Fig. 3

Extracellular versus intracellular Ca²⁺ in platelet activation on TiO₂ and on glass. a Platelets isolated in the calcium-containing buffer (2 mM CaCl₂) were incubated with bare (i and iii) or TiO₂-coated (ii and iv) glass cover slips for 10 min (i and ii) and 3 h (iii and iv), followed by extensive washing with the same buffer and incubation with antibodies specific against the platelet marker CD41a (green) and activation marker CD62P (P-selectin, red). The expression of P-selectin was observed on the majority of platelets both after 10 min and 3 h of contact with either surface. bHistograms Platelets isolated in the nominally calcium-free buffer were treated with BAPTA-AM (100 µM) for 30 min to chelate the intracellular Ca²⁺, and analyzed by flow cytometry. They do not express CD62P (left, red) or CD63 (right, red) upon stimulation with an agonist (TRAP). Untreated stimulated platelets do (pink). Non-stimulated platelets do not express CD62P or CD63 independently of the BAPTA-AM treatment (green and turquoise). Sample auto-fluorescence recorded in the absence of antibodies is shown in black. Plot: % of CD62P (blue)- and % of CD63 (red)- positive platelets as determined with flow cytometry by analyzing the histograms such as those shown on the left upon stimulation with 70 µM TRAP as a function of the BAPTA-AM concentration. Each point represents an average of six experiments, three of which were performed in the Ca-free HEPES buffer and three—in the citrate buffer; no differences were detected between the two sets. This plot demonstrates that the chelation of intracellular Ca²⁺ affects the expression of both of these markers in the same way. c BAPTA-AM treated platelets were incubated with the glass and TiO₂ surfaces and analyzed for CD41a (green) and CD62P or CD63 (red) expression by confocal microscopy. No spreading was observed on either of the two surfaces after 10 min or after 3 h of incubation (see Figure S3 in Supporting Information). Some CD62P expression is evident in platelets incubated on glass (i and iii). On the contrary, no CD62P expression was observed in platelets incubated on TiO₂ (ii and iv). Some CD63 expression is evident on both glass (v and vii) and TiO₂ (vi and viii) surfaces