NK₁stimulates SP-mediated activation of platelets. (A) RNA isolated from human platelets (Pl; lanes 2, 4, 6) and HEL (H, human erythroleukemia cell line; lanes 1, 3, 5) was subjected to RT-PCR, resulting in the amplification of bands of the sizes 495, 449, and 528 bp representing NK₁, NK₂, and NK₃, respectively. (B) Whole cell lysates from platelets, U373MG, and HEL cells were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted to detect NK₁. (C) Flow cytometry was used to detect NK₁ protein on the surface of unstimulated or thrombin-stimulated (0.1 U/mL) platelets. Relative quantification was calculated using geometric mean data. Values shown are mean ± SEM (n = 3); **P < .02 (t test). (D) Platelets preincubated for 2 minutes with increasing concentrations of anti-NK₁ antibody or control antibody (Ctrl) were stimulated with SP (7.5 μM) for 90 seconds (arrow indicates addition of agonist). Aggregation was monitored by turbidometric aggregometry. Traces are representative of 3 separate experiments. (E) Platelets from NK₁-deficient mice and litter-matched controls were stimulated for 90 seconds with increasing concentrations of SP. The percentage of single platelets remaining in suspension was determined. Values shown are mean percentage of single platelets compared with basal ± SEM (n = 6). *P < .003 with respect to basal (within); **P < .05 between control and knock out.