Pierre Marche

Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets

The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin.

Abnormal phosphoinositide metabolism and protein phosphorylation in platelets from a patient with the grey platelet syndrome.

Washed platelets isolated from one patient suffering from the inherited grey platelet syndrome were studied during thrombin‐induced activation. The agonist‐induced changes in (i) morphology, (ii) typical functional cell responses, (iii) membrane phospholipid metabolism and protein phosphorylation were studied and compared with the changes obtained with normal platelets. The morphology of the platelets as visualized by electron microscopy confirmed the almost total absence of intracellular alpha‐granules and marked vacuolization. During thrombin stimulation the morphological changes were clearly delayed as compared to normal platelets, the granule centralization and aggregation occurring only 15s after thrombin addition instead of 5 s in normal platelets. After 15 s, however, even though no alpha‐granules were observed, a ring‐like structure occurred centrally, indicating that they are not a prerequisite for this reaction. The whole release reaction, i.e. liberation of [14C]ser‐otonin from dense granules and β‐n‐acetylglucosaminidase activity from lysosomes, and the thromboxane synthesis were delayed and remained lower than in normal platelets. No thrombin‐induced phosphatidyl 4,5‐bisphosphate breakdown was measurable on 32P‐prelabelled platelets although [32P]phosphatidate formation occurred normally. Phosphorylation time courses of myosin light chain (P20) and of protein P43 (mol wt 43 000) markedly differed from those of controls, being less than half of the normal during the first 15 s and remaining subnormal even after complete aggregation. These results suggest that in platelets devoid of alpha‐granules a deficient transmembrane signalling system is likely responsible for the impaired physiological responses.

Influence of alpha- and beta-adrenoceptors on thrombin-induced serotonin release in rat platelets

This work was designed to investigate the influence of rat platelet adrenoceptors on the early thrombin-induced serotonin release. In washed platelets prelabeled with [3H]-serotonin, adrenaline and isoproterenol both inhibited, in a dose-dependent manner, the early thrombin-induced secretion of serotonin. Inhibitory responses of both adrenaline and isoproterenol were blocked in the presence of beta-adrenoceptor antagonists, suggesting that the catecholamine acted solely through beta-adrenoceptors. However, isoproterenol inhibited the thrombin-induced serotonin release to a much greater extent than the catecholamine, suggesting that the alpha 2-component of adrenaline might account for the difference observed between the two compounds. Our observation that selective alpha 2-adrenoceptor antagonists as yohimbine and rauwolscine potentiated the inhibitory effect of adrenaline to a level close to that observed with isoproterenol, lends support to the above hypothesis. This latter result suggested that, conversely, alpha 2-adrenergic compounds might exert a counteracting effect on a full beta-adrenoceptor mediated inhibition. Although synthetic alpha 2-adrenergic agents failed to influence isoproterenol inhibitory effect, our study shows that prestimulation of beta-adrenoceptors by isoproterenol, followed by addition of adrenaline or noradrenaline markedly diminished the inhibitory effect of isoproterenol to a level close to that which characterized the inhibition observed with catecholamines, when tested alone. Our work favours the hypothesis that, in rat platelets, early after platelet stimulation, catecholamines might counteract a beta-adrenoceptor- mediated inhibition, through alpha 2-adrenoceptor sites.

Cell Membrane in Hypertension

Several alterations in membrane transport systems are observed in rat and human hypertension. Na+ flux changes are numerous, and cellular homeostasis to Na+ loading is impaired. Transmembrane Ca2+ movements are also numerous but clearly defined by a reduction in Ca2+ binders, a hypersensitivity of membrane phospholipase C, possible increased Ca2+ leak and reduced sensitivity of the Ca2+-pump to calmodulin. The resulting Ca2+ increase within arterial cells can be responsible for increased contractility and tone, leading to hypertension. These functional alterations in membrane transport can be secondary to a few well-defined membrane defects of genetic origin or to a diffuse structural perturbation in membranes involving lipid changes.

Serotonin release and phosphoinositide breakdown in thrombin-induced activation of human platelets.

