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ABSTRACT: Cerebral venous sinus thrombosis (CVST) is an uncommon venous thromboembolic event accounting for <1% of strokes resulting in brain parenchymal injuries. JAK2V617F mutation, the most frequent driving mutation of myeloproliferative neoplasms, has been reported to be associated with worse clinical outcomes in patients with CVST. We investigated whether hematopoietic JAK2V617F expression predisposes to specific pathophysiological processes and/or worse prognosis after CVST. Using an in vivo mouse model of CVST, we analyzed clinical, biological, and imaging outcomes in mice with hematopoietic-restricted Jak2V617F expression, compared with wild-type Jak2 mice. In parallel, we studied a human cohort of JAK2V617F-positive or -negative CVST. Early after CVST, mice with hematopoietic Jak2V617F expression had increased adhesion of platelets and neutrophils in cerebral veins located in the vicinity of CVST. On day 1, Jak2V617F mice had a worse outcome characterized by significantly more frequent and severe intracranial hemorrhages (ICHs) and higher mortality rates. Peripheral neutrophil activation was enhanced, as indicated by higher circulating platelet-neutrophil aggregates, upregulated CD11b expression, and higher myeloperoxydase plasma level. Concurrently, immunohistological and brain homogenate analysis showed higher neutrophil infiltration and increased blood-brain barrier disruption. Similarly, patients with JAK2V617F-positive CVST tended to present higher thrombotic burden and had significantly higher systemic immune-inflammation index, a systemic thromboinflammatory marker, than patients who were JAK2V617F-negative. In mice with CVST, our study corroborates that Jak2V617F mutation leads to a specific pattern including increased thrombotic burden, ICH, and mortality. The exacerbated thromboinflammatory response, observed both in mice and patients positive for JAK2V617F, could contribute to hemorrhagic complications.
Assuntos
Inflamação , Janus Quinase 2 , Mutação , Trombose dos Seios Intracranianos , Animais , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Camundongos , Trombose dos Seios Intracranianos/genética , Humanos , Prognóstico , Inflamação/genética , Modelos Animais de Doenças , Masculino , Feminino , Neutrófilos/metabolismoRESUMO
BACKGROUND: The recruitment of activated factor VIII (FVIII) at the surface of activated platelets is a key step toward the burst of thrombin and fibrin generation during thrombus formation at the site of vascular injury. It involves binding to phosphatidylserine and, possibly, to fibrin-bound αIIbß3. Seminal work had shown the binding of FVIII to resting platelets, yet without a clear understanding of a putative physiological relevance. OBJECTIVES: To characterize the effects of FVIII-platelet interaction and its potential modulation of platelet function. METHODS: FVIII was incubated with washed platelets. The effects on platelet activation (spontaneously or triggered by collagen and thrombin) were studied by flow cytometry and light transmission aggregometry. We explored the involvement of downstream pathways by studying phosphorylation profiles (Western blot). The FVIII-glycoprotein (GP) VI interaction was investigated by ELISA, confocal microscopy, and proximity ligation assay. RESULTS: FVIII bound to the surface of resting and activated platelets in a dose-dependent manner. FVIII at supraphysiological concentrations did not induce platelet activation but rather specifically inhibited collagen-induced platelet aggregation and altered glycoprotein VI (GPVI)-dependent phosphorylation. FVIII, freed of its chaperone protein von Willebrand factor (VWF), interacted in close proximity with GPVI at the platelet surface. CONCLUSION: We showed that VWF-free FVIII binding to, or close to, GPVI modulates platelet activation in vitro. This may represent an uncharacterized negative feedback loop to control overt platelet activation. Whether locally activated FVIII concentrations achieved during platelet accumulation and thrombus formation at the site of vascular injury in vivo are compatible with such a function remains to be determined.
