at pH 2 did not show any difference. Therefore, the current study confirms the importance of multicompartmental dissolution testing for weak bases as observed for other case examples but also the impact of excipients on duodenal and jejunal in vivo behavior.

scholarly journals Repeated social defeat exaggerates fibrin-rich clot formation in FeCl3-induced arterial thrombosis mouse model by enhancing NETs formation via modulation of neutrophil functional properties

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
T Sugimoto ◽  
H Yamada ◽  
H Kubota ◽  
D Miyawaki ◽  
M Saburi ◽  
...  
Keyword(s):  
Functional Properties ◽  
Arterial Thrombosis ◽  
Social Defeat ◽  
Arterial Injury ◽  
Physical Contact ◽  
Clot Formation ◽  
Positive Staining ◽  
Double Positive ◽  
The Impact

Abstract Background and objective Depression is an independent risk factor of cardiovascular disease (CVD). We have recently shown that repeated social defeat (RSD) precipitates depressive-like behaviors in apoE−/− mice and exaggerates atherosclerosis development by enhancing neutrophil extracellular traps (NETs) formation. Here, we investigated the impact of RSD on arterial thrombosis. Methods and results Eight-week-old male WT mice were exposed to RSD by housing with a larger CD-1 mouse in a shared home cage. They were subjected to vigorous physical contact daily for 10 consecutive days. Control mice were housed in the same gage without physical contact. After social interaction test to confirm depressive-like behaviors, defeated mice (19 of 31) and control mice (12 of 14) were underwent arterial injury at 10 wks of age. A filter paper saturated with 10% FeCl3 was applied on the adventitial surface of left carotid artery for 3 min and analyzed 3 hrs later. The volume of thrombi was comparable between the two groups. However, fibrinogen/fibrin-positive areas in immunofluorescent images significantly increased in defeated mice (27.8% vs. 48.8%, p<0.01). The number of Ly-6G-positive cells in thrombi was markedly higher in defeated mice (144/mm2 vs. 878/mm2, p<0.05). Further, Ly-6G-positive cells were almost accumulated at the inner surface of injured artery, which were co-localized with neutrophil elastase, Cit-H3, and CD41-positive staining. Treatment with DNase I completely diminished the exaggerated fibrin-rich clot formation in defeated mice to an extent similar to that in control mice (25.7% vs. 22.3%, p = ns), without affecting the volume of thrombi and accumulation of Ly-6G-positive cells. Given that platelet aggregations induced by ADP or collagen were comparable between the two groups, neutrophil functional properties primarily contribute to the exaggerated fibrin-rich clot formation in defeated mice. We then examined neutrophil subset and vulnerability to NETs formation. At 3 hrs after FeCl3 application, the numbers of immature neutrophils (Ly6Glo/+CXCR2-) were comparable between the two groups in both bone marrow (BM) and peripheral blood (PB). In contrast, the number of PB mature neutrophils (Ly6G+CXCR2+) was markedly higher in defeated mice than control mice (580±68 /μl vs. 1265±114, p<0.01). We next examined in vitro NETs formation upon PMA in BM mature neutrophils by FACS and nucleic acid staining. The percentage of double-positive cells (Cit-H3, MPO) was significantly higher in defeated mice (7.5% vs. 10.2%, p<0.05), as well as SYTOX green-positive cells expelling DNA fibers (8.1% vs. 11.8%, p<0.05). Conclusions Our findings demonstrate for the first time that repeated social defeat enhances fibrin-rich clot formation after arterial injury by enhancing NETs formation via modulation of neutrophil functional properties, suggesting that NETosis could be a new therapeutic target in depression-related CVD development. Funding Acknowledgement Type of funding source: None

Abstract 356: Adhesion of Staphylococcus Epidermidis to Fibrinogen Alters Clot Formation Kinetics and Ultimate Stiffness

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Carolyn Vitale ◽  
Tianhui Ma ◽  
Michael J Solomon ◽  
J. Scott VanEpps
Keyword(s):  
Stationary Phase ◽  
Deposition Rate ◽  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Fibrin Clot ◽  
Exponential Phase ◽  
Automated Image Analysis ◽  
Clot Formation ◽  
Coagulation Proteins

