ADP causes subsecond changes in protein phosphorylation of platelets.

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched-flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.