Assuntos
Plaquetas , Fator VIII , Ativação Plaquetária , Agregação Plaquetária , Glicoproteínas da Membrana de Plaquetas , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Plaquetas/metabolismo , Fosforilação , Fator VIII/metabolismo , Colágeno/metabolismo , Ligação Proteica , Citometria de Fluxo , Trombina/metabolismo , Relação Dose-Resposta a Droga , Microscopia ConfocalRESUMO
The association between endometriosis and autoimmune diseases is well known, however no acquired platelet function defect has been described so far. We describe the case of two patients with endometriosis associated with an antiplatelet glycoprotein VI (anti-GPVI) antibody. The two women with deep pelvic endometriosis associated with secondary infertility presented a mild bleeding tendency, a deficient platelet aggregation response to collagen, convulxin or CRP and a severe GPVI deficiency. Immunoblot revealed a combined FcRγ deficiency but no indication of GPVI cleavage. In the first case, platelet count was normal and an anti-GPVI IgG was detected in plasma. A first corticosteroids administration normalized in vitro platelet functions but further administrations were unsuccessful. Three IVF attempts failed. Conservative laparoscopic surgery was carried out after antifibrinolytic treatment without bleeding. The second case presented with a history of moderate thrombocytopenia and a weak anti-GPVI in the context of infertility and autoimmune disease, the Sjögren syndrome resolved after corticosteroids and hydroxychloroquine treatment. Acquired GPVI deficiencies are rare. It would be useful to determine whether the association with endometriosis is coincidental or not by more systematic investigations. It does not seem that in these patients, GPVI deficiency is associated with an increased risk of bleeding.
What is the context? ⢠Evidence for an immune system dysfunction is reported in endometriosis and the association between endometriosis and autoimmune diseases is well known.⢠No autoimmune platelet function defect has been described so far.What is new?⢠We report two unrelated patients with endometriosis-associated infertility presenting a platelet glycoprotein VI deficiency due to an autoantibody.⢠In both cases, a deficient platelet aggregation response to collagen, convulxin or CRP and a severe GPVI deficiency were observed.⢠Immunoblot revealed no indication of GPVI cleavage.What is the impact? ⢠Our observation raises the question whether GPVI could be a preferential target for the development of anti-GPVI autoantibodies associated with endometriosis.⢠It does not seem that in these patients, GPVI deficiency is associated with an increased risk of severe bleeding disorder.
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Endometriose , Infertilidade , Humanos , Feminino , Glicoproteínas da Membrana de Plaquetas , Endometriose/complicações , Endometriose/tratamento farmacológico , Anticorpos , Contagem de Plaquetas , PlaquetasRESUMO
SIGNIFICANCE STATEMENT: Kidney-derived thrombopoietin (TPO) increases myeloid cell and platelet production during antibody-mediated chronic kidney disease (AMCKD) in a mouse model, exacerbating chronic thromobinflammation in microvessels. The effect is mirrored in patients with extracapillary glomerulonephritis associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases.Neutralization of TPO in mice normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. The findings suggest that TPO is a relevant biomarker and a promising therapeutic target for patients with CKD and other chronic thromboinflammatory diseases. BACKGROUND: Chronic thromboinflammation provokes microvascular alterations and rarefaction, promoting organ dysfunction in individuals with various life-threatening diseases. Hematopoietic growth factors (HGFs) released by the affected organ may sustain emergency hematopoiesis and fuel the thromboinflammatory process. METHODS: Using a murine model of antibody-mediated chronic kidney disease (AMCKD) and pharmacological interventions, we comprehensively monitored the response to injury in the circulating blood, urine, bone marrow, and kidney. RESULTS: Experimental AMCKD was associated with chronic thromboinflammation and the production of HGFs, especially thrombopoietin (TPO), by the injured kidney, which stimulated and skewed hematopoiesis toward myelo-megakaryopoiesis. AMCKD was characterized by vascular and kidney dysfunction, TGF ß -dependent glomerulosclerosis, and microvascular rarefaction. In humans, extracapillary glomerulonephritis is associated with thromboinflammation, TGF ß -dependent glomerulosclerosis, and increased bioavailability of TPO. Analysis of albumin, HGF, and inflammatory cytokine levels in sera from patients with extracapillary glomerulonephritis allowed us to identify treatment responders. Strikingly, TPO neutralization in the experimental AMCKD model normalized hematopoiesis, reduced chronic thromboinflammation, and ameliorated renal disease. CONCLUSION: TPO-skewed hematopoiesis exacerbates chronic thromboinflammation in microvessels and worsens AMCKD. TPO is both a relevant biomarker and a promising therapeutic target in humans with CKD and other chronic thromboinflammatory diseases.
