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1.
Platelets ; 34(1): 2139365, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36325627

RESUMO

Activated platelets possess procoagulant activity expressing on their surface phosphatidylserine (PS), a substrate for assembling coagulation complexes. We examined the effects of platelets activated by different agonists on fibrin formation and thrombin generation and compared these effects with each other and with PS expression. Modified plasma recalcification assay was developed to assess platelet effects on fibrin formation. Washed human platelets were left intact or activated by A23187 ionophore, collagen, arachidonic acid, ADP or TRAP (Thrombin Receptor Activating Peptide) and spun down in 96-well plates. Plasma was then added, recalcified, and fibrin formation was monitored by light absorbance. Platelets prepared in the same way were tested for their effect on thrombin generation. PS expression was evaluated by flow cytometry using annexin V staining. Platelets significantly accelerated fibrin formation and thrombin generation. They shortened lag phase and increased maximum rate of plasma clotting, and increased peak and maximum rate of thrombin generation. In both tests platelets were presumably activated by endogenous thrombin formed in plasma after triggering coagulation reactions. However, pretreatment with exogenous agonists additionally increased platelet procoagulant activity. It reached the maximum after incubation with A23187, being lower with collagen and arachidonic acid and minimum with ADP and TRAP (the latter might be ineffective due to competition with endogenous thrombin). The effects of platelets activated by different agonists on fibrin formation and thrombin generation correlate with each other and correspond to PS expression on their surface.


Why was the study done? Platelets and blood coagulation system interact with each other in hemostasis and intravascular thrombosis.Direct platelet effects on fibrin formation (plasma clotting), the final stage of blood coagulation cascade, have been insufficiently studied.The work is aimed at developing a method for studying platelet participation in fibrin formation in blood plasma and investigating the influence of platelet agonists on this reaction.What is new? Platelets significantly accelerate fibrin formation and their activation with various agonists (thrombin, collagen, arachidonic acid) enhances these effects.Effects of platelets on fibrin formation correlated with their ability to stimulate thrombin generation in blood plasmaEffects of platelets on fibrin formation and thrombin generation correlated with the level of phosphatidylserine exposure on their surfaceWhat is the impact? This study provides further evidence that platelet procоagulant effects on fibrin formation should be considered in investigations of platelet involvement in hemostatic and thrombotic reactions and in the evaluation of the efficacy of antiplatelet drugs.


Assuntos
Plaquetas , Trombina , Humanos , Trombina/farmacologia , Trombina/metabolismo , Plaquetas/metabolismo , Fibrina/metabolismo , Calcimicina/metabolismo , Calcimicina/farmacologia , Ácido Araquidônico/farmacologia , Ácido Araquidônico/metabolismo , Fosfatidilserinas/metabolismo , Colágeno/farmacologia , Colágeno/metabolismo , Difosfato de Adenosina/farmacologia , Difosfato de Adenosina/metabolismo
2.
Int J Mol Sci ; 23(21)2022 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-36361742

RESUMO

Transglutaminase (TGM) isoform catalyze the cross-linking reaction of identical or different substrate proteins. Eosinophil has been recognized in chronic rhinosinusitis with nasal polyps (CRSwNP) forming tissue eosinophil in nasal polyp (NP), and TGM isoforms are suggested to be associated with a critical role in asthma and other allergic conditions. The aim of this study was to reveal the association of specific TGM isoform with both the tissue eosinophil infiltration deeply concerning with the intractable severity of CRSwNP and the fibrin polymerization ability of TGM isoform associated with the tissue eosinophil infiltration, which lead to NP formation and/or maintenance in CRSwNP. NP tissues (CRSwNP group) and uncinate process (UP) (control group) were collected from patients with CRSwNP and control subjects. We examined: (1) the expression level of TGM isoforms by using a real-time polymerase chain reaction (PCR) and the comparison to the issue eosinophil count in the CRSwNP group, (2) the location of specific TGM isoform in the mucosal tissue using immunohistochemistry, (3) the inflammatory cell showing the colocalization of specific TGM isoform in Laser Scanning Confocal Microscopy (LSCM) imaging, and (4) the fibrin polymerase activity of specific TGM isoform using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A certain level of TGM 1, 2, 3, 5 expression was present in both the CRSwNP group and the control group. Only TGM 1 expression showed a positive significant correlation with the tissue eosinophil count in the CRSwNP group. The localization of TGM 1 in NP (CRSwNP) laid mainly in a submucosal layer as inflammatory cells and was at the cytoplasm in the tissue eosinophil. Fibrin polymerase activity of TGM 1 showed the same polymerase ability of factor XIIIA. TGM 1 might influence the NP formation and/or maintenance in CRSwNP related to the tissue eosinophil infiltration, which formed fibrin mesh composing NP stroma.


