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. 2016 Mar 22;291(20):10586–10601. doi: 10.1074/jbc.M115.699058

FIGURE 1.

FIGURE 1.

PMPs bind Gas6 and protein S. Scanning electron micrographs of unstimulated isolated human platelets and human platelets stimulated with a calcium ionophore to induce MP formation are shown. A, Scale bars, 5 μm (left and middle panel) and 100 nm (right panel). The MP size distribution was evaluated by nanotracking analysis and showed a mean size of just below 200 nm (B). PMPs were evaluated for phosphatidylserine exposure by measuring their ability to bind fluorescent lactadherin using flow cytometry (C). PMPs were incubated with increasing amounts of Gas6 and protein S before fluorescent antibodies (Ab) were added. Binding was monitored by flow cytometry (D and E). PMPs were treated with or without phospholipase A2 (PLA2) before Gas6 binding was evaluated by flow cytometry (F). Gas6 was pre-incubated with liposomes composed of PS, PE, and PC or PE and PC alone before incubation with PMPs. Gas6 binding was evaluated with flow cytometry (G). PMPs were incubated with γ-carboxylated (own Gas6) and non-γ-carboxylated Gas6 (RnD Gas6), and the amount of bound protein was evaluated with an antibody recognizing both Gas6 variants using flow cytometry (H). Gas6 was incubated with PMPs in the presence or absence of 5 mm EDTA to measure the calcium dependence of the Gas6-MP interaction (I). PMPs were incubated with 250 ng/ml Gas6 in the presence of increasing amounts of protein S after which fluorescent Gas6-recognizing antibodies were added. The amount of PMP-associated Gas6 was evaluated using flow cytometry (J). The expression of TAMs on the surface of the platelets was evaluated using flow cytometry (K). sAxl at increasing concentrations was pre-incubated with Gas6 before adding PMPs, and the amount of MP-bound Gas6 was evaluated using flow cytometry (L). Data in D–J and L are shown as mean and S.D. of three individual experiments. C and K shown representative histograms from at least three individual experiments. ns, not significant; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. MFI, mean fluorescent intensity.