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Glomerulonefrite , Insuficiência Renal Crônica , Trombose , Humanos , Camundongos , Animais , Trombopoetina/metabolismo , Trombopoetina/farmacologia , Receptores de Trombopoetina , Inflamação , Tromboinflamação , Hematopoese/fisiologia , Anticorpos/farmacologia , Rim/metabolismo , Insuficiência Renal Crônica/etiologia , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Platelet glycoprotein VI (GPVI) is attracting interest as a potential target for the development of new antiplatelet molecules with a low bleeding risk. GPVI binding to vascular collagen initiates thrombus formation and GPVI interactions with fibrin promote the growth and stability of the thrombus. In this study, we show that glenzocimab, a clinical stage humanized antibody fragment (Fab) with a high affinity for GPVI, blocks the binding of both ligands through a combination of steric hindrance and structural change. A cocrystal of glenzocimab with an extracellular domain of monomeric GPVI was obtained and its structure determined to a resolution of 1.9 Å. The data revealed that (1) glenzocimab binds to the D2 domain of GPVI, GPVI dimerization was not observed in the crystal structure because glenzocimab prevented D2 homotypic interactions and the formation of dimers that have a high affinity for collagen and fibrin; and (2) the light variable domain of the GPVI-bound Fab causes steric hindrance that is predicted to prevent the collagen-related peptide (CRP)/collagen fibers from extending out of their binding site and preclude GPVI clustering and downstream signaling. Glenzocimab did not bind to a truncated GPVI missing loop residues 129 to 136, thus validating the epitope identified in the crystal structure. Overall, these findings demonstrate that the binding of glenzocimab to the D2 domain of GPVI induces steric hindrance and structural modifications that drive the inhibition of GPVI interactions with its major ligands.
Assuntos
Glicoproteínas da Membrana de Plaquetas , Trombose , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Colágeno/metabolismo , Trombose/tratamento farmacológico , Trombose/etiologia , Trombose/prevenção & controle , Fibrina/metabolismoRESUMO
Immediate reocclusion after mechanical thrombectomy (MT) for acute ischemic stroke (AIS) is a rare but devastating condition associated with poor functional outcome. The aim of this study was to gain insights into the mechanisms underlying immediate reocclusion, and to evaluate the efficacy and safety of the glycoprotein IIb/IIIa antagonist abciximab, for its treatment. Clinical data were collected from April 2015 to April 2019 in a monocentric prospective registry of AIS patients treated by MT. All patients with immediate reocclusion were retrospectively selected and subdivided into 2 groups according to abciximab treatment status. In vitro, the separate and combined effects of abciximab and alteplase on clot formation in whole blood under flow conditions were further investigated in microfluidic chambers. From 929 MT-treated patients, 21 had post-MT immediate reocclusion. Abciximab treatment in reocclusion patients (n = 10) led to higher rate of final recanalization (p < .001) while it did not increase bleeding complications. Flow chamber experiments revealed that, in contrast to alteplase, abciximab efficiently limits thrombus accretion from flowing blood by blocking platelet aggregation. Our results underscore a key role for platelet aggregation and the potential of Glycoprotein IIb/IIIa antagonists as a rescue therapy in post-MT immediate reocclusion.