Assuntos
Pólipos Nasais , Rinite , Sinusite , Humanos , Pólipos Nasais/patologia , Eosinófilos/metabolismo , Rinite/patologia , Fibrina/metabolismo , Polimerização , Sinusite/metabolismo , Transglutaminases/genética , Transglutaminases/metabolismo , Doença Crônica
3.
Sci Rep ; 12(1): 18829, 2022 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-36335251

RESUMO

Intramuscular injection of anemoside B4 (AB4) has a superior therapeutic effect on clinical mastitis in lactating cows. Here, we explored AB4's effect on milk whey in clinical mastitis-affected cows using proteomics. Among fifty clinical mastitis cows received AB4 administration (0.05 ml/kg/day, for 7 days), twelve healed cows were selected and marked as group T. Twelve clinically heathy cows received the same dose of saline for 7 days, marked as group C. Collected milk whey of group T before and after AB4 administration marked as T1 and T2, respectively. The milk whey of group C after saline injection marked as C1. Milk whey protein changes were detected using tandem mass tag-based quantitative proteomic. We identified 872 quantifiable proteins in the samples. Among them, 511 proteins between T1 and C1, and 361 proteins between T2 and T1 were significantly altered. T1 than C1 had significantly more proteins associated with inflammatory damage and trans-endothelial migration of leukocytes, whereas these proteins were reduced in T2 treated with AB4. Compared with C, proteins associated with fibrin clot degradation and complement system activation were downregulated in T1 but upregulated in T2. In summary, AB4 can exert its therapeutic effect on clinical mastitis in cows mainly by reducing inflammatory damage, activating the complement system, inhibiting trans-endothelial migration of leukocytes, and promoting degradation of milk fibrin clots.


Assuntos
Mastite Bovina , Leite , Animais , Bovinos , Feminino , Fibrina/metabolismo , Lactação , Mastite Bovina/tratamento farmacológico , Mastite Bovina/metabolismo , Leite/metabolismo , Proteínas do Leite/metabolismo , Proteômica , Soro do Leite/metabolismo , Proteínas do Soro do Leite/farmacologia , Proteínas do Soro do Leite/metabolismo
4.
PLoS One ; 17(10): e0275956, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36301961

RESUMO

Fibrin clot structure/function contributes to cardiovascular disease. We examined sulfur-containing metabolites as determinants of fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to outcomes in coronary artery disease (CAD) patients. Effects of B-vitamin/folate therapy on CLT and Absmax were studied. Plasma samples were collected from 1,952 CAD patients randomized in a 2 x 2 factorial design to (i) folic acid, vitamins B12, B6; (ii) folic acid, vitamin B12; (iii) vitamin B6; (iv) placebo for 3.8 years in the Western Norway B-Vitamin Intervention Trial. Clot lysis time (CLT) and maximum absorbance (Absmax) were determined using a validated turbidimetric assay. Acute myocardial infarction (AMI) and mortality were assessed during a 7-year follow-up. Data were analyzed using bivariate and multiple regression. Survival free of events was studied using Kaplan Mayer plots. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox proportional hazards models. Baseline urinary homocysteine (uHcy)-thiolactone and plasma cysteine (Cys) were significantly associated with CLT while plasma total Hcy was significantly associated with Absmax, independently of fibrinogen, triglycerides, vitamin E, glomerular filtration rate, body mass index, age, sex plasma creatinine, CRP, HDL-C, ApoA1, and previous diseases. B-vitamins/folate did not affect CLT and Absmax. Kaplan-Meier analysis showed associations of increased baseline CLT and Absmax with worse outcomes. In Cox regression analysis, baseline CLT and Absmax (>cutoff) predicted AMI (CLT: HR 1.58, 95% CI 1.10-2.28; P = 0.013. Absmax: HR 3.22, CI 1.19-8.69; P = 0.021) and mortality (CLT: HR 2.54, 95% CI 1.40-4.63; P = 0.002. Absmax: 2.39, 95% CI 1.17-4.92; P = 0.017). After adjustments for other prognostic biomarkers these associations remained significant. Cys and uHcy-thiolactone, but not tHcy, were significant predictors of AMI in Cox regression models that included CLT. Conclusions uHcy-thiolactone and plasma Cys are novel determinants of CLT, an important predictor of adverse CAD outcomes. CLT and Absmax were not affected by B-vitamin/folate therapy, which could account for the lack of efficacy of such therapy in CAD. Trial registration: URL: http://clinicaltrials.gov. Identifier: NCT00354081.


