Correlation of the effects of NO on the thrombin-induced shape change and protein phosphorylation. [³²P]-prelabeled, aspirin-treated, and gel-filtered human platelets in the presence of CP/CPK were stimulated with thrombin (0.015 U/mL) and NO (1.7 × 10^-10 mol NO/mL/min). Shape change was continuously monitored by light transmission (A traces i-iii). Aliquots were withdrawn (arrows 1-12) for determination of VASP Ser157 phosphorylation (B) and [³²P]-protein phosphorylation (C). (A) Trace i shows the effect of thrombin (without NO infusion); trace ii is the effect of intermittent NO infusion to the platelet suspension combined with addition of thrombin; and trace iii is the effect of intermittent NO infusion to the platelet suspension (without thrombin stimulation). (B) Western immunoblot of proteins separated by 5% to 15% linear gradient SDS-PAGE, probed with mouse anti-VASP. The numbers indicate the following: (1) thrombin (0.2 U/mL) for 5 minutes without stirring; (2) control (stirred for 1 minute); (3) thrombin from 1 to 3 minutes; (4) NO infusion from 0 to 1 minute; (5) NO infusion from 0 to 3 minutes, and thrombin stimulation from 1 to 3 minutes; (6) NO infusion from 0 to 3 minutes, and thrombin stimulation from 1 to 6 minutes; (7) NO infusion from 0 to 3 minutes, and thrombin stimulation from 1 to 8 minutes; (8) NO infusion from 0 to 3 minutes, thrombin stimulation from 1 to 10 minutes, and NO infusion from 8 to 10 minutes; (9) NO infusion from 0 to 3 minutes; (10) NO infusion from 0 to 3 minutes, stop NO infusion from 3 to 6 minutes; (11) NO infusion from 0 to 3 minutes, stop NO infusion from 3 to 8 minutes; (12) NO infusion from 0 to 3 minutes, stop NO infusion from 3 to 8 minutes, and NO infusion from 8 to 10 minutes. (C) Aliquots from the same samples as in panel B were separated by 5% to 15% linear gradient SDS-PAGE and [³²P]-protein phosphorylation were visualized by autoradiography. The results shown are from a single experiment representative of 3.