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Abciximab/uso terapêutico , Administração Intravenosa/métodos , AVC Isquêmico/tratamento farmacológico , AVC Isquêmico/cirurgia , Inibidores da Agregação Plaquetária/uso terapêutico , Trombectomia/métodos , Abciximab/farmacologia , Doença Aguda , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/farmacologiaRESUMO
Cerebral venous sinus thrombosis (CVST) is an uncommon cause of stroke resulting in parenchymal injuries associated with heterogeneous clinical symptoms and prognosis. Therefore, an experimental animal model is required to further study underlying mechanisms involved in CVST. This study is aimed at developing a novel murine model suitable and relevant for evaluating injury patterns during CVST and studying its clinical aspects. CVST was achieved in C57BL/6J mice by autologous clot injection into the superior sagittal sinus (SSS) combined with bilateral ligation of external jugular veins. Clot was prepared ex vivo using thrombin before injection. On days 1 and 7 after CVST, SSS occlusion and associated-parenchymal lesions were monitored using different modalities: in vivo real-time intravital microscopy, magnetic resonance imaging (MRI), and immuno-histology. In addition, mice were subjected to a neurological sensory-motor evaluation. Thrombin-induced clot provided fibrin- and erythrocyte-rich thrombi that lead to reproducible SSS occlusion at day 1 after CVST induction. On day 7 post-CVST, venous occlusion monitoring (MRI, intravital microscopy) showed that initial injected-thrombus size did not significantly change demonstrating no early spontaneous recanalization. Microscopic histological analysis revealed that SSS occlusion resulted in brain edema, extensive fibrin-rich venular thrombotic occlusion, and ischemic and hemorrhagic lesions. Mice with CVST showed a significant lower neurological score on post-operative days 1 and 7, compared to the sham-operated group. We established a novel clinically CVST-relevant model with a persistent and reproducible SSS occlusion responsible for symptomatic ischemic and hemorrhagic lesions. This method provides a reliable model to study CVST physiopathology and evaluation of therapeutic new regimens.
Assuntos
Trombose dos Seios Intracranianos , Animais , Modelos Animais de Doenças , Imageamento por Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Trombose dos Seios Intracranianos/diagnóstico por imagem , Seio Sagital SuperiorRESUMO
Not available.
Assuntos
Anticorpos Monoclonais Humanizados , Plaquetas , Hemostasia , Inflamação , Glicoproteínas , HumanosRESUMO
BACKGROUND: Carboxypeptidase U (CPU, CPB2, TAFIa) is a potent attenuator of fibrinolysis. The inhibition of CPU is thus an interesting strategy for improving thrombolysis. OBJECTIVES: The time course of CPU generation and proCPU consumption were assessed in an experimental rat model of acute ischemic stroke (AIS). In addition, the effects of the selective CPU inhibitor AZD9684 on CPU kinetics, microvascular thrombosis (MT), and AIS outcome were evaluated. METHODS: Rats were subjected to transient middle cerebral artery occlusion (tMCAO) and received recombinant tissue-type plasminogen activator (tPA), a specific CPU inhibitor (AZD9684), combination therapy of tPA and AZD9684, or saline for 1 hour using a randomized treatment regime. CPU and proCPU levels were determined at five time points and assessed in light of outcome parameters (a.o.: infarct volume and fibrin[ogen] deposition as a measure for MT). RESULTS: Clear activation of the CPU system was observed after AIS induction, in both saline- and tPA-treated rats. Maximal CPU activities were observed at treatment cessation and were higher in tPA-treated animals compared to the saline group. Concomitant proCPU consumption was more pronounced in tPA-treated rats. AZD9684 suppressed the CPU activity and reduced fibrin(ogen) deposition, suggesting a reduction of MT. Nonetheless, a significant decrease in infarct volume was not observed. CONCLUSIONS: A pronounced activation of the CPU system was observed during tMCAO in rats. Selective inhibition of CPU with AZD9684 was able to reduce fibrin(ogen) deposition and brain edema, suggesting a reduction of MT but without a significant effect on final infarct volume.
Assuntos
Isquemia Encefálica , Carboxipeptidase B2 , Acidente Vascular Cerebral , Trombose , Animais , Fibrinólise , Ratos , Acidente Vascular Cerebral/tratamento farmacológico , Trombose/tratamento farmacológico , Ativador de Plasminogênio TecidualRESUMO
OBJECTIVE: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kß, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator). CONCLUSIONS: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.