Assuntos
Doença da Artéria Coronariana , Infarto do Miocárdio , Trombose , Complexo Vitamínico B , Humanos , Fibrina/metabolismo , Prognóstico , Vitamina B 12 , Ácido Fólico , Homocisteína , Infarto do Miocárdio/tratamento farmacológico , Trombose/tratamento farmacológico , Fatores de Risco
5.
Int J Mol Sci ; 23(20)2022 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-36293273

RESUMO

INTRODUCTION: The in vitro culture of primordial follicles is the only available option for preserving fertility in prepubertal girls with malignant tumors. The cultivation of primordial follicles in scaffolds as artificial ovaries is a promising approach for this. METHODS: Dissociated follicles were placed into an artificial ovarian scaffold composed of fibrinogen and thrombin. The follicles were cultured in a dish dedicated to live cell imaging and observed for growth using immunofluorescence and development via optical microscopy. The morphology of the follicles in the scaffold was three-dimensionally reconstructed using the Imaris software. Growth and development were also quantified. RESULTS: The morphology of artificial ovaries began to degrade over time. Within approximately 7 days, primordial follicles were activated and grew into secondary follicles. A comparison of optical and confocal microscopy results revealed the superior detection of live cells using confocal microscopy. The three-dimensional reconstruction of the confocal microscopy data enabled the automatic enumeration and evaluation of the overall morphology of many follicles. CONCLUSIONS: The novel artificial ovary-enabled primordial follicles to enter the growth cycle after activation and grow into secondary follicles. The use of a fibrin scaffold as a carrier preserves the developmental potential of primordial germ cells and is a potentially effective method for preserving fertility in prepubertal girls.


Assuntos
Preservação da Fertilidade , Neoplasias , Humanos , Feminino , Ovário/metabolismo , Preservação da Fertilidade/métodos , Trombina/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Bioengenharia , Neoplasias/metabolismo
6.
Int J Mol Sci ; 23(20)2022 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-36293367

RESUMO

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.


Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas , Trombose , Camundongos , Animais , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Inibidores da Agregação Plaquetária/metabolismo , Actomiosina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Plaquetas/metabolismo , Trombose/metabolismo , Fibrinogênio/metabolismo , Proteína Quinase C/metabolismo , Difosfato de Adenosina/metabolismo , Fibrina/metabolismo , Fosfatidilinositóis/metabolismo
7.
Int J Mol Sci ; 23(19)2022 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-36232775

