Low ectonucleotidase activity and increased neutrophil-platelet aggregates in patients with antiphospholipid syndrome.

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.

Calprotectin Impairs Platelet Survival in Patients With Primary Antiphospholipid Syndrome.

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.

Taxifolin inhibits NETosis through activation of Nrf2 and provides protective effects in models of lupus and antiphospholipid syndrome.

OBJECTIVES: Taxifolin (dihydroquercetin) is a bioactive plant flavonoid that exhibits anti-inflammatory and anti-oxidative properties. We hypothesized that taxifolin might be an effective dietary supplement to ameliorate symptoms arising from thrombo-inflammatory diseases such as lupus and antiphospholipid syndrome (APS). METHODS: We used in vitro assays and a mouse model to determine mechanisms by which taxifolin inhibits neutrophil extracellular trap (NET) formation (i.e., NETosis) and venous thrombosis in lupus and APS. RESULTS: At doses ranging from 0.1 to 1 µg/ml, taxifolin inhibited NETosis from control neutrophils stimulated with autoantibodies isolated from lupus and APS patients, and its suppressive effects were mitigated by blocking the antioxidant transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). Furthermore, taxifolin at a dose as low as 20 mg/kg/day reduced in vivo NETosis in thrombo-inflammatory mouse models of lupus and APS while also significantly attenuating autoantibody formation, inflammatory cytokine production, and large-vein thrombosis. CONCLUSION: Our study is the first to demonstrate the protective effects of taxifolin in the context of lupus and APS. Importantly, our study also suggests a therapeutic potential to neutralize neutrophil hyperactivity and NETosis that could have relevance to a variety of thrombo-inflammatory diseases.

Inhibition of neutrophil extracellular trap formation alleviates vascular dysfunction in type 1 diabetic mice.

While neutrophil extracellular traps (NETs) have previously been linked to some diabetes-associated complications, such as dysfunctional wound healing, their potential role in diabetic vascular dysfunction has not been studied. Diabetic Akita mice were crossed with either Elane-/- or Pad4-/- mice to generate NET-deficient diabetic mice. By 24 weeks of age, Akita aortae showed markedly impaired relaxation in response to acetylcholine, indicative of vascular dysfunction. Both Akita-Elane-/- mice and Akita-Pad4-/- mice had reduced levels of circulating NETs and improved acetylcholine-mediated aortic relaxation. Compared with wild-type aortae, the thromboxane metabolite TXB2 was roughly 10-fold higher in both intact and endothelium-denuded aortae of Akita mice. In contrast, Akita-Elane-/- and Akita-Pad4-/- aortae had TXB2 levels similar to wild type. In summary, inhibition of NETosis by two independent strategies prevented the development of vascular dysfunction in diabetic Akita mice. Thromboxane was up-regulated in the vessel walls of NETosis-competent diabetic mice, suggesting a role for neutrophils in driving the production of this vasoconstrictive and atherogenic prostanoid.

Ginger intake suppresses neutrophil extracellular trap formation in autoimmune mice and healthy humans.

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.

Prevalence of Antiphospholipid Antibodies and Association With Incident Cardiovascular Events.

Importance: The prevalence of antiphospholipid antibodies (aPL) and their association with future atherosclerotic cardiovascular disease (ASCVD) risk has yet to be thoroughly investigated. Objective: To determine the association between measurements of aPL at a single time point and ASCVD risk in a diverse population. Design, Setting, and Participants: This cohort study measured 8 aPL (anticardiolipin [aCL] IgG/IgM/IgA, anti-beta-2 glycoprotein I [aß2GPI] IgG/IgM/IgA, and antiphosphatidylserine/prothrombin [aPS/PT] IgG/IgM) by solid-phase assays in plasma from participants of the Dallas Heart Study (DHS) phase 2, a multiethnic, population-based cohort study. Blood samples were collected between 2007 and 2009. The median follow-up was 8 years. Statistical analysis was performed from April 2022 to January 2023. Main Outcomes and Measures: Associations of aPL with future ASCVD events (defined as first nonfatal myocardial infarction, first nonfatal stroke, coronary revascularization, or death from cardiovascular cause) were assessed by Cox proportional hazards models, adjusting for known risk factors, medications, and multiple comparisons. Results: Among the 2427 participants (mean [SD] age, 50.6 [10.3] years; 1399 [57.6%] female; 1244 [51.3%] Black, 339 [14.0%] Hispanic, and 796 [32.8%] White), the prevalence of any positive aPL tested at a single time point was 14.5% (353 of 2427), with approximately one-third of those detected at a moderate or high titer; aCL IgM had the highest prevalence (156 individuals [6.4%]), followed by aPS/PT IgM (88 [3.4%]), aß2GPI IgM (63 [2.6%]), and aß2GPI IgA (62 [2.5%]). The IgA of aCL (adjusted hazard ratio [HR], 4.92; 95% CI, 1.52-15.98) and aß2GPI (HR, 2.91; 95% CI, 1.32-6.41) were independently associated with future ASCVD events. The risk further increased when applying a positivity threshold of at least 40 units (aCL IgA: HR, 9.01 [95% CI, 2.73-29.72]; aß2GPI IgA: HR, 4.09 [95% CI, 1.45-11.54]). Levels of aß2GPI IgA negatively correlated with cholesterol efflux capacity (r = -0.055; P = .009) and positively correlated with circulating oxidized LDL (r = 0.055; P = .007). aß2GPI IgA-positive plasma was associated with an activated endothelial cell phenotype as evidenced by increased surface expression of surface E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Conclusions and Relevance: In this population-based cohort study, aPL detectable by solid-phase assays were present in a substantial proportion of adults; positive aCL IgA and aß2GPI IgA at a single time point were independently associated with future ASCVD events. Longitudinal studies with serial aPL measurements are needed to further explore these findings.

Understanding the Pathophysiology of Thrombotic APS through Animal Models.

Antiphospholipid syndrome (APS) is a leading acquired cause of thrombotic events, with a notable tendency to promote thrombosis in vascular beds of all sizes, including both arterial and venous circuits. While pathogenic antiphospholipid antibodies circulate at relatively stable levels in blood, thrombosis tends to manifest as discrete and acute events, suggesting the requirement for a "second hit." While this two-hit model is generally accepted, much remains to be learned about exactly how antiphospholipid antibodies predispose to thrombosis in vivo and exactly how this predisposition interacts with the second hit. To this end, investigators have turned to animal models. Numerous approaches for modeling APS in animals have been described to date, each with potential advantages and disadvantages. This review will attempt to describe the most common APS models employed so far while discussing some pros and cons of each. Mechanisms of thrombotic APS that have thus far been explored in animal models will also be briefly addressed.