Bacterial infection is known to increase the risk for thromboembolism. The mechanism underlying this correlation remains largely unknown. We recently showed that the common pathogen Staphylococcus epidermidis retards clot formation, increases clot elasticity and generates a heterogeneous clot structure that remodels over time. Here, we elucidate the mechanism of this process by evaluating the capacity for S. epidermidis to bind to fibrinogen as a function of its growth phase. We hypothesized that the effect of S. epidermidis on a fibrin clot is related to its propensity toward biofilm formation. Therefore, stationary phase (biofilm-like) S. epidermidis will have a more robust effect on clot kinetics and elasticity than exponential phase (planktonic). Furthermore, this difference is mediated by increased adhesion to fibrinogen. Rheometry was used to evaluate the formation and resultant elasticity of fibrin clots with exponential or stationary phase S. epidermidis . A functional in vitro model was developed to evaluate adhesion of S. epidermidis to a fibrinogen coated surface in a continuously flowing environment. Fluorescent labeled exponential and stationary phase S. epidermidis were visualized flowing through a parallel plate microfluidic chamber past immobilized fibrinogen. Images were obtained every 3 seconds for 30 min. Bacterial deposition rate and mean adhesion time were quantified by automated image analysis. A paired Student’s t-test was used for statistical analysis. Stationary phase S. epidermidis retards clot formation and increases resultant elasticity while exponential phase only slightly reduces elasticity. The bacterial deposition rate onto fibrinogen was significantly (p=0.03) greater for stationary phase (1741 ± 1513 cells/cm 2 · sec -1 ) vs exponential phase (676 ± 270 cells/cm 2 · sec -1 ). The average adhesion time however was similar for exponential and stationary phase cells. Coagulation proteins can provide a framework for bacterial adhesion, biofilm formation and infection. In turn infected thrombi with (biofilm-like) bacteria are stiffer which correlates to more frequent bacterial binding to fibrinogen. This provides a potential molecular mechanism for infection mediated thromboembolic events.

Stabilization but no functional influence of HIF-1α expression in the intestinal epithelium during Salmonella Typhimurium infection

2022 ◽  
Author(s):  
Laura Robrahn ◽  
Aline Dupont ◽  
Sandra Jumpertz ◽  
Kaiyi Zhang ◽  
Christian H. Holland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intestinal Epithelium ◽  
Bacterial Infections ◽  
Inflammatory Diseases ◽  
Protein Stabilization ◽  
Inflammatory Factors ◽  
Intestinal Epithelial ◽  
Data Set ◽  
The Impact

The hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and to ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we could infer significant activation of HIF-1 after oral infection of mice with Salmonella Typhimurium. Immunohistochemistry and western blot analysis confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a -deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced non-canonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact on inflammatory gene expression, bacterial spread or disease outcome. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro , HIF-1α-deficient macrophages showed an overall impaired transcription of mRNA encoding pro-inflammatory factors, however, intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

Polyphosphate Enhances Fibrin Clot Structure.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 403-403
Author(s):  
Stephanie A. Smith ◽  
James H. Morrissey
Keyword(s):  
High Energy ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Length Ratio ◽  
Factor Xiiia ◽  
Clot Lysis ◽  
Cross Linking ◽  
Inorganic Polyphosphate ◽  
Calcium Dependent ◽  
Clot Structure

Abstract Introduction: Inorganic polyphosphate (polyP) is a negatively charged polymer of phosphate units linked by high energy phosphoanhydride bonds. Dense granules of human platelets contain polyP which is released in response to thrombin stimulation. We recently reported that polyphosphate is a potent hemostatic regulator, accelerating blood clotting by activating the contact pathway and promoting the activation of factor V. Our previous studies found that polyP did not affect the time to clot formation when plasma was clotted with thrombin, however, suggesting that polyP exerts its procoagulant actions upstream of thrombin. We now report that polyP enhances fibrin clot structure. Methods: Purified fibrinogen and polyP were preincubated for 15 min in multiwell plates in buffer containing CaCl2, after which clotting was initiated by adding 0.1 to 8 nM thrombin and fibrin clot formation was evaluated by quantifying the change in turbidity (A405). Mass-length ratios were calculated from scans of A400 to A800. The effect of polyP on fibrinolysis was examined by adding 8 nM plasmin to the reaction mixtures immediately prior to thrombin. Scanning electron microscopy (SEM) was employed to visualize clot structure, and time courses of covalent fibrin cross-linking were assessed by SDS-PAGE. Results: PolyP had no effect on time to clot formation, but clots formed in the presence of polyP had markedly (up to threefold) higher turbidity than clots formed in the absence of polyP (see figure), irrespective of thrombin concentration. The increased turbidity in the presence of polyP was calcium-dependent and was enhanced when fibrinogen, CaCl2, and polyP were preincubated for up to 15 min prior to initiation of clotting with thrombin. PolyP increased the mass-length ratio of fibrin, and SEM confirmed that fibers formed with polyP were thicker than those formed without polyP. The ability of polyP to enhance fibrin clot turbidity was independent of factor XIIIa activity, and polyP did not alter the rate or extent of covalent fibrin cross-linking by factor XIIIa. When plasmin was included in clotting reactions containing polyP, mean times to 50% clot lysis were 28.5 ± 0.8 min for clots without polyP but 120.4 ± 5.6 min for clots with polyP. Conclusions: PolyP alters polymerization of fibrin, resulting in fibers of higher mass-length ratio that are lysed more slowly. This effect is calcium-dependent and is enhanced by preincubation of fibrinogen with calcium and polyP. Release of polyP from activated platelets or infectious microorganisms may therefore enhance fibrin clot structure. Figure Figure