Assuntos
Afibrinogenemia/sangue , Plaquetas/efeitos dos fármacos , Fibrinogênio/metabolismo , Fragmentos Fab das Imunoglobulinas/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Trombose/tratamento farmacológico , Afibrinogenemia/diagnóstico , Afibrinogenemia/genética , Plaquetas/metabolismo , Simulação por Computador , Fibrinogênio/genética , Fibrinolíticos/farmacologia , Humanos , Cinética , Microscopia de Vídeo , Modelos Biológicos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Transdução de Sinais , Estresse Mecânico , Trombina/metabolismo , Trombose/sangue , Trombose/diagnóstico , Trombose/genéticaRESUMO
BACKGROUND: Ligneous conjunctivitis (LC) is a rare disorder associated with plasminogen deficiency characterized by chronic fibrin deposits in the eyelids. All patients with plasminogen deficiency do not develop LC, whose underlying mechanisms remain unknown. OBJECTIVE: We investigated whether fibrinolytic activity was correlated with phenotype and/or genotype in patients suffering from LC and their relatives. METHODS: Plasminogen activity/antigen levels and PLG mutations were determined in 10 patients with LC, 17 of their asymptomatic relatives, and 10 healthy individuals used as a control group. Plasma fibrinolytic activity was evaluated using three different assays: (1) tissue-plasminogen activator (t-PA) front lysis, (2) cell-based urokinase-dependent euglobulin clot lysis (ECLT) at the surface of corneal cells, and (3) urokinase-dependent plasminogen activation. RESULTS: Plasminogen activity varied from <10 to 40% in patients, 36 to 105% in relatives, and >80% in control healthy individuals. Homozygous K19E mutation was associated with normal antigenic plasminogen levels. In front-lysis experiments, all patients had a lower fibrinolysis rate as compared with their relatives and to control individuals. The cell-based ECLT and plasminogen activation assay demonstrated that urokinase-mediated fibrinolysis was not impaired in patients with homozygous K19E mutation compared with the other mutants. CONCLUSION: We confirm that plasminogen levels fail to predict LC occurrence. In these conditions, t-PA clot lysis front is useful to predict clinical outcome in plasminogen deficiency. Moreover, we provide evidence that occurrence of LC overlaps quantitative and qualitative plasminogen deficiencies. The homozygous K19E mutation is associated with isolated impaired t-PA-mediated fibrinolysis compared with other mutants.
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Testes de Coagulação Sanguínea , Conjuntivite/diagnóstico , Olho/metabolismo , Fibrina/metabolismo , Fibrinólise , Plasminogênio/deficiência , Dermatopatias Genéticas/diagnóstico , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Conjuntivite/genética , Conjuntivite/metabolismo , Feminino , Predisposição Genética para Doença , Hereditariedade , Heterozigoto , Homozigoto , Humanos , Cinética , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Plasminogênio/genética , Plasminogênio/metabolismo , Valor Preditivo dos Testes , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/metabolismo , Ativador de Plasminogênio Tecidual/metabolismo , Adulto JovemRESUMO
Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Pegylated-interferon alpha (IFN) and hydroxyurea (HU) are commonly used to treat MPN, but their effect on hemostasis has not yet been studied. The aim of our study was to determine whether IFN and HU impact the biological hemostatic profile of MPN patients by studying markers of endothelial, platelet, and coagulation activation. A total of 85 patients (50 polycythemia vera and 35 essential thrombocythemia) were included: 28 treated with IFN, 35 with HU, and 22 with no cytoreductive drug (non-treated, NT). Von Willebrand factor, shear-induced platelet aggregation, factor VIII coagulant activity (FVIII:C), fibrinogen, and thrombin generation with and without exogenous thrombomodulin were significantly higher in IFN-treated patients compared to NT patients, while protein S anticoagulant activity was lower. In 10 patients in whom IFN therapy was discontinued, these hemostatic biomarkers returned to the values observed in NT patients, strongly suggesting an impact of IFN therapy on endothelial and coagulation activation. Overall, our study shows that treatment with IFN is associated with significant and reversible effects on the biological hemostatic profile of MPN patients. Whether they could be associated with an increased thrombotic risk remains to be determined in further randomized clinical studies.
