Impact of human and mouse sPLA₂s on platelet MPs.

<p>(A) MPs from human platelets (stimulated with collagen) labeled with the CMFDA cell tracker were incubated for 1 and 6 hours at 37°c in absence or in presence of indicated concentrations of human recombinant sPLA₂ IIA, V, X, or 1µg/ml of the inactive mutant V H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and were compared to the untreated conditions (dotted line). Data are mean ± SEM of 5 independent experiments presented as % of untreated (control) (B) MPs from mouse platelets (stimulated with collagen), identified using YFP as fluorescent tracker, were incubated 1 and 6 hours at 37°c, in absence or in presence of indicated concentrations of mouse recombinant sPLA₂ IIA, V, X, or 1µg/ml of the inactive mutant X H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to determine the concentrations of YFP⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and then compared to the untreated conditions (dotted line). Data are mean ± SEM of 5 independent experiments presented as % of untreated (control). (C) MPs from human platelets labeled with the CMFDA cell tracker and obtained following stimulation with collagen, thrombin or HA-IgG were incubated 6 hours at 37°c in absence or in presence of indicated concentration of human recombinant sPLA₂ IIA, V and X and 1µg/ml of the inactive mutant sPLA₂ V H48Q. Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CD41⁺ MPs (middle panel), of annexin-V⁺ MPs (right panel) and then compared to the untreated conditions (dotted line). Data are mean ± SEM of 3 independent experiments presented as % of untreated (control). (D) MPs from human platelets (stimulated with collagen) labeled with the CMFDA cell tracker were incubated 6 hours at 37°c in PFP of C57BL6 (supplemented or not with 1µg/ml of recombinant human sPLA₂ IIA) or transgenic mice expressing the human sPLA₂ IIA (Tg). Fluorochrome-conjugated antibodies directed against CD41 and fluorochrome-conjugated annexin-V were used to assess the quantities of CMFDA⁺ MPs (left panel), of CMFDA⁺ CD41⁺ MPs (middle panel) and CMFDA⁺ annexin-V⁺ MPs (right panel). Data are mean ± SEM of 3 independent experiments. (E) Concentrations of Annexin-V⁺ MPs and CD41⁺ MPs present in the synovial fluids of RA patients determined by high sensitivity flow cytometry and correlated to the concentration of human sPLA₂ IIA assayed (in the same synovial fluids) by time-resolved immunofluorescence analysis. * P< .05; # P< .01; § P< .001.