scholarly journals OLT1177, a β-sulfonyl nitrile compound, safe in humans, inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation

2018 ◽  
Vol 115 (7) ◽  
pp. E1530-E1539 ◽  
Author(s):  
Carlo Marchetti ◽  
Benjamin Swartzwelter ◽  
Fabia Gamboni ◽  
Charles P. Neff ◽  
Katrin Richter ◽  
...  
Keyword(s):  
Human Blood ◽  
Nlrp3 Inflammasome ◽  
Inflammatory Diseases ◽  
Plasma Concentrations ◽  
Metabolic Cost ◽  
Inflammasome Activation ◽  
Potassium Efflux ◽  
Caspase 1 ◽  
Healthy Humans

Activation of the NLRP3 inflammasome induces maturation of IL-1β and IL-18, both validated targets for treating acute and chronic inflammatory diseases. Here, we demonstrate that OLT1177, an orally active β-sulfonyl nitrile molecule, inhibits activation of the NLRP3 inflammasome. In vitro, nanomolar concentrations of OLT1177 reduced IL-1β and IL-18 release following canonical and noncanonical NLRP3 inflammasome activation. The molecule showed no effect on the NLRC4 and AIM2 inflammasomes, suggesting specificity for NLRP3. In LPS-stimulated human blood-derived macrophages, OLT1177 decreased IL-1β levels by 60% and IL-18 by 70% at concentrations 100-fold lower in vitro than plasma concentrations safely reached in humans. OLT1177 also reduced IL-1β release and caspase-1 activity in freshly obtained human blood neutrophils. In monocytes isolated from patients with cryopyrin-associated periodic syndrome (CAPS), OLT1177 inhibited LPS-induced IL-1β release by 84% and 36%. Immunoprecipitation and FRET analysis demonstrated that OLT1177 prevented NLRP3-ASC, as well as NLRP3-caspase-1 interaction, thus inhibiting NLRP3 inflammasome oligomerization. In a cell-free assay, OLT1177 reduced ATPase activity of recombinant NLRP3, suggesting direct targeting of NLRP3. Mechanistically, OLT1177 did not affect potassium efflux, gene expression, or synthesis of the IL-1β precursor. Steady-state levels of phosphorylated NF-κB and IkB kinase were significantly lowered in spleen cells from OLT1177-treated mice. We observed reduced IL-1β content in tissue homogenates, limited oxidative stress, and increased muscle oxidative metabolism in OLT1177-treated mice challenged with LPS. Healthy humans receiving 1,000 mg of OLT1177 daily for 8 d exhibited neither adverse effects nor biochemical or hematological changes.

scholarly journals Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma

2020 ◽  
Author(s):  
Shejuan An ◽  
Jeannie Camarillo ◽  
Tina Huang ◽  
Daphne Li ◽  
Juliette Morris ◽  
...  
Keyword(s):  
Cell Lines ◽  
Radiation Treatment ◽  
Diffuse Intrinsic Pontine Glioma ◽  
Response To Treatment ◽  
Wild Type ◽  
Post Translational Modification ◽  
Pontine Glioma ◽  
Epigenetic Signatures ◽  
The Impact

Abstract Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n=12) and cell lines (n=7).Results: We found 29.2% and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro. Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.

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