RESUMO

Quickly developing precision medicine and patient-oriented treatment strategies urgently require novel technological solutions. The randomly cell-populated scaffolds usually used for tissue engineering often fail to mimic the highly anisotropic characteristics of native tissue. In this work, an ultrasound standing-wave-based tissue engineering acoustophoretic (TEA) set-up was developed to organize murine mesenchymal stromal cells (mMSCs) in an in situ polymerizing 3-D fibrin hydrogel. The resultant constructs, consisting of 17 cell layers spaced at 300 µm, were obtained by continuous wave ultrasound applied at a 2.5 MHz frequency. The patterned mMSCs preserved the structured behavior within 10 days of culturing in osteogenic conditions. Cell viability was moderately increased 1 day after the patterning; it subdued and evened out, with the cells randomly encapsulated in hydrogels, within 21 days of culturing. Cells in the structured hydrogels exhibited enhanced expression of certain osteogenic markers, i.e., Runt-related transcription factor 2 (RUNX2), osterix (Osx) transcription factor, collagen-1 alpha1 (COL1A1), osteopontin (OPN), osteocalcin (OCN), and osteonectin (ON), as well as of certain cell-cycle-progression-associated genes, i.e., Cyclin D1, cysteine-rich angiogenic inducer 61 (CYR61), and anillin (ANLN), when cultured with osteogenic supplements and, for ANLN, also in the expansion media. Additionally, OPN expression was also augmented on day 5 in the patterned gels cultured without the osteoinductive media, suggesting the pro-osteogenic influence of the patterned cell organization. The TEA set-up proposes a novel method for non-invasively organizing cells in a 3-D environment, potentially enhancing the regenerative properties of the designed anisotropic constructs for bone healing.


Assuntos
Células-Tronco Mesenquimais , Osteogênese , Animais , Diferenciação Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ciclina D1/metabolismo , Cisteína/metabolismo , Fibrina/metabolismo , Humanos , Hidrogéis/metabolismo , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Osteocalcina/metabolismo , Osteonectina/metabolismo , Osteopontina/metabolismo , Engenharia Tecidual/métodos , Tecidos Suporte
8.
Int J Implant Dent ; 8(1): 39, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36184700

RESUMO

PURPOSE: To compare the release of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF-I) and interleukin 1ß (IL-1ß) of plasma rich in growth factors (PRGF) and leucocyte platelet-rich fibrin (L-PRF) and to evaluate their biological implication in osteoblasts. METHODS: Blood from 3 healthy volunteers was processed into PRGF, immediate L-PRF (L-PRF 0') and L-PRF 30 min after collection (L-PRF-30') and a control group. Growth factors release were analyzed at 7 times by ELISA. Cell proliferation, collagen-I synthesis and alkaline phosphatase activity were assessed in primary cultures of human osteoblasts. RESULTS: A slower controlled release of IGF-I, VEGF and PDGF was observed in the PRGF group at day 14. A higher synthesis of type I collagen was also quantified in PRGF. L-PRF released significantly higher amounts of IL-1ß, that was almost absent in the PRGF. CONCLUSIONS: The addition of leukocytes dramatically increases the secretion of proinflammatory cytokines, which are likely to negatively influence the synthesis of type I collagen and alkaline phosphatase (ALP) by osteoblasts.


Assuntos
Fibrina Rica em Plaquetas , Fosfatase Alcalina/metabolismo , Colágeno Tipo I/metabolismo , Citocinas/metabolismo , Preparações de Ação Retardada/metabolismo , Fibrina/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Interleucina-1beta/metabolismo , Leucócitos , Osteoblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fibrina Rica em Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
9.
Int J Numer Method Biomed Eng ; 38(11): e3652, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36167948

RESUMO

Fibrin is an important product of the coagulation cascade, and plays an eminent role in platelet stabilization. Since coagulation cascade models typically involve the reaction kinetics of dozens of proteins, which will incur burdensome computational costs when coupled to blood flow in complex geometries, researchers often ignore this process when constructing thrombosis models. However, previous studies have shown that fundamental aspects of coagulation can be reproduced with simpler models, which motivated us to obtain a reduced-order model of fibrin generation through a systematic approach. Therefore, we introduced a semi-automatic framework to perform model-reduction of cascade reactions in this study, which consisted of two processes. Specifically, the retained protein species and cascade reactions were determined based on published studies and simulation results from the full cascade model, while the optimal reaction rates for the new cascade network were determined using a genetic algorithm. The framework has been applied to a 19-species coagulation model that triggers fibrin generation in internal fields via reactive boundaries, and a 10-species reduced-order model was obtained to reproduce the kinetics of fibrinogenesis in the full cascade model at different boundary tissue factor concentrations. This reduced-order model of fibrinogenesis would be valuable for thrombosis modeling that considers both the coagulation cascade and platelet activity. Furthermore, the framework proposed herein can also be applied to the reductions of other cascade reaction models.


