Sushila Krishnamurthi

Weak inhibition of protein kinase C coupled with various non-specific effects make sphingosine an unsuitable tool in platelet signal transduction studies

The effects of sphingosine, the newly described inhibitor of the enzyme protein kinase C, on human platelet activation, were studied in order to gain further information on the role of protein kinase in platelet responses. Concentrations of the drug (5-20 microM) which had little effect on protein kinase C activation as measured by the phosphorylation of the 45 kDa and 20 kDa protein substrates induced by phorbol 12-myristate 13-acetate (PMA) and thrombin, strongly inhibited platelet aggregation induced by these agonists, as well as aggregation induced by ADP and ionomycin, which caused no detectable protein kinase C activation or 5-hydroxy[14C]tryptamine[( 14C]5HT) secretion. At approx. 10-fold higher concentrations (150-200 microM), sphingosine had significant inhibitory effects on PMA and thrombin-induced 45 kDa and 20 kDa protein phosphorylation. However, at these high concentrations, the drug caused extensive membrane damage/leakiness as suggested by the substantial release of [14C]5HT and [3H]adenine from pre-loaded platelets (50-70% release of both markers), and the total quenching of quin2 fluorescence by Mn2+ in the presence of the drug. Due to the increased membrane leakiness in the presence of the drug, an apparent potentiation of agonist-induced intracellular Ca2+ elevations in quin2-loaded platelets, as well as an increase in quin2 fluorescence with the drug alone (more than 50 microM) were also observed. Despite this, however, thrombin-induced [3H]arachidonate release was severely reduced in the presence of sphingosine, underlining the inhibitory effects at the membrane level. It is concluded that the weak, if any, inhibitory effects on protein kinase C at concentrations not affecting membrane integrity, as well as the inhibitory effects of sphingosine on platelet aggregation, make it an unsuitable compound as a tool for studies on platelet stimulus-response coupling.

Lack of inhibition of thrombin‐induced rise in intracellular Ca2+ levels and 5‐hydroxytryptamine secretion by 1‐oleoyl‐2‐acetylglycerol in human platelets

The effect of 1‐oleoyl‐2‐acetylglycerol (OAG) on the thrombin‐induced rise in intracellular Ca2+ levels ([Ca2+]i) and 5‐hydroxy[14C]tryptamine ([14C]5HT) secretion was studied. In washed human platelets prelabelled with [14C]5HT and quin 2, OAG (10–50 ) induced no significant aggregation, [14C]5HT secretion or rise in [Ca2+]i in the presence or absence of fibrinogen. However, addition of OAG (10–50 ) 10 s to 5 min before or 10–60 s after addition of threshold concentrations of thrombin ( < 0.03 ) resulted in a significant potentiation of aggregation and [14C]5HT secretion without any effect on the thrombin‐induced rise in [Ca2+]i. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced [14C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Ca2+]i nor the extent of [14C]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrombin‐induced [Ca2+]i mobilisation but can synergize with low concentrations of thrombin in potentiating [14C]5HT secretion even at basal [Ca2+]i.

Na+/H+ exchange is not necessary for protein kinase C-mediated effects in platelets

The role of Na+/H+ exchange in protein kinase C‐mediated effects in platelets was investigated by studying the effect of removal of extracellular Na+ ([Na+]e) on the different responses induced by phorbol 12‐myristate 13‐acetate (PMA) and 1,2‐ dioctanoylglycerol (diC8). None of the responses studied, namely, protein phosphorylation, translocation of enzyme activity to the membrane fraction, potentiatory and inhibitory effects on platelet activation ([Ca2+]i, arachidonate and granule release) showed an absolute dependence on [Na+]e. With the exception of dense‐granule release, which was clearly potentiated by the removal of [Na+]e and showed a negative correlation with exchanger activity, the other effects of PMA and diC8 were not affected by [Na+]e removal. It is concluded that Na+/H+ exchange is not essential for protein kinase C activation in platelets.

Extracellular Na+ removal enhances granule secretion in platelets 3-evidence that Na+/H+ exchange is inhibitory to secretion induced by some agonists

The effect of extracellular Na+ ([Na+]e) removal on agonist‐induced granule secretion in platelets in relation to [ph]i and [Ca2+]i changes was investigated. Substitution of [Na+]e with choline+ of K+ resulted in a significant enhancement of 5HT secretion induced by thrombin, collagen, U46619 and the protein kinase C activators, PMA and diC8. Increases in [Ca2+]i induced by thrombin and U46619 were slightly inhibited or unaffected in these buffers, but [pH]i increases induced by thrombin, U46619, PMA and diC8 were abolished and a drop in [pH]i (0.05–0.1 units below resting) was observed. Although preincubation with potassium acetate produced a big drop in [pH]i and greatly increased secretion with all the agonists, particularly in the absence of [Na+]e, clear evidence that [pH]i rises due to Na+/H+ exchange are inhibitory to secretion was obtained only with thrombin. Thus, (i) NH4Cl, which restored the increase in [pH]i in the absence of [Na+]e reduced the potentiated secretory response to thrombin, (ii) no increase in thrombin‐induced secretion was observed when Na+ was replaced with Li+, which allowed a normal increase in [pH]i and (iii) ethyl isopropyl amiloride (EIPA) abolished the [pH]i rise and potentiated thrombin‐induced secretion. With collagen and U46619, the results suggest that removal of [Na+]e per se rather than inhibition of Na+/H+ exchange results in enhanced secretion. It is concluded that [Na+]e per se and [pH]i elevations via Na+/H+ exchange both have important inhibitory roles in the control of platelet granule secretion.