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OBJECTIVES: Thrombi responsible for large vessel occlusion (LVO) in the setting of acute ischemic stroke (AIS) are characterized by a low recanalization rate after IV thrombolysis. To test whether AIS thrombi have inherent common features that limit their susceptibility to thrombolysis, we analyzed the composition and ultrastructural organization of AIS thrombi causing LVO. METHODS: A total of 199 endovascular thrombectomy-retrieved thrombi were analyzed by immunohistology and scanning electron microscopy (SEM) and subjected to ex vivo thrombolysis assay. The relationship between thrombus organization and thrombolysis resistance was further investigated in vitro using thrombus produced by recalcification of citrated whole blood. RESULTS: SEM and immunohistology analyses revealed that, although AIS thrombus composition and organization was highly heterogeneous, AIS thrombi shared a common remarkable structural feature in the form of an outer shell made of densely compacted thrombus components including fibrin, von Willebrand factor, and aggregated platelets. In vitro thrombosis experiments using human blood indicated that platelets were essential to the formation of the thrombus outer shell. Finally, in both AIS and in vitro thrombi, the thrombus outer shell showed a decreased susceptibility to tissue plasminogen activator-mediated thrombolysis as compared to the thrombus inner core. INTERPRETATION: Irrespective of their etiology and despite their heterogeneity, intracranial thrombi causing LVO have a core shell structure that influences their susceptibility to thrombolysis.
Assuntos
Isquemia Encefálica/patologia , Resistência a Medicamentos , Trombose Intracraniana/patologia , Acidente Vascular Cerebral/patologia , Trombectomia , Idoso , Idoso de 80 Anos ou mais , Plaquetas/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/cirurgia , Eritrócitos/metabolismo , Armadilhas Extracelulares/metabolismo , Feminino , Fibrina/metabolismo , Fibrinolíticos/uso terapêutico , Humanos , Imuno-Histoquímica , Técnicas In Vitro , Trombose Intracraniana/tratamento farmacológico , Trombose Intracraniana/metabolismo , Trombose Intracraniana/cirurgia , Leucócitos/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Pessoa de Meia-Idade , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/cirurgia , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/uso terapêutico , Fator de von Willebrand/metabolismoRESUMO
OBJECTIVES: Behçet's disease (BD) is a chronic systemic vasculitis. Thrombosis is a frequent and life-threatening complication. The pathogenesis of BD is poorly understood and evidence supporting a role for primed neutrophils in BD-associated thrombotic risk is scant. To respond to inflammatory insults, neutrophils release web-like structures, known as neutrophil extracellular traps (NETs), which are prothrombotic. We evaluated the role of NETs and markers of NETs in BD. METHODS: Blood samples were collected from patients with BD, according to the International Study Group Criteria for Behçet's disease, and healthy donors (HD). NET components, including cell-free DNA (CfDNA) and neutrophil enzymes myeloperoxidase (MPO), were assessed in serum or in purified neutrophils from patients with BD and HD. RESULTS: Patients with active BD had elevated serum cfDNA levels and MPO-DNA complexes compared with patients with inactive BD and to HD. In addition, levels of cfDNA and MPO-DNA complexes were significantly higher in patients with BD with vascular involvement compared with those without vascular symptoms. Purified neutrophils from patients with BD exhibited spontaneous NETosis compared with HD. Thrombin generation in BD plasma was significantly increased and positively correlated with the levels of MPO-DNA complexes and cfDNA. Importantly, DNAse treatment significantly decreased thrombin generation in BD plasma but not in HD plasma. In addition, biopsy materials obtained from patients with BD showed NETs production in areas of vasculitic inflammation and thrombosis. CONCLUSIONS: Our data show that NETs and markers of NETS levels are elevated in patients with BD and contribute to the procoagulant state. Targeting NETs may represent a potential therapeutic target for the reduction or prevention of BD-associated thrombotic risk.
Assuntos
Síndrome de Behçet/sangue , Armadilhas Extracelulares/metabolismo , Neutrófilos/metabolismo , Adulto , Síndrome de Behçet/patologia , Biomarcadores/sangue , Feminino , Humanos , Masculino , Neutrófilos/patologia , Índice de Gravidade de DoençaRESUMO
Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor-coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.
