Increased platelet reactivity as measured by plasma glycoprotein VI in gout.

Patients with gout have an increased risk of cardiovascular events. The glycoprotein VI (GPVI) receptor is found exclusively on platelets and megakaryocytes, is proteolytically cleaved upon platelet activation, and detectable in plasma as soluble GPVI (sGPVI). Therefore, elevated sGPVI is a marker of platelet activation and a risk marker for cardiovascular events. The aim of this study was to assess platelet activation, as measured by plasma sGPVI level in gout. Blood samples were taken from patients with gout or osteoarthritis, and from healthy volunteers. Demographic and clinical data were collected for all participants. Blood samples were processed as double-spun platelet-poor plasma. Plasma sGPVI levels were measured using enzyme-linked immunosorbent assay. Mann-Whitney U test was used to compare groups. In total, 91 patients were included, 27 during gout flare, 41 with intercritical gout, 23 with osteoarthritis, and 53 healthy controls. Median (interquartile range) sGPVI levels were 6.51 ng/mL (4.52, 8.41) in gout flare, 3.58 ng/mL (2.11, 5.55) in intercritical gout, 2.73 ng/mL (2.17, 3.72) in osteoarthritis, and 2.19 ng/mL (1.72, 3.31) in healthy controls. Plasma sGPVI levels in both gout groups were significantly increased compared to healthy controls (p < 0.005 for each), in gout flare compared to osteoarthritis (p < 0.005), and in gout flare compared to intercritical gout (p = 0.001). There was no significant difference in sGPVI levels in gout patients with and without tophi or in those prescribed colchicine. We conclude that patients with gout exhibit platelet hyperactivity as demonstrated by elevated sGPVI levels. Platelet activation is exacerbated in gout, especially during gout flares.

Soluble glycoprotein VI, a specific marker of platelet activation is increased in the plasma of subjects with seropositive rheumatoid arthritis.

OBJECTIVES: Anti-citrullinated protein antibodies (ACPA) have been shown to cause platelet activation in vitro, through the low-affinity immunoglobulin G (IgG) receptor (FcγRIIa) on platelets. Platelet activation via engagement of FcγRIIa results in proteolytic cleavage and shedding of platelet specific glycoprotein VI (GPVI) which can be detected in the plasma as soluble GPVI (sGPVI). We hypothesized that plasma levels of sGPVI would be increased among patients with seropositive RA as a consequence of antibody-induced platelet activation and GPVI shedding. METHODS: Samples from 84 patients with RA (65 seropositive and 19 seronegative) and 67 healthy controls were collected prospectively and analysed for sGPVI using a standardised ELISA. RESULTS: Patients with seropositive RA had significantly higher levels of sGPVI compared to seronegative RA and controls. Median (IQR) sGPVI levels were 4.2 ng/ml (3.2, 8.0) in seropositve RA, 2.2 ng/ml (1.5, 3.5) in seronegative RA and 2.2 ng/ml (1.6, 3.4) in controls (p<0.0001). sGPVI levels correlated with ACPA titres (r = 0.32, p = 0.0026) and with RF titres (r = 0.48, p<0.0001). CONCLUSION: Plasma sGPVI, a specific marker of platelet activation is increased among patients with seropositive RA.

Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells.

BACKGROUND: Osteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation. METHODS: Primary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR). RESULTS: We demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1ß and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule S100A8 and MMP1 in a Syk-dependent manner and that synovial fluid from OA patients together with BCP crystals exacerbates these effects. CONCLUSIONS: We identify Syk and PI3K as key signalling molecules activated by BCP crystals prior to inflammatory cytokine and DAMP expression and therefore propose that Syk and PI3K represent potential targets for the treatment of BCP-related pathologies.