4,4'-Di-isothiocyanatostilbene-2,2'-disulphonic 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, Ca2+ influx and protein kinase C (PKC) activation leading to Na+/H+ exchange, 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 platelet responses mediated via alpha 2A-adrenergic receptors, using 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS), which is known to bind to this type of receptor. Addition of DIDS (2-20 microM) induced (i) a rise in cytosolic pH of 0.23 +/- 0.05 pH unit (n = 5) as detected by BCECF fluorescence, 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 81,072 +/- 7293 (n = 3) binding sites for 125I-fibrinogen per platelet, and (iv) irreversible aggregation. These responses occurred without changes in cytosolic [Ca2+], secretion of dense-granule contents and enhanced phosphoinositide metabolism, and were not affected by inhibition of thromboxane A2 generation (30 microM indomethacin). The alpha 2A-adrenergic-receptor antagonists oxymetazoline (500 microM) and yohimbine (1 mM) completely abolished DIDS-induced responses. Inhibition of PKC (1 microM staurosporine) prevented phosphorylation of the 47 kDa protein, the increase in Na+/H+ exchange and exposure of fibrinogen-binding sites. Thus our present data suggest that activation of PKC is an early event in DIDS-induced platelet activation via the alpha 2A-adrenergic receptor, which precedes any of the other known signal-transducing sequences.
Department of Hematology, University Hospital Utrecht, The Netherlands.
R
R
Nieuwland
G
G
Van Willigen
J W
JW
Akkerman
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D002352:Carrier Proteins / Q000187:drug effects* / Q000378:metabolism; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000187:drug effects; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D010712:Phosphatidic Acids / Q000096:biosynthesis; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects; D011493:Protein Kinase C / Q000378:metabolism*; D011942:Receptors, Adrenergic, alpha / Q000187:drug effects* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D010712:Phosphatidic Acids; D011942:Receptors, Adrenergic, alpha; D017923:Sodium-Hydrogen Exchangers; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D005340:Fibrinogen; D011493:Protein Kinase C; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid