Male BALB/c mice (8-10 week old, weight 20-22 g) were housed in standard environmental conditions. All experiments were conducted in accordance with the Bulgarian Food Safety Agency Guidelines №352 06.01.2012 and approved by the Animal Care Committee at the Institute of Microbiology, Sofia.

Mice were were divided into following groups (n=10 each in 2 separate experiments): non-arthritic, ZIA injected i.a. with 180 mg zymosan A from Saccharomyces cerevisiae (Sigma-Aldrich, Germany) and ZIA+CVF (Sigma-Aldrich, injected intraperitoneally with 10 ng/g body weight of CVF, 72 and 24 h before i.a. injection of 180 mg zymosan.

The paws were embedded in paraffin, serial sections were cut and mounted on glass slides. The sections were deparaffinised and after blocking of endogene peroxidase activity were incubated for 1 h with PE-conjugated anti-mouse CD62P antibody (BioLegend, San Diego, USA) and stained with with DAB solution kit (3 ,3 diaminobenzididne kit, Sigma-Aldrich). The number of cells stained positive for the examined protein was determined by imaging system software (ImageJ 1.42; Research Services Branch, NIH, USA). Isotype rabbit antibody waw used as specific control.

Data were analyzed by two way ANOVA, using InStat3.0 and GraphicPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Differences were considered significant when P<0.05.

The results demonstrated that the number of CD62P positive cells increased in CVF-treated group compared to mice with ZIA at the active stage of arthritis (day 18). Giant megakaryocytes with already formed platelets were found in CVF group (Figure 1A, B and F). Such cells were absent in non-arthritic synovium (Figure 1C). The progression of arthritis resulted in significant elevation of megakaryocyte number in ZIA mice especially at the sites of synovial inflammation at the chronic phase (day 30) markedly inhibited in CVF group (Figure 1D, E and F). At day 18 the number of CD62P positive cells in cartilage was higher in CVF-treated mice compared to ZIA mice (Figure 2A, B and F) and such cells were almost absent in non-arthritic group (Figure 2C). Single positive cells were found in arthritic group at late stage of ZIA elevated in CVF group (Figure 2D, E and F).

Megakaryocytes can modulate the differentiation of osteogenic cells by the secretion of regulatory proteins, including osteocalcin, osteonectin, bone morphogenetic proteins, and OPG. The increased high local concentrations of MKs have a direct effect on osteoblastogenesis mediated by direct cell-to-cell contact of MKs and osteoblasts12. Therefore, MKs could influence bone formation and bone remodeling in a result of osteoblast proliferation and development or inhibition of OC development. There is such possibility if viable MKs persist for a longer duration in the bone marrow13. Complement activation might increase bone resorption via enhancing osteoclastogenesis, while recruitment of osteoblast precursors might favour bone formation.

Present data show that ZIA is an appropriate model to reveal the role of MKs in arthritis pathology. CD62P marker was used as it is expressed by megacaryocytes and platelets, thus we observed positive cells at different stages of MK maturation. Considering the abundance of MK in the synovium and cartilage, it is reasonable to assume that platelets by releasing cytokines and chemokines may recruit more inflammatory cells like neutrophils to the sites of inflammation. The effect of CVF-treatment differed in regard to active and established phases of ZIA. MKs, osteoblasts, and osteoclasts may interact with each other in their natural environment during joint inflmmation, as dysregulation of the MK lineage corresponds with bone pathology both in mice and in humans. Further studies are necessary to test these possibilities and to elucidate the participation of complement in MK-dependent mechanisms.