Background/Purpose
Recently we showed that FXa reactive IgG in patients with the antiphospholipid syndrome (APS) displayed higher avidity binding to FXa and had greater functional effects upon the coagulant functions of FXa compared to patients with anti-FXa IgG isolated from patients with systemic lupus erythematosus (SLE) without APS. FXa signalling has been observed in many cell types via activation of protease-activated receptors (PAR) which stimulate an increase in intracellular calcium (Ca2+) levels. Given that cellular responses elicited through activation of PARs are important in influencing pathways responsible for inflammation, tissue repair and haemostasis, FXa-reactive IgG may be important in the pathogenesis of SLE and APS. Therefore, we characterised the interaction between FXa and human umbilical vein endothelial cells (HUVEC) and the cellular effects of anti-FXa IgG isolated from patients with APS and SLE without APS.
Methods
IgG was purified from patients with APS (n=14) and SLE/no APS (n=14) who had medium or high serum levels of anti-FXa IgG and healthy controls (HC) (n=8). The effect of IgGs on FXa-mediated intracellular calcium release was measured using the fluorescent image plate reader (FLIPR). HUVECs were seeded in 96-well plates, for 48 hours until confluent and then incubated with Fluo-4AM calcium binding dye for one hour. Alterations in intracellular calcium release were monitored for 10 minutes following stimulation with α-Thrombin (Thr) or FXa +/- IgGs. PAR inhibitors, agonist peptides (AP), hydroxychloroquine (HCQ), fluvastatin and desensitisation experiments were also performed to characterise responses. Results
We observed a dose-dependent induction of Ca2+ transients by FXa in HUVEC. FXa was a weaker agonist than Thr but the kinetics of response was different with a longer lag-time and duration. APS IgG significantly potentiated the FXa-induced Ca2+ release compared to SLE/no APS IgG (p=0.04), HC IgG (p<0.05) and FXa alone (p<0.05). FXa-mediated Ca2+ release was significantly reduced by PAR-1 inhibitors. When PAR-1 receptors were desensitised by Thr (10 nM) and cells were stimulated by PAR-2 AP, PAR-2 response was reduced by 47.5%. When Thr-desensitised cells were stimulated by FXa, response was reduced by 66% and residual response was not inhibited by PAR-1 or completely by 2 inhibitors. When cells were treated with: Antistasin, a specific FXa inhibitor; Hydroxychloroquine; or fluvastatin – FXa induced and IgG potentiated intracellular Ca2+ release was significantly reduced.
Conclusion
We confirmed FXa stimulation of HUVEC to be mediated mainly via PAR1 and 2 dependent signalling mechanisms that may be acting as a unit and this effect was enhanced by FXa-reactive APS-IgG. These effects were blocked by a FXa inhibitor which raises the exciting prospect that anti-FXa IgG positivity may be used as a novel biomarker to predict response to treatment with FXa inhibitors in patients with APS. Despite the need for the specificity to be investigated further, results also suggest that HCQ and fluvastatin may be beneficial in patients with APS.
Disclosure:
B. Artim-Esen,
None;
N. Smoktunowicz,
None;
V. M. Ripoll,
None;
C. Pericleous,
None;
R. Chambers,
None;
I. Mackie,
None;
D. Isenberg,
None;
A. Rahman,
None;
Y. Ioannou,
None;
I. Giles,
None.
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-cellular-effects-of-anti-factor-xa-antibodies-isolated-from-patients-with-antiphospholipid-syndrome-are-inhibited-by-factorxa-inhibitors-hydroxychloroquine-and-fluvastatin/