Phenotype of Platelet Is Altered and Associated with Disease Activity of Rheumatoid Arthritis

Platelets play a major role in the hemostasis physiologically through their activation. Activated platelets acquire functional abilities such as adhesion, morphological changes, and releasing humoral factors or microparticles (MPs). Recent reports have been shown that platelets and MPs include unique humoral factors such as chemokines and cytokines. We hypothesized that platelets and MPs can be vectors for transferring these factors to the lesions through circulation and contribute to disease processes of systemic diseases. To clarify the role of platelets in the pathogenesis of connective tissue diseases (CTDs), especially in rheumatoid arthritis (RA), we examined the phenotype and activation status of circulating platelets and the association with clinical characteristics.

Fifty-seven patients with RA were involved and 21 systemic sclerosis (SSc), which has different characteristic compared to RA in CTDs, and 16 healthy individuals (HI) were examined as controls. Platelet rich plasma was used to detect fractions of platelets and MPs by flow cytometry. Fraction of platelets and MPs were defined by the size using standardized Megamix® beads. For the phenotypic analysis of platelets, surface expression of CD62P (P-selectin), membrane-bound TGF (transforming growth factor)-beta, CD147 (emmprin), CD142 (tissue factor), CD31 (platelet endothelial cell adhesion molecule (PECAM)-1) on CD41+CD45-CD14- platelets was examined. For the activation status of platelets, the ratio of CD62P or PAC1-positive to -negative platelets for the activation and the ratio of MPs to platelets as a marker for the release of MPs. Clinical parameters at blood drawing were retrospectively obtained and correlation was examined.

We first examined the phenotype of circulating platelets in RA and controls to have a general picture of the difference. Proportion of CD62P+ platelets were higher in both RA and SSc compared to HI (0.054 ± 0.032, 0.060 ± 0.030 versus 0.021 ± 0.006, P = 0.0005, P < 0.0002, respectively). Interestingly, CD147+ platelets were significantly higher in RA (0.61 ± 0.05 versus 0.29 ± 0.01, P = 0.0004), whereas not only proportion of CD147+ platelets (0.69 ± 0.11) but that of TGF-beta+ platelets (0.073 ± 0.021 versus 0.014± 0.006) were higher in SSc compared to healthy controls (P = 0.0001, P = 0.0001, respectively). Thus, phenotype of platelets is altered in RA compared to HI or SSc. Then, we focused on RA and activation status of circulating platelets was further examined by expression of activation markers and release of MPs. Proportion of activated platelets such as CD62P (0.047 ± 0.041 versus 0.012 ± 0.007) and PAC1 (0.075 ± 0.072 versus 0.013 ± 0.011) and that of MPs (0.034 ± 0.06 versus 0.21 ± 0.03) were higher in RA compared to HI (P < 0.001, P < 0.004, P < 0.00001, respectively). In patients with RA, proportion of activated platelets (CD62P+ or PAC1+ platelets) and that of MPs were correlated each other (r = 0.57, P < 0.0001; r = 0.36, P < 0.004, respectively). When activation status of platelets was compared to clinical parameters, ratio of CD62P+ platelets and that of MPs were positively correlated with clinical parameters associated with disease activity of patients with RA, such as serum CRP (r = 0.36, P < 0.006 and r = 0.29, P < 0.03), erythrocyte sedimentation rate (r = 0.31, P < 0.02 and r = 0.35, P < 0.009) and composite disease markers such as Simplified Disease Activity Index (SDAI) (r = 0.28, P < 0.04 and r = 0.31, P < 0.02) and Clinical Disease Activity Index (CDAI) (r = 0.28, P < 0.04 and r = 0.32, P < 0.02), but interestingly, there was no correlation between proportion of PAC1+ platelets and clinical parameters. No correlation was observed with age, sex, HAQ-DI, nor MMP-3. Furthermore, six patients with RA who were in active phase and treated with methotrexate and/or biologics and achieved improvement of disease activity at 12 week after the treatment were compared. The ratio of CD62+ platelets and that of MPs were decreased along with the improvement of disease. (P < 0.03, P < 0.03, respectively).

In conclusion, circulating platelets in RA were activated and their activation status is correlated with the inflammatory markers and disease activity, suggesting that circulating platelets reflects and can contribute to the process of systemic chronic inflammation in RA. Phenotype of platelets can be a novel biomarker for the disease.