Assuntos
Coagulação Sanguínea , Trombose , Humanos , Coagulação Sanguínea/fisiologia , Fibrina/metabolismo , Plaquetas/metabolismo , Algoritmos
10.
J Thromb Haemost ; 20(12): 2873-2886, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36111375

RESUMO

BACKGROUND: Obesity predisposes individuals to metabolic syndrome, which increases the risk of cardiovascular diseases, non-alcoholic fatty liver disease (NAFLD), and type 2 diabetes. A pathological manifestation of obesity is the activation of the coagulation system. In turn, extravascular fibrin(ogen) deposits accumulate in adipose tissues and liver. These deposits promote adiposity and downstream sequelae by driving pro-inflammatory macrophage function through binding the leukocyte integrin receptor αM ß2 . OBJECTIVES: An unresolved question is whether conversion of soluble fibrinogen to a crosslinked fibrin matrix is required to exacerbate obesity-driven diseases. METHODS: Here, fibrinogen-deficient/depleted mice (Fib- or treated with siRNA against fibrinogen [siFga]), mice expressing fibrinogen that cannot polymerize to fibrin (FibAEK ), and mice deficient in the fibrin crosslinking transglutaminase factor XIII (FXIII-) were challenged with a high-fat diet (HFD) and compared to mice expressing a mutant form of fibrinogen lacking the αM ß2 -binding domain (Fib𝛾390-396A ). RESULTS AND CONCLUSIONS: Consistent with prior studies, Fib𝛾390-396A mice were significantly protected from increased adiposity, NAFLD, hypercholesterolemia, and diabetes while Fib- and siFga-treated mice gained as much weight and developed obesity-associated pathologies identical to wildtype mice. FibAEK and FXIII- mice displayed an intermediate phenotype with partial protection from some obesity-associated pathologies. Results here indicate that fibrin(ogen) lacking αM ß2 binding function offers substantial protection from obesity and associated disease that is partially recapitulated by preventing fibrin polymer formation or crosslinking of the wildtype molecule, but not by reduction or complete elimination of fibrinogen. Finally, these findings support the concept that fibrin polymerization and crosslinking are required for the full implementation of fibrin-driven inflammation in obesity.


Assuntos
Afibrinogenemia , Diabetes Mellitus Tipo 2 , Hemostáticos , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Fibrina/metabolismo , Polímeros , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Fibrinogênio/genética , Fibrinogênio/metabolismo , Fator XIII/metabolismo , Obesidade , Dieta
11.
Int J Mol Sci ; 23(17)2022 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-36077472

RESUMO

The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.


Assuntos
Fator de Iniciação 4E em Eucariotos , Peixe-Zebra , Animais , Proliferação de Células , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fibrina/metabolismo , Coração , Miócitos Cardíacos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
12.
Int J Mol Sci ; 23(18)2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36142125

RESUMO

Platelet and coagulation activation are highly reciprocal processes driven by multi-molecular interactions. Activated platelets secrete several coagulation factors and expose phosphatidylserine, which supports the activation of coagulation factor proteins. On the other hand, the coagulation cascade generates known ligands for platelet receptors, such as thrombin and fibrin. Coagulation factor (F)Xa, (F)XIIIa and activated protein C (APC) can also bind to platelets, but the functional consequences are unclear. Here, we investigated the effects of the activated (anti)coagulation factors on platelets, other than thrombin. Multicolor flow cytometry and aggregation experiments revealed that the 'supernatant of (hirudin-treated) coagulated plasma' (SCP) enhanced CRP-XL-induced platelet responses, i.e., integrin αIIbß3 activation, P-selectin exposure and aggregate formation. We demonstrated that FXIIIa in combination with APC enhanced platelet activation in solution, and separately immobilized FXIIIa and APC resulted in platelet spreading. Platelet activation by FXIIIa was inhibited by molecular blockade of glycoprotein VI (GPVI) or Syk kinase. In contrast, platelet spreading on immobilized APC was inhibited by PAR1 blockade. Immobilized, but not soluble, FXIIIa and APC also enhanced in vitro adhesion and aggregation under flow. In conclusion, in coagulation, factors other than thrombin or fibrin can induce platelet activation via GPVI and PAR receptors.


