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ABSTRACT: Many patients with antiphospholipid syndrome had decreased ectonucleotidase activity on neutrophils and platelets, which enabled extracellular nucleotides to trigger neutrophil-platelet aggregates. This phenotype was replicated by treating healthy neutrophils and platelets with patient-derived antiphospholipid antibodies or ectonucleotidase inhibitors.
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Síndrome Antifosfolipídica , Humanos , Neutrófilos , Anticorpos Antifosfolipídeos , PlaquetasRESUMO
Our study aimed to evaluate the presence, clinical associations, and potential mechanistic roles of non-criteria antiphospholipid antibodies (aPL) and circulating calprotectin, a highly stable marker of neutrophil extracellular trap release (NETosis), in pediatric APS patients. We found that 79% of pediatric APS patients had at least one non-criteria aPL at moderate-to-high titer. Univariate logistic regression demonstrated that positive anti-beta-2 glycoprotein I domain 1 (anti-D1) IgG (p = 0.008), anti-phosphatidylserine/prothrombin (aPS/PT) IgG (p < 0.001), and aPS/PT IgM (p < 0.001) were significantly associated with venous thrombosis. Positive anti-D1 IgG (p < 0.001), aPS/PT IgG (p < 0.001), and aPS/PT IgM (p = 0.001) were also associated with non-thrombotic manifestations of APS, such as thrombocytopenia. Increased levels of calprotectin were detected in children with APS. Calprotectin correlated positively with absolute neutrophil count (r = 0.63, p = 0.008) and negatively with platelet count (r = -0.59, p = 0.015). Mechanistically, plasma from pediatric APS patients with high calprotectin levels impaired platelet viability in a dose-dependent manner.
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Anticorpos Antifosfolipídeos , Síndrome Antifosfolipídica , Humanos , Criança , Biomarcadores , beta 2-Glicoproteína I , Imunoglobulina G , Imunoglobulina M , Protrombina , Complexo Antígeno L1 LeucocitárioRESUMO
Beta-2 glycoprotein I (ß2GPI) is a phospholipid-binding plasma protein and prominent autoantigen in antiphospholipid syndrome (APS). Here, we tested the hypothesis that ß2GPI might bind to not only phospholipids, but also cell-free DNA and neutrophil extracellular traps (NETs). We report that ß2GPI interacts with cell-free DNA from different species, as well as NETs, in a dose-dependent manner, retarding their migration in an agarose-gel electrophoretic mobility shift assay. We confirm the direct binding interaction of ß2GPI with DNA and NETs by ELISA. We also demonstrate that ß2GPI colocalizes with NET strands by immunofluorescence microscopy. Finally, we provide evidence that ß2GPI-DNA complexes can be detected in the plasma of APS patients, where they positively correlate with an established biomarker of NET remnants. Taken together, our findings indicate that ß2GPI interacts with DNA and NETs, and suggest that this interaction may play a role as a perpetuator and/or instigator of autoimmunity in APS.
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OBJECTIVES: Neutrophils and neutrophil extracellular traps (NETs) contribute to the vascular complications of multiple diseases, but their role in systemic sclerosis (SSc) is understudied. We sought to test the hypothesis that NETs are implicated in SSc vasculopathy and that treatment with prostacyclin analogs may ameliorate SSc vasculopathy not only through vasodilation but also by inhibiting NET release. METHODS: Blood from 125 patients with SSc (87 diffuse cutaneous SSc and 38 limited cutaneous SSc) was collected at a single academic medical center. Vascular complications such as digital ulcers, pulmonary artery hypertension, and scleroderma renal crisis were recorded. The association between circulating NETs and vascular complications was determined using in vitro and ex vivo assays. The impact of the synthetic prostacyclin analog epoprostenol on NET release was determined. RESULTS: Neutrophil activation and NET release were elevated in patients with SSc-associated vascular complications compared to matched patients without vascular complications. Neutrophil activation and NETs positively correlated with soluble E-selectin and VCAM-1, circulating markers of vascular injury. Treatment of patients with digital ischemia with a synthetic prostacyclin analog boosted neutrophil cyclic AMP, which was associated with the blunting of NET release and reduced NETs in circulation. CONCLUSION: Our study demonstrates an association between NETs and vascular complications in SSc. We also identified the potential for an additional therapeutic benefit of synthetic prostacyclin analogs, namely to reduce neutrophil hyperactivity and NET release in SSc patients.
