Background/Purpose: We have previously reported that antiphospholipid antibodies (aPL) activate neutrophils and thereby exaggerate neutrophil extracellular trap release (NETosis), which potentially contributes to the thrombotic events inherent to antiphospholipid syndrome (APS). Recent evidence suggests that the second messenger cyclic AMP (cAMP) suppresses NETosis. Here, we hypothesized that surface adenosine receptors (which trigger cAMP formation in neutrophils) serve as an endogenous counterpoint to thrombo-inflammatory disease, and that pharmacological agonism of adenosine receptors may therefore mitigate the thrombotic manifestations of APS. In particular, our data led us to investigate the adenosine 2A receptor and its downstream pathways.

Methods: For in vitro studies, control neutrophils were prepared from healthy volunteers and stimulated with (i) total IgG pooled from primary APS patients, (ii) affinity-purified anti-β₂GPI antibodies, or (iii) phorbol 12-myristate 13-acetate (as a positive control). NETs were quantified by chromogenic detection of the enzymatic activity of NET-associated myeloperoxidase, or by SytoxGreen-based fluorescence measurement of extracellular DNA. For in vivo studies, thrombosis was triggered by surgical restriction of blood flow through the inferior vena cava, with resulting macroscopic thrombi assessed 48 hours later (a model of thrombosis that our group and others has shown to be accentuated by NETs).

Results: We began by assessing the effect of cAMP on aPL-mediated NETosis. 8-Br-cAMP (a cell-permeable, degradation-resistant cAMP derivative) significantly inhibited NETosis in dose-dependent fashion. As extracellular adenosine receptors are key regulators of neutrophil cAMP levels, we assessed agonism of the adenosine 2A, 2B, and 3 receptors. While CGS21680 (A2A agonist) suppressed NETosis with nanomolar potency, BAY60-6583 (A2B agonist) and 2-Cl-IB-MECA (A3 agonist) had minimal effect, even at micromolar concentrations. The antithrombotic agent dipyridamole is known to potentiate adenosine-mediated signaling by (i) increasing extracellular concentrations of adenosine, and (ii) interfering with the breakdown of cAMP. Indeed, dypridamole suppressed aPL-mediated NETosis in a dose-dependent manner. Finally, in a mouse model of macroscopic venous thrombosis, CGS21680 (A2A agonist) was highly effective in mitigating thrombosis (3-fold reduction in thrombus weight; p=0.028 with 15 mice per group).

Conclusion: We demonstrate for the first time that adenosine 2A receptor agonism can attenuate aPL-mediated NETosis in vitro and venous thrombosis in mice. Furthermore, dipyridamole (a drug known to potentiate adenosine-mediated signaling) phenocopies adenosine 2A receptor agonism in terms of suppressing in vitro NETosis. Studies are underway to fully characterize relevant signaling pathways in neutrophils, and to assess the therapeutic potential of dipyridamole against aPL-accelerated thrombosis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/adenosine-receptor-agonism-protects-against-antiphospholipid-antibody-mediated-netosis-and-venous-thrombosis/