Figure 3

(A) IgG (used at 200 μg/ml) effect on FXa (150 nM)-induced Ca²⁺ release. ****p < 0.0001 when APS-IgG (191.5 ± 11.3%) compared to FXa-only control (78.7 ± 6.9%) and to HC IgG (87.1 ± 3.7%), *p = 0.02 when compared to SLE IgG (152.1 ± 11.5%), dotted line represents IgG alone. Each bar represents mean ± standard error of mean (SEM) of n = 14 APS IgG, n = 14 SLE IgG and n = 8 HC IgG (B) Antistasin inhibited IgG potentiated FXa-calcium mediated responses. IgG that potently inhibited Ca²⁺ release were selected (shown as APS1 and APS2). Comparison of calcium release: by APS1 with inhibited vs unhibited FXa: ****p < 0.0001, by APS2 with inhibited vs uninhibited FXa: *p = 0.03. Potentiation of FXa-mediated release by IgG is also significant: APS1 ***p = 0.0008, APS2 *p = 0.03. Plotted with mean ± SEM (C) Inhibition of IgG potentiated FXa-induced Ca²⁺ release by PAR inhibitors and blocking antibodies. IgG that potently inhibited Ca²⁺ release were selected (shown as APS1 and APS2). ***p = 0.0006 APS1+RWJ-58259 vs APS1, **p < 0.05 APS1 + GB83/PAR 1 BL AB/ PAR2 BL AB vs APS1; ****p < 0.0001 APS2+RWJ-58259 vs APS2, **p < 0.05 APS1 + GB83/PAR 1 BL AB, *p = 0.01 APS2 + PAR2 BL AB vs APS2; Potentiation of FXa-mediated release by IgG is significant: APS1 **p = 0.005, APS2 *p = 0.04. Plotted with mean ± standard error of mean. APS: Antiphospholipid syndrome; ATAP2: PAR-1 blocking antibody; FXa: Factor Xa; GB83: PAR-2 selective antagonist; HC: Healthy control; ns: non-significant; RWJ–58259: PAR-1 antagonist; SAM11: PAR-2 blocking antibody; SLE: Systemic lupus erythematosus.