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Epoprostenol , Armadilhas Extracelulares , Escleroderma Sistêmico , Humanos , Armadilhas Extracelulares/efeitos dos fármacos , Armadilhas Extracelulares/metabolismo , Feminino , Masculino , Escleroderma Sistêmico/tratamento farmacológico , Pessoa de Meia-Idade , Epoprostenol/análogos & derivados , Epoprostenol/uso terapêutico , Epoprostenol/farmacologia , Adulto , Idoso , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Neutrófilos/imunologia , Ativação de Neutrófilo/efeitos dos fármacos , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/etiologiaRESUMO
OBJECTIVE: While thrombosis and pregnancy loss are the best-known clinical features of antiphospholipid syndrome (APS), many patients also exhibit "extra-criteria" manifestations, such as thrombocytopenia. The mechanisms that drive APS thrombocytopenia are not completely understood, and no clinical biomarkers are available for predicting antiphospholipid antibody (aPL)-mediated thrombocytopenia. Calprotectin is a heterodimer of S100A8 and S100A9 that is abundant in the neutrophil cytoplasm and released upon proinflammatory neutrophil activation. Here, we sought to evaluate the presence, clinical associations, and potential mechanistic roles of circulating calprotectin in a cohort of primary APS and aPL-positive patients. METHODS: Levels of circulating calprotectin were determined in plasma by the QUANTA Flash chemiluminescent assay. A viability dye-based platelet assay was used to assess the potential impact of calprotectin on aPL-mediated thrombocytopenia. RESULTS: Circulating calprotectin was measured in 112 patients with primary APS and 30 aPL-positive (without APS criteria manifestations or lupus) patients as compared to patients with lupus (without APS), patients with unprovoked venous thrombosis (without aPL), and healthy controls. Levels of calprotectin were higher in patients with primary APS and aPL-positive patients compared to healthy controls. After adjustment for age and sex, calprotectin level correlated positively with absolute neutrophil count (r = 0.41, P < 0.001), positively with C-reactive protein level (r = 0.34, P = 0.002), and negatively with platelet count (r = -0.24, P = 0.004). Mechanistically, we found that calprotectin provoked aPL-mediated thrombocytopenia by engaging platelet surface toll-like receptor 4 and activating the NLRP3-inflammasome, thereby reducing platelet viability in a caspase-1-dependent manner. CONCLUSION: These data suggest that calprotectin has the potential to be a functional biomarker and a new therapeutic target for APS thrombocytopenia.
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Síndrome Antifosfolipídica , Plaquetas , Complexo Antígeno L1 Leucocitário , Trombocitopenia , Humanos , Síndrome Antifosfolipídica/sangue , Feminino , Complexo Antígeno L1 Leucocitário/sangue , Masculino , Pessoa de Meia-Idade , Adulto , Trombocitopenia/sangue , Plaquetas/metabolismo , Biomarcadores/sangue , Receptor 4 Toll-Like/sangue , Anticorpos Antifosfolipídeos/sangueRESUMO
Neutrophil hyperactivity and neutrophil extracellular trap release (NETosis) appear to play important roles in the pathogenesis of the thromboinflammatory autoimmune disease known as antiphospholipid syndrome (APS). The understanding of neutrophil metabolism has advanced tremendously in the past decade, and accumulating evidence suggests that a variety of metabolic pathways guide neutrophil activities in health and disease. Our previous work characterizing the transcriptome of APS neutrophils revealed that genes related to glycolysis, glycogenolysis, and the pentose phosphate pathway (PPP) were significantly upregulated. Here, we found that neutrophils from patients with APS used glycolysis more avidly than neutrophils from people in the healthy control group, especially when the neutrophils were from patients with APS with a history of microvascular disease. In vitro, inhibiting either glycolysis or the PPP tempered phorbol myristate acetate- and APS IgG-induced NETosis, but not NETosis triggered by a calcium ionophore. In mice, inhibiting either glycolysis or the PPP reduced neutrophil reactive oxygen species production and suppressed APS IgG-induced NETosis ex vivo. When APS-associated thrombosis was evaluated in mice, inhibiting either glycolysis or the PPP markedly suppressed thrombosis and circulating NET remnants. In summary, these data identify a potential role for restraining neutrophil glucose flux in the treatment of APS.