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Proteínas de Bactérias/farmacologia , Heparina/efeitos adversos , Imunoglobulina G/metabolismo , Fator Plaquetário 4/metabolismo , Streptococcus pyogenes/enzimologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/metabolismo , Animais , Fibrina/genética , Fibrina/metabolismo , Heparina/administração & dosagem , Humanos , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/genética , Receptores de IgG/metabolismo , Trombocitopenia/genética , Trombocitopenia/patologiaRESUMO
Objective- Despite the high clinical relevance of thrombolysis, models for its study in human flowing blood are lacking. Our objective was to develop a microfluidic model for comparative evaluation of thrombolytic therapeutic strategies. Approach and Results- Citrated human blood was supplemented with 3,3'-dihexyloxacarbocyanine iodide and Alexa Fluor 647 fibrinogen conjugate, recalcified, and perfused for 3 to 4 minutes at venous or arterial wall shear rate in microfluidic flow chambers coated with collagen and tissue factor to generate nonocclusive fluorescent thrombi. A second perfusion was performed for 10 minutes with rhodamine-6G-labeled citrated whole blood, supplemented or not with r-tPA (recombinant tissue-type plasminogen activator), fluorescein isothiocyanate-conjugated r-tPA, and Alexa Fluor 568 plasminogen conjugate. Plasminogen and r-tPA bound to preformed thrombi and r-tPA caused a concentration-dependent decrease in thrombus fibrin content (up to 50% reduction at 15 µg/mL r-tPA) as assessed by fluorescence microscopy. Fibrinolysis was confirmed by measurement of D-dimers in the output flow. Remarkably, despite ongoing fibrinolysis, new platelets continued to be recruited to the thrombus under lysis. Under the arterial condition, combining r-tPA with hirudin enhanced fibrinolysis but did not prevent the recruitment of new platelets, which was, however, prevented by antiplatelet agents (ticagrelor or the GPVI [glycoprotein VI]-blocking antigen-binding fragment 9O12). Conclusions- Our microfluidic thrombolysis model is suitable for studying thrombolysis and testing the efficacy of drugs used in combination with r-tPA. Real-time analysis of fibrin and platelets during r-tPA-mediated fibrinolysis at arterial or venous flow conditions showed that platelets continue to accumulate during fibrinolysis. Such platelet accumulation may impair r-tPA-mediated recanalization.
Assuntos
Plaquetas/efeitos dos fármacos , Fibrinólise/efeitos dos fármacos , Fibrinolíticos/farmacologia , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Terapia Trombolítica/métodos , Trombose/tratamento farmacológico , Ativador de Plasminogênio Tecidual/farmacologia , Anticoagulantes/farmacologia , Plaquetas/metabolismo , Feminino , Fibrina/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Humanos , Masculino , Inibidores da Agregação Plaquetária/farmacologia , Trombose/sangue , Fatores de TempoRESUMO
The ephrin transmembrane receptor family of tyrosine kinases is involved in platelet function. We report the first EPHB2 variant affecting platelets in 2 siblings (P1 and P2) from a consanguineous family with recurrent bleeding and normal platelet counts. Whole-exome sequencing identified a c.2233C>T variant (missense p.R745C) of the EPHB2 gene. P1 and P2 were homozygous for this variant, while their asymptomatic parents were heterozygous. The p.R745C variant within the tyrosine kinase domain was associated with defects in platelet aggregation, αIIbß3 activation, and granule secretion induced by G-protein-coupled receptor (GPCR) agonists and convulxin, as well as in thrombus formation on collagen under flow. In contrast, clot retraction, flow-dependent platelet adhesion, and spreading on fibrinogen were only mildly affected, indicating limited effects on αIIbß3 outside-in signaling. Most importantly, Lyn, Syk, and FcRγ phosphorylation, the initial steps in glycoprotein VI (GPVI) platelet signaling were drastically impaired in the absence of platelet-platelet contact, indicating a positive role for EPHB2 in GPVI activation. Likewise platelet activation by PAR4-AP showed defective Src activation, as opposed to normal protein kinase C activity and Ca2+ mobilization. Overexpression of wild-type and R745C EPHB2 variant in RBL-2H3 (rat basophilic leukemia) cells stably expressing human GPVI confirmed that EPHB2 R745C mutation impaired EPHB2 autophosphorylation but had no effect on ephrin ligand-induced EPHB2 clustering, suggesting it did not interfere with EPHB2-ephrin-mediated cell-to-cell contact. In conclusion, this novel inherited platelet disorder affecting EPHB2 demonstrates this tyrosine kinase receptor plays an important role in platelet function through crosstalk with GPVI and GPCR signaling.
