4,4'-DI-ISOTHIOCYANATOSTILBENE-2,2'-DISULFONIC ACID (DIDS) ACTIVATES PROTEIN-KINASE-C AND NA+ H+ EXCHANGE IN HUMAN PLATELETS VIA ALPHA(2A)-ADRENERGIC RECEPTORS/

Most agonists stimulate platelets by inducing Ca2+ mobilization, Ca2influx and protein kinase C (PKC) activation leading to Na+/H+ exchang e, exposure of fibrinogen-binding sites and aggregation. In contrast, previous studies showed that adrenaline induces exposure of fibrinogen -binding sites and aggregation without appreciable changes in cystolic Ca2+ content or PKC activity. In the present study we investigated pl atelet responses mediated via alpha2A-adrenergic receptors, using 4,4' -di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), which is know n to bind to this type of receptor. Addition of DIDS (2-20 muM) induce d (i) a rise in cytosolic pH of 0.23 + 0.05 pH unit (n = 5) as detecte d by BCECF fluorscence, due to activation of the Na+/H+ exchanger, (ii ) a 3.5-4-fold increase in the phosphorylation of the 47 kDa protein, a major substrate of PKC, (iii) exposure of 81072 +/- 7293 (n = 3) bin ding sites for I-125-fibrinogen per platelet, and (iv) irreversible ag gregation. These responses occurred without changes in cytosolic Ca2!, secretion of dense-granule contents and enhanced phosphoinositide m etabolism, and were not affected by inhibition of thromboxane A, gener ation (30 muM indomethacin). The alpha2A-adrenergic-receptor antagonis ts oxymetazoline (500 muM) and yohimbine (1 mM) completely abolished D IDS-induced responses. Inhibition of PKC (1 muM staurosporine) prevent ed phosphorylation of the 47 kDa protein, the increase in Na+/H+ excha nge and exposure of fibrinogen-binding sites. Thus our present data su ggest that activation of PKC is an early event in DIDS-induced platele t activation via the alpha2A-adrenergic receptor, which precedes any o f the other known signal-transducing sequences.