Fig. 1

Flow cytometry analysis of purified platelets. Platelets isolated from sodium citrate-anticoagulated blood were re-suspended in a nominally Ca²⁺-free buffer and analyzed by flow cytometry [33, 53, 55, 56, 82, 83]. Results of one representative experiment are shown as percentages of positive events out of a total of 10,000 events are shown on the plots. Average values from several experiments are shown in Figure S2 in the Supporting Information. a The scatter profile shows one population of cells, 95 % of which stained positive for the platelet-specific marker CD41a (platelet transmembrane glycoprotein GPIIb). This is consistent with what is expected of isolated quiescent platelets that are free of contaminants such as erythrocytes and white blood cells [56, 82, 83]. Platelets stain positive for CD62P (b) and CD63 (c) upon addition of 70 µM TRAP (green and pink histograms) but negative in its absence (orange and turquoise histograms). Expression of CD62P and CD63 in TRAP-activated platelets is independent of the extracellular Ca²⁺: the pink (absence of Ca²⁺) and the green (presence of Ca²⁺) histograms nearly overlap; this is consistent with previous findings [56, 57]. Isotype controls are negative in the presence and in the absence of TRAP (light purple and grey). Sample auto-fluorescence recorded in the absence of antibodies is shown in black. d Platelets expose PS upon activation with 70 µM TRAP as judged by the binding of APC-labeled annexin A5 in the presence of extracellular calcium (green histogram). Platelets that were not activated with TRAP do not expose PS (turquoise histogram) even when Ca²⁺ is present (orange histogram). Annexin A5 binding is Ca²⁺-dependent; therefore, it is not possible to check whether PS is expressed on the surface of TRAP-activated platelets in the absence of Ca²⁺ with this reagent. Black sample auto-fluorescence in the absence of APC-annexin A5. e Platelets activated with 10 µM PMA express the active form of GPIIb/IIIa, as judged by the binding of PAC1 antibody in the presence of Ca²⁺ (green histogram) [54, 82]. No binding is detected in platelets that were not activated by PMA (orange and turquoise histogram). The inactive-to-active conformation change in GPIIb/IIIa is Ca²⁺-dependent. The Ca²⁺ requirement for PAC1 antibody binding is well-established in the literature and was not tested in our experiments [53, 83, 86]. Isotype controls are negative in the presence and in the absence of PMA (light purple and grey)