okadaic-acid and 1-2-dihexanoylglycerol

The primary phase of ADP-induced aggregation of human platelets does not involve appreciable formation of thromboxane A2 or release of granule contents; lack of formation of inositol trisphosphate has also been noted. Because these responses of platelets to ADP differ so markedly from their responses to other aggregating agents, the roles in ADP-induced aggregation of diacylglycerol, protein kinase C, increases in cytosolic [Ca2+], phosphorylation of pleckstrin (47 kDa) and phosphatases 1 and 2a were investigated. Washed human platelets, prelabelled with [14C]5-hydroxytryptamine and suspended in Tyrode solution (2 mM Ca2+, 1 mM Mg2+), were used for comparisons between the aggregation induced by 2-4 microM ADP, in the presence of fibrinogen, and that induced by 0.05 units/ml thrombin. The diacylglycerol kinase inhibitor 6-(2-[(4-fluorophenyl)phenyl-methylene]-1-piperidinylethyl)-7-meth yl-5H-thiazolo[3,2-a]-pyrimidin-5-one (R59022; 25 microM) had no, or only a slight, enhancing effect on ADP-induced aggregation, but potentiated thrombin-induced responses to a much greater extent. 1,2-Dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol (25 microM) added with or 30-90 s before ADP greatly potentiated aggregation without formation of thromboxane; staurosporine, an inhibitor of protein kinase C, reduced this potentiation. Staurosporine (25 nM) did not inhibit ADP-induced aggregation, although it strongly inhibited thrombin-induced aggregation and release of [14C]5-hydroxytryptamine. All these observations indicate little or no dependence of primary ADP-induced aggregation on the formation of diacylglycerol or on the activation of protein kinase C. At 2-4 microM, ADP did not significantly increase the phosphorylation of pleckstrin (studied with platelets prelabelled with [32P]orthophosphate), but 1,2-dihexanoyl-sn-glycerol- induced phosphorylation of pleckstrin was increased by ADP. Surprisingly, the diacylglycerols strongly inhibited the ADP-induced rise in cytosolic [Ca2+] concurrently with potentiation of ADP-induced aggregation; thus the extent of primary aggregation is independent of the level to which cytosolic [Ca2+] rises. Incubation of platelets with 1,2-dihexanoyl-sn-glycerol or 1-oleoyl-2-acetyl-sn-glycerol for several minutes reversed their potentiating effects on aggregation, and inhibition was observed. Incubation of platelets with okadaic acid, an inhibitor of phosphatases 1 and 2a, inhibited ADP- and thrombin-induced aggregation; although th

Topics: Adenosine Diphosphate; Alkaloids; Blood Platelets; Diacylglycerol Kinase; Diglycerides; Enzyme Activation; Ethers, Cyclic; Humans; Okadaic Acid; Phosphotransferases; Platelet Aggregation; Protein Kinase C; Pyrimidinones; Serotonin; Staurosporine; Thiazoles; Thrombin; Type C Phospholipases

Phosphorylation of the Na+/H+ exchanger in human platelets is apparently controlled by the balancing activities of protein kinase C (PKC) and protein phosphatase (PP). To explore cellular expressions of these activities, we have examined the impact of modulation of PKC and PP on Na+/H+ exchange activity, its pHi set point and intracellular pH (pHi). These parameters were followed spectrofluorimetrically in BCECF-loaded platelets. Phorbol 12-myristate 13-acetate (PMA) and dihexanoylglycerol (DHG), which stimulate PKC, and okadaic acid, which inhibits PP 1 and 2A, elevate the measured parameters in concert, while staurosporine, which inhibits protein kinases, had opposite effects. The stimulatory and inhibitory effects are similarly very rapid, being discerned within seconds. It is concluded that: (a) phosphorylation of the Na+/H+ exchanger is the common origin of the diverse effects of PMA, DHG, okadaic acid and staurosporine, (b) Na+/H+ exchange properties are tightly regulated by phosphorylation and dephosphorylation, and (c) the exchanger plays a major role in pHi regulation in platelets.

Topics: Alkaloids; Blood Platelets; Carrier Proteins; Cytoplasm; Diglycerides; Ethers, Cyclic; Fluoresceins; Humans; Hydrogen-Ion Concentration; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Sodium-Hydrogen Exchangers; Staurosporine; Tetradecanoylphorbol Acetate