EDTA inhibits collagen-induced ATP+ADP secretion and tyrosine phosphorylation in platelets independently of Mg 2+ chelation and decrease in pH

The effects of Mg2+ and EDTA on collagen-induced platelet dense granule secretion and protein tyrosine phosphorylation were studied in the presence and absence of various inhibitors of autocrine agonists that are released from secretory granules. Addition of EDTA to gel-filtered platelets in Tyrode's solution caused a decrease in pH of 0.7-0.9 pH units, due to chelation of Mg2+, and a marked inhibition of cATP+ ADP secretion induced by collagen ( 25 mug/ml). Lowering pH of the platelet suspension to the same extent by HCl had no effect on secretion which also was the same in the absence of exogenous Mg2+ as in its presence. Similarly, secretion induced by collagen per se, i.e., isolated from autocrine agonists by inhibitors, which was about 50% less than with autocrine agonists, was not affected by the presence or absence of exogenous Mg2+. The level of tyrosine phosphorylation of several proteins in resting platelets was higher in the absence of Mg2+ than in its presence, but the onset of collagen-induced phosphorylation and dephosphorylation was more rapid in the absence of Mg2+. Tyrosine phosphorylation of p38 was specifically enhanced in the presence of inhibitors of autocrine phosphorylation and even more enhanced in the absence of Mg2+. EDTA inhibited the protein phosphorylation to the same extent in the presence as in the absence of exogenous Mg2+. These results show that EDTA inhibits collagen-induced dense granule secretion neither through chelation of Mg2+ nor lowering of pH, and thus probably through other effects on the platelet by the EDTA(2-) species that are reflected in protein tyrosine phosphorylation signalling.