Protein phosphorylation during tannin-mediated activation of human platelets

Experiments to explore human platelet protein phosphorylation changes and 5-hydroxytryptamine (5-HT) secretion after challenge with cotton bract tannin were performed. Quantitative changes in sodium phosphate phosphorus 32 incorporation in two platelet proteins of 19 kilodaltons (kd) and 47 kd were assessed by measuring protein band densities on autoradiographs of dried polyacrylamide gels. Secretion of 5-HT was assessed by 14C-5-HT release. Results show that tannin causes increases in phosphorylation of discrete platelet proteins that begin in less than 2 seconds. These increases are maximal in 1 minute for the 47 kd protein and in 3 minutes for the 19 kd protein. Fifty percent of maximum response required less than 2 seconds for both of these proteins, and 50% of maximum 5-HT secretion required 48 seconds. Dose-response studies comparing 0 to 50 micrograms/ml tannin with 0 to 1 U/ml human alpha-thrombin showed that tannin caused 5-HT secretion and protein phosphorylation changes that were very similar to those induced by human alpha-thrombin. Fifty micrograms per milliliter of tannin caused increases in 19 kd protein phosphorylation and 47 kd protein phosphorylation to 312% +/- 34% (SEM) and 204% +/- 13% of control, respectively (n = 14). One unit per milliliter of thrombin induced changes of 350% +/- 40% and 221% +/- 17% of control in the 19 kd and 47 kd proteins, respectively. Release of 5-HT by tannin and thrombin was 61% +/- 3% and 69% +/- 3% of total cellular 5-HT, respectively. Indomethacin had little inhibitory effect on activation by these two different agents.(ABSTRACT TRUNCATED AT 250 WORDS)