Non-criteria antiphospholipid antibodies and calprotectin as potential biomarkers in pediatric antiphospholipid syndrome

Abstract

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.

Keywords: Antiphospholipid syndrome; Calprotectin; Non-criteria antiphospholipid antibodies; Pediatric; Thrombocytopenia.

Figure 1:. Calprotectin in plasma of pediatric antiphospholipid syndrome (APS) patients.

A, Levels of calprotectin were measured in patients with pediatric APS, pediatric venous thromboembolism (VTE), pediatric systemic lupus erythematosus (SLE), and healthy pediatric controls. Calprotectin levels in pediatric APS patients were compared with healthy controls by the Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s method; *p<0.05. B, Platelet count of pediatric APS patients negatively correlates with plasma calprotectin concentration according to Spearman’s correlation coefficient analysis.

Figure 2:. Effect of calprotectin on platelet viability.

A, Schematic illustration of platelet viability assay. Image created at www.biorender.com. B,D, Healthy donor platelets were incubated with 5% plasma from pediatric APS patients with low calprotectin (APS-Low-CP), pediatric APS patients with high calprotectin (APS-High-CP), or pediatric controls. Platelet viability was evaluated by flow cytometry using Calcein-AM dye (B) and annexin V FITC (D). The percentage of non-viable platelets and externalized phosphatidyl serine (PS), respectively, were then compared between groups by the Kruskal-Wallis test adjusted for multiple comparisons by Dunn’s method (*p<0.05, **p<0.01, ns=not significant). C,E, Calprotectin levels from pediatric APS patients were strongly correlated with the percentage of non-viable platelets evaluated by Calcein-AM dye (C; r=0.71, p=0.0182) and annexin V FITC (E; r=0.75, p=0.0098).