Assuntos
Plaquetas/patologia , Mutação de Sentido Incorreto , Ativação Plaquetária , Receptor EphB2/genética , Adolescente , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Criança , Feminino , Humanos , Masculino , Linhagem , Adesividade Plaquetária , Agregação Plaquetária , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptor EphB2/metabolismo , Transdução de Sinais , Adulto JovemRESUMO
BACKGROUND AND PURPOSE: Neutrophil Extracellular Traps (NETs) are DNA extracellular networks decorated with histones and granular proteins produced by activated neutrophils. NETs have been identified as major triggers and structural factors of thrombosis. A recent study designated extracellular DNA threads from NETs as a potential therapeutic target for improving tissue-type plasminogen activator (tPA)-induced thrombolysis in acute coronary syndrome. The aim of this study was to assess the presence of NETs in thrombi retrieved during endovascular therapy in patients with acute ischemic stroke (AIS) and their impact on tPA-induced thrombolysis. METHODS: We analyzed thrombi from 108 AIS patients treated with endovascular therapy. Thrombi were characterized by hematoxylin/eosin staining, immunostaining, and ex vivo enzymatic assay. Additionally, we assessed ex vivo the impact of deoxyribonuclease 1 (DNAse 1) on thrombolysis of AIS thrombi. RESULTS: Histological analysis revealed that NETs contributed to the composition of all AIS thrombi especially in their outer layers. Quantitative measurement of thrombus NETs content was not associated with clinical outcome or AIS pathogenesis but correlated significantly with endovascular therapy procedure length and device number of passes. Ex vivo, recombinant DNAse 1 accelerated tPA-induced thrombolysis, whereas DNAse 1 alone was ineffective. CONCLUSIONS: This study suggests that thrombus NETs content may be responsible for reperfusion resistance, including mechanical or pharmacological approaches with intravenous tPA, irrespectively of their etiology. The efficacy of a strategy involving an administration of DNAse 1 in addition to tPA should be explored in the setting of AIS. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT02907736.
Assuntos
Isquemia Encefálica , Procedimentos Endovasculares , Armadilhas Extracelulares/metabolismo , Acidente Vascular Cerebral , Terapia Trombolítica , Ativador de Plasminogênio Tecidual/administração & dosagem , Isquemia Encefálica/sangue , Isquemia Encefálica/terapia , Feminino , Humanos , Masculino , Acidente Vascular Cerebral/sangue , Acidente Vascular Cerebral/terapia , Trombose/sangue , Trombose/terapiaAssuntos
Síndrome da Aderência Leucocítica Deficitária/terapia , Alelos , Biomarcadores , Gerenciamento Clínico , Fator VIIa/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Humanos , Síndrome da Aderência Leucocítica Deficitária/diagnóstico , Síndrome da Aderência Leucocítica Deficitária/genética , Proteínas de Membrana/genética , Mutação , Proteínas de Neoplasias/genética , Fenótipo , Proteínas Recombinantes/uso terapêutico , Resultado do TratamentoRESUMO
Glycoprotein VI, a major platelet activation receptor for collagen and fibrin, is considered a particularly promising, safe antithrombotic target. In this study, we show that human glycoprotein VI signals upon platelet adhesion to fibrinogen. Full spreading of human platelets on fibrinogen was abolished in platelets from glycoprotein VI- deficient patients suggesting that fibrinogen activates platelets through glycoprotein VI. While mouse platelets failed to spread on fibrinogen, human-glycoprotein VI-transgenic mouse platelets showed full spreading and increased Ca2+ signaling through the tyrosine kinase Syk. Direct binding of fibrinogen to human glycoprotein VI was shown by surface plasmon resonance and by increased adhesion to fibrinogen of human glycoprotein VI-transfected RBL-2H3 cells relative to mock-transfected cells. Blockade of human glycoprotein VI with the Fab of the monoclonal antibody 9O12 impaired platelet aggregation on preformed platelet aggregates in flowing blood independent of collagen and fibrin exposure. These results demonstrate that human glycoprotein VI binds to immobilized fibrinogen and show that this contributes to platelet spreading and platelet aggregation under flow.