Assuntos
Selectina-P , Glicoproteínas da Membrana de Plaquetas , Plaquetas/metabolismo , Fator XIIIa/metabolismo , Fibrina/metabolismo , Hirudinas/metabolismo , Hirudinas/farmacologia , Selectina-P/metabolismo , Fosfatidilserinas/metabolismo , Ativação Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteína C/metabolismo , Receptor PAR-1/metabolismo , Quinase Syk/metabolismo , Trombina/metabolismo , Trombina/farmacologia
13.
PLoS Comput Biol ; 18(9): e1010414, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36107837

RESUMO

Thrombin is an enzyme produced during blood coagulation that is crucial to the formation of a stable clot. Thrombin cleaves soluble fibrinogen into fibrin, which polymerizes and forms an insoluble, stabilizing gel around the growing clot. A small fraction of circulating fibrinogen is the variant γA/γ', which has been associated with high-affinity thrombin binding and implicated as a risk factor for myocardial infarctions, deep vein thrombosis, and coronary artery disease. Thrombin is also known to be strongly sequestered by polymerized fibrin for extended periods of time in a way that is partially regulated by γA/γ'. However, the role of γA/γ'-thrombin interactions during fibrin polymerization is not fully understood. Here, we present a mathematical model of fibrin polymerization that considered the interactions between thrombin, fibrinogen, and fibrin, including those with γA/γ'. In our model, bivalent thrombin-fibrin binding greatly increased thrombin residency times and allowed for thrombin-trapping during fibrin polymerization. Results from the model showed that early in fibrin polymerization, γ' binding to thrombin served to localize the thrombin to the fibrin(ogen), which effectively enhanced the enzymatic conversion of fibrinogen to fibrin. When all the fibrin was fully generated, however, the fibrin-thrombin binding persisted but the effect of fibrin on thrombin switched quickly to serve as a sink, essentially removing all free thrombin from the system. This dual role for γ'-thrombin binding during polymerization led to a paradoxical decrease in trapped thrombin as the amount of γ' was increased. The model highlighted biochemical and biophysical roles for fibrin-thrombin interactions during polymerization and agreed well with experimental observations.


Assuntos
Fibrina , Trombina , Fibrina/metabolismo , Fibrinogênio/metabolismo , Modelos Teóricos , Polimerização , Trombina/metabolismo
14.
Clin Epigenetics ; 14(1): 117, 2022 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-36127710

RESUMO

BACKGROUND: DNA 5-hydroxymethylcytosine (5hmC) is produced by dynamic 5mC oxidation process contributing to tissue specification, and loss of 5hmC has been reported in multiple cancers including genitourinary cancers. However, 5hmC is also cell-type specific, and its variability may exist between differentiated tumor cells and cancer stem cells. Thus, cancer-associated changes in 5hmC may be contributed by distinct sets of tumor cells within the tumor tissues. RESULTS: Here, we applied a sensitive immunoprecipitation-based method (hMeDIP-seq) to analyze 5hmC changes during genitourinary carcinogenesis (including prostate, urothelial and kidney). We confirmed the tissue-specific distribution of 5hmC in genitourinary tissues and identified regional gain and global loss of 5hmC coexisting in genitourinary cancers. The genes with gain of 5hmC during tumorigenesis were functionally enriched in regulating stemness and hypoxia, whereas were associated with poor clinical prognosis irrespective of their differences in tumor type. We identified that gain of 5hmC occurred in soft fibrin gel-induced 3D tumor spheres with a tumor-repopulating phenotype in two prostate cancer cell lines, 22RV1 and PC3, compared with conventional two-dimensional (2D) rigid dishes. Then, we defined a malignant signature derived from the differentially hydroxymethylated regions affected genes of cancer stem-like cells, which could predict a worse clinical outcome and identified phenotypically malignant populations of cells from prostate cancer tumors. Notably, an oxidation-resistant vitamin C derivative, ascorbyl phosphate magnesium, restored 5hmC and killed the cancer stem cell-like cells leading to apoptosis in prostate cancer cell lines. CONCLUSIONS: Collectively, our study dissects the regional gain of 5hmC in maintaining cancer stem-like cells and related to poor prognosis, which provides proof of concept for an epigenetic differentiation therapy with vitamin C by 5hmC reprogramming.