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Síndrome Antifosfolipídica , Armadilhas Extracelulares , Glucose , Glicólise , Neutrófilos , Via de Pentose Fosfato , Neutrófilos/metabolismo , Neutrófilos/imunologia , Humanos , Animais , Camundongos , Síndrome Antifosfolipídica/imunologia , Síndrome Antifosfolipídica/metabolismo , Síndrome Antifosfolipídica/tratamento farmacológico , Armadilhas Extracelulares/metabolismo , Armadilhas Extracelulares/imunologia , Masculino , Feminino , Glucose/metabolismo , Trombose/metabolismo , Trombose/imunologia , Trombose/patologia , Trombose/genética , Adulto , Espécies Reativas de Oxigênio/metabolismo , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: This study assessed patient-reported outcomes (PROs) in individuals with persistently positive antiphospholipid antibodies (aPL) to better understand how living with aPL may affect their quality of life. METHODS: Patients completed Patient-Reported Outcomes Measurement Information System Physical Function (PF) and Cognitive Function (CF) Short Forms as well as the pain intensity (PI) rating (scale of 1-10). Patients were characterized for demographics, clinical manifestations of antiphospholipid syndrome (APS), cardiovascular risk factors, laboratory test results, and medication usage. Multivariate modeling was done via linear regression. RESULTS: Of 139 patients, 89 had primary APS, 21 had secondary APS, and 29 had persistent aPL without meeting clinical criteria for APS. The average T scores (±SD) for PF and CF were 45.4 ± 9.2 and 48.6 ± 11.6, respectively; the average for PI was 3.0 ± 2.6. Approximately half of the patients (47%) endorsed at least mild impairment in PF (T score < 45). Mean PF, CF, and PI did not differ between diagnostic groups. Individuals who endorsed more impairment on one measure also tended to endorse more impairment on another (Pearson r = 0.43-0.59). In the multivariate models, age, smoking, pain medications, and serotonergic medications were associated with impairment in at least one PRO domain. The Damage Index for APS was significantly correlated with both PF and CF. CONCLUSION: Individuals living with APS endorsed more impairment in PF (and potentially CF) than expected for the general population. The relationship between certain medications and PROs warrants further study, as does the longitudinal trajectory of these and other PROs.
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We previously reported that treatment of mice with 6-gingerol, the most abundant phytochemical in ginger root, leads to phosphodiesterase inhibition that counteracts neutrophil hyperactivity in models of antiphospholipid syndrome (APS) and lupus. Here, we explored the extent to which oral intake of a whole-ginger extract would similarly impact neutrophils in both autoimmune mice and healthy humans. In vitro, a solubilized ginger extract was able to attenuate neutrophil extracellular trap formation (NETosis) by human neutrophils through a mechanism that was dependent upon the cyclic AMP-dependent kinase, protein kinase A. When mice with features of either APS or lupus were administered a ginger extract orally, they demonstrated reduced circulating NETs, as well as the tempering of other disease outcomes, such as large-vein thrombosis (APS) and autoantibody production (lupus). In a pilot clinical trial, which was validated in a second cohort, daily intake of a ginger supplement for 7 days by healthy volunteers boosted neutrophil cAMP, inhibited NETosis in response to disease-relevant stimuli, and reduced circulating plasma NET levels. In summary, this work demonstrates that ginger intake restrains neutrophil hyperactivity in autoimmune mouse models and that ginger consumption by healthy individuals makes their neutrophils more resistant to NETosis.