Factor Xa Mediates Calcium Flux in Endothelial Cells and is Potentiated by Igg From Patients With Lupus and/or Antiphospholipid Syndrome

Abstract

Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca²⁺). Therefore, we measured alterations in Ca²⁺ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca²⁺ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects' IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca²⁺ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca²⁺ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca²⁺ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.

Figure 1

HUVEC response to FXa, thrombin and PAR agonist peptides. Fluo-4 –loaded HUVEC were stimulated by PAR-1 and PAR-2 agonists and fluorescence intensity readings recorded continuously for 10 minutes as described. Data are shown as representative traces of intracellular Ca²⁺ release upon stimulation with (A) Thrombin (10 nM); (B) FXa (150 nM) (C) PAR-1 agonist peptide TFLLR and PAR-2 agonist peptide SLIGKV. (D) Concentration-response curves in HUVECs following Thrombin (3–30 nM) and FXa (30–150 nM) stimulation.

Figure 2

(A) FXa-mediated calcium responses are inhibited by specific FXa inhibitor Antistasin related peptide at concentrations 25, 50 and 100 μM. (B) Inhibition of FXa-induced Ca²⁺ release by PAR inhibitors and blocking antibodies. (C) Inhibition of thrombin-induced Ca²⁺ release by PAR inhibitors and Antistasin. Data are plotted with mean ± standard error of mean (SEM), 1-way ANOVA **p < 0.05, ***p < 0.001 when compared to FXa alone at all concentrations compared to FXa or thrombin alone. Dotted line represents control buffer. ATAP2: PAR-1 blocking antibody; FXa: Factor Xa; GB83: PAR-2 selective antagonist; RWJ–58259: PAR-1 antagonist; SAM11: PAR-2 blocking antibody, ns: non-significant.

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.

Figure 4

Effect of drugs on FXa (150 nM)-mediated Ca²⁺ signaling. Cells were treated with HCQ and fluvastatin for 20 hours and stimulated with FXa 150 nM (A) Hydroxychloroquine (HCQ) (used at 1.25, 2.5, 5, 10 μg/ml) *p = 0.02, **p = 0.001, ***p < 0.001 for comparison of FXa stimulation of untreated cells vs 2.5, 5, 10 μg/ml HCQ treated HUVEC respectively. (B) Fluvastatin (used at 1.25, 2.5, 5, 10 μM) ****p < 0.0001 for comparison of FXa stimulation of untreated cells vs 2.5, 5, 10 μM fluvastatin treated HUVEC respectively. (C) Inhibition of IgG-potentiated intracellular Ca²⁺ release in HCQ 5 μg/ml and fluvastatin 10 μM treated cells. IgG that mediated the strongest effect on Ca²⁺ release was selected. FXa + APS IgG vs FXa-only stimulation in untreated cells, *p = 0.03; FXa + APS IgG in untreated cells vs HCQ treated cells, *p = 0.01, vs fluvastatin treated cells **p = 0.009. Plotted with mean ± standard error of mean. Dotted line represents drug alone in graphs A and B.

Figure 5

Affinity purification of anti-FXa IgG and effects on FXa-mediated intracellular signaling. The anti-(a)FXa sub-fraction was affinity purified from n = 3 patients with the hightest FXa binding. (A) Gel showing flowthrough of serum passed through the Heparin column (lane 2), wash-step (lane 3), purification through aFXa column (lane 4) and subsequent IgG purification by Sepharose protein G (lane 5). (B) A graph showing the aFXa activity of IgG fractions: column 1, flow-through of heparin column; column 2, fraction containing IgG non-specifically bound to heparin; and column 3, affinity purified aFXa IgG. The values are OD at 450 nm. (C) Representative (n = 1) western blot and (D) densitometric analysis (n = 3) showing that FXa reactive whole IgG and affinity purified aFXa IgG significantly (p < 0.05) upregulates phosphorylation of NFkB in comparison to FXa alone or IgG alone.