Assuntos
Neoplasias da Próstata , Neoplasias Urogenitais , 5-Metilcitosina/análogos & derivados , Ácido Ascórbico/farmacologia , Carcinogênese , DNA/metabolismo , Metilação de DNA , Fibrina/metabolismo , Humanos , Magnésio , Masculino , Fosfatos , Neoplasias da Próstata/genética , Neoplasias Urogenitais/genética
15.
Cardiovasc Diabetol ; 21(1): 190, 2022 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-36131342

RESUMO

BACKGROUND: Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. METHODS: Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. RESULTS: Our long COVID cohort's symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. CONCLUSION: Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.


Assuntos
COVID-19 , Diabetes Mellitus Tipo 2 , Trombose , Biomarcadores , Proteína C-Reativa/metabolismo , COVID-19/complicações , Diabetes Mellitus Tipo 2/complicações , Fibrina/metabolismo , Fibrinogênio/metabolismo , Humanos , Interleucina-6 , Calicreína Plasmática , Fator Plaquetário 4 , Proteômica , Trombose/diagnóstico , alfa 2-Antiplasmina , Fator de von Willebrand/análise
16.
Biofabrication ; 14(4)2022 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-36108605

RESUMO

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.


Assuntos
Neoplasias da Mama , Células T Invariantes Associadas à Mucosa , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Citocinas/metabolismo , Feminino , Fibrina/metabolismo , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-13/metabolismo , Ligantes , Antígenos de Histocompatibilidade Menor/metabolismo , Células T Invariantes Associadas à Mucosa/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T , Microambiente Tumoral
17.
J Clin Invest ; 132(20)2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36006736

RESUMO

Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.


Assuntos
Fator XIIIa , Infecções Estreptocócicas , Androgênios/metabolismo , Animais , Fator XIIIa/metabolismo , Feminino , Fibrina/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Masculino , Mastócitos/metabolismo , Camundongos , Infecções Estreptocócicas/genética , Streptococcus agalactiae/metabolismo , Transglutaminases/metabolismo
18.
Thromb Res ; 218: 112-129, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36037547

RESUMO

The collagen receptor glycoprotein VI (GPVI) drives strong platelet activation, however its role at later stages of clotting remains less clear. Controlled timing of addition of anti-human GPVI Fab (clone E12) with microfluidic venous whole blood flow over collagen (± lipidated tissue factor, TF) produced distinct effects on platelets, fibrin, P-selectin exposure, and phosphatidylserine (PS) exposure. On collagen alone, Fab present initially potently reduced platelet deposition on collagen, while Fab added 90 s after initial platelet deposition, stopped subsequent platelet accumulation (despite the absence of fibrin). With thrombin generation via TF, Fab added at either t = 0 or 90 s had no effect on platelet deposition. However, Fab added initially, but not at 90-s, blocked fibrin formation. Gly-Pro-Arg-Pro ablated fibrin formation without effect on platelet accumulation (regardless of Fab added at t = 0 or 90 s), indicating thrombin signaling can suffice over GPVI signaling. Still, Fab moderately reduced P-selectin exposure with thrombin present and fibrin absent. On collagen/TF, Fab present initially ablated PS exposure, but had no effect when added 30 to 90-s later. The thrombin generated via PS exposure had an important role in driving platelet deposition in the presence of Fab, since inhibition of PS via annexin V binding in the presence of Fab significantly inhibited platelet deposition. We conclude GPVI signaling in the first platelet layer on collagen dictates thrombin and fibrin production, but the role of GPVI at subsequent times after formation of the first monolayer is obscured by thrombin-induced signaling.


