Lack of detection of P2X_1delprotein or ADP evoked P2X responses in human platelets. (A) Ten μg platelet total protein from 13 healthy human donors was analyzed by Western blotting (i) and (ii) as a control. Samples of recombinant P2X₁WT and P2X_1del receptors in HEK293 cells are shown. P2X₁WT receptors were detected in the platelets from each donor; the P2X_{1 del} receptor, however, was below the limit of detection. (B) To overcome the possibility that P2X_{1 del} receptor was not detected because of the weak expression level in platelets, dilution of one of the platelet protein samples (donor 4) was analyzed by Western blotting. The band corresponding to the P2X₁ WT receptor was still detectable when only 0.625 μg platelet total proteins were separated (the small box represents the 3 last lanes for which the autoradiography has been submitted to a longer exposure). Therefore, if P2X_{1 del} receptor is also expressed in human platelets, the ratio P2X_1del/P2X₁WT is less than 1:24. (C) [Ca²⁺]_i measurements in suspensions of fura-2-loaded human platelets, as indicated by the 340/380 fluorescence excitation ratio, in response to either 10 μM αβmeATP or 10 μM hexokinase-treated ADP (arrow). Traces from 2 donors (10 and 11 in panel Aii) are shown at 2 different time scales and are representative of the results from 13 donors. The cuvette temperature was set at 13°C to allow clear separation of P2X and P2Y receptor-evoked responses, as described in Mahaut-Smith et al.⁹