Thrombin Causes Subsecond Changes in Protein Phosphorylation of Platelets

We have developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20and 40-kd proteins has been analyzed during the first five seconds of platelet response to thrombin from 0.1 to 5.0 U/mi and compared with the progress of aggregation and serotonin secretion. The onset time for aggregation and phosphorylation of both proteins was less than one second, although with lowest (<0.5 U/mi) thrombin levels, a lag of up to 0.6 seconds occurred before 40K phosphorylation increased. The thrombin sensitivity of aggregation and 20K phosphoryla-