Assuntos
Trombina , Tromboplastina , Anexina A5 , Colágeno/metabolismo , Colágeno/farmacologia , Fibrina/metabolismo , Humanos , Microfluídica , Selectina-P/metabolismo , Fosfatidilserinas , Glicoproteínas da Membrana de Plaquetas , Receptores de Colágeno/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismo
19.
Cell Mol Gastroenterol Hepatol ; 14(5): 1077-1101, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35926777

RESUMO

BACKGROUND & AIMS: Fulminant viral hepatitis (FVH) is a life-threatening disease, but its pathogenesis is not fully understood. Neutrophil extracellular traps (NETs) were an unrecognized link between inflammation and coagulation, which are 2 main features of FVH. Here, we investigated the role and mechanism of NETs in the pathogenesis of FVH. METHODS: A mouse model of FVH was established by murine hepatitis virus strain-3 infection. Liver leukocytes of infected or uninfected mice were used for single-cell RNA sequencing and whole-transcriptome sequencing. NETs depletion was achieved using DNase 1. Acetaminophen was used to establish a mouse model of non-virus-caused acute liver failure. Clinically, NETs-related markers in liver, plasma, and peripheral neutrophils were assessed in patients with hepatitis B virus (HBV)-related acute liver injury. RESULTS: Increased hepatic NETs formation was observed in murine hepatitis virus strain-3-infected mice, but not in acetaminophen-treated mice. NETs depletion improved the liver damage and survival rate in FVH by inhibiting hepatic fibrin deposition and inflammation. An adoptive transfer experiment showed that neutrophil-specific fibrinogen-like protein 2 (FGL2) promoted NETs formation. FGL2 was found to directly interact with mucolipin 3, which regulated calcium influx and initiated autophagy, leading to NETs formation. Clinically, increased plasma NETs level was associated with coagulation dysfunction in patients with HBV acute liver injury. Colocalization of FGL2, NETs, and fibrin in liver was observed in these patients. CONCLUSIONS: NETs aggravated liver injury in FVH by promoting fibrin deposition and inflammation. NETs formation was regulated by the FGL2-mucolipin 3-autophagy axis. Targeting NETs may provide a new strategy for the treatment of FVH.


Assuntos
Armadilhas Extracelulares , Hepatite Viral Animal , Hepatite Viral Humana , Vírus da Hepatite Murina , Camundongos , Animais , Hepatite Viral Animal/metabolismo , Hepatite Viral Animal/patologia , Camundongos Endogâmicos BALB C , Acetaminofen/efeitos adversos , Cálcio/metabolismo , Vírus da Hepatite Murina/metabolismo , Fibrinogênio/genética , Fibrinogênio/metabolismo , Hepatite Viral Humana/complicações , Modelos Animais de Doenças , Inflamação , Autofagia , Fibrina/metabolismo , Desoxirribonucleases/metabolismo
20.
Biophys J ; 121(17): 3271-3285, 2022 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-35927957

RESUMO

Thrombosis, resulting in occlusive blood clots, blocks blood flow to downstream organs and causes life-threatening conditions such as heart attacks and strokes. The administration of tissue plasminogen activator (t-PA), which drives the enzymatic degradation (fibrinolysis) of these blood clots, is a treatment for thrombotic conditions, but the use of these therapeutics is often limited due to the time-dependent nature of treatment and their limited success. We have shown that clot contraction, which is altered in prothrombotic conditions, influences the efficacy of fibrinolysis. Clot contraction results in the volume shrinkage of blood clots, with the redistribution and densification of fibrin and platelets on the exterior of the clot and red blood cells in the interior. Understanding how these key structural changes influence fibrinolysis can lead to improved diagnostics and patient care. We used a combination of mathematical modeling and experimental methodologies to characterize the process of exogenous delivery of t-PA (external fibrinolysis). A three-dimensional (3D) stochastic, multiscale model of external fibrinolysis was used to determine how the structural changes that occur during the process of clot contraction influence the mechanism(s) of fibrinolysis. Experiments were performed based on modeling predictions using pooled human plasma and the external delivery of t-PA to initiate lysis. Analysis of fibrinolysis simulations and experiments indicate that fibrin densification makes the most significant contribution to the rate of fibrinolysis compared with the distribution of components and degree of compaction (p < 0.0001). This result suggests the possibility of a certain fibrin density threshold above which t-PA effective diffusion is limited. From a clinical perspective, this information can be used to improve on current therapeutics by optimizing timing and delivery of lysis agents.


Assuntos
Trombose , Ativador de Plasminogênio Tecidual , Plaquetas/fisiologia , Fibrina/metabolismo , Fibrinólise/fisiologia , Humanos , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tecidual/farmacologia
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