The release reaction and the metabolism of inositol lipids were studied in parallel in washed human platelets following the activation by low doses of thrombin. The breakdown of phosphatidylinositol 4,5-bisphosphate (PI-P2) was accompanied by the release of serotonin. These events preceded the breakdown of the other phosphoinositides and the release of alpha-granules and lysosome constituents, respectively. The secretion of serotonin probably is triggered by products of thrombin-induced activation of the phospholipase C directed against PI-P2.

Influence of adrenoceptors on thrombin-induced phosphoinositide metabolism in rat platelets

Stimulation of washed rat platelets with thrombin resulted in an increased turnover of phosphoinositides. Adrenaline and isoproterenol both inhibited thrombin-induced phosphatidic acid formation in a dose-dependent manner. Inhibitory responses of both compounds were blocked by a beta-adrenoceptor antagonist. However, isoproterenol was a more potent inhibitor than adrenaline. Addition of a selective alpha2-adrenoceptor antagonist potentiated the inhibitory effect of adrenaline up to the level observed with isoproterenol. Prestimulation of beta-adrenoceptors with isoproterenol, followed by addition of adrenaline (or noradrenaline) markedly diminished the inhibitory effect induced by the full beta-adrenoceptor agonist. Our results indicate that, in rat platelets, catecholamines are able to counteract, via alpha2-receptors, the beta-adrenoceptor-mediated inhibition of thrombin-induced phosphatidic acid formation. This suggests that catecholamines, by controlling cAMP level, may modulate phospholipase C activity and thereby platelet reactivity.

Defective phosphoinositide metabolism in primary hypertension

An increase in free cytosolic calcium content has been reported in essential hypertension. Since within the membrane, the phosphoinositides participate in the control of cell calcium homeostasis, we investigated whether impaired phosphoinositide metabolism could account for the calcium handling abnormality observed in hypertensives. In erythrocyte membranes of hypertensives the activity of kinases involved in polyphosphoinositide formation appears to be impaired and could be related to the alteration in calcium binding capacity and ATP-dependent calcium transport. In platelets of hypertensives, the hyperactivity of phospholipase C (observed even in the absence of calcium in the external medium) is likely to be responsible for the hypersensitivity of cells to various agonists. These observations are consistent with the hypothesis that in cells from hypertensives, a membrane defect linked to phosphoinositide metabolism is involved in the overall calcium handling defect.

Enhanced turnover of phosphatidylcholine in platelets of hypertensive rats. Possible involvement of a phosphatidylcholine-specific phospholipase C.

In an attempt to determine the mechanism involved in the hyperreactivity of platelets in primary hypertension, the dynamic behavior of phospholipids was investigated in quiescent platelets of spontaneously hypertensive rats (SHR) compared to normotensive controls. By using 32Pi, [methyl-3H]choline or [3H]glycerol as the radioactive precursors, the labeling of phosphatidylcholine (PC) was shown to be markedly enhanced (10-20-times) in SHR compared to controls. This difference between SHR and controls could not be ascribed to differences either in the actual amount of PC or in the uptake of various labels, suggesting that PC turnover was markedly enhanced in platelets of SHR. The [methyl-3H]choline labeling of phosphocholine and of CDP-choline was twice as high in SHR as in controls; chase experiments showed that when the label disappeared from phosphocholine, it was rapidly converted to PC. The results indicated that in rat platelets, PC synthesis occurred mainly via the CDP-choline pathway, and suggested that CTP:phosphocholine cytidylyltransferase was the rate-limiting step; they also indicated that the activity of this enzyme and that of choline kinase might be enhanced in SHR platelets compared to Wistar-Kyoto rat (WKY) platelets, and may thus be responsible for the enhanced PC synthesis. From these results, the existence of a PC-specific phospholipase C activity involved in PC turnover in SHR platelets can be envisaged.

The Mg2+-activated phosphatidylinositol 4,5-bisphosphate-specific phosphomonoesterase of erythrocyte membrane

The possibility that the well documented PI breakdown [l-3] might itself be secondary to the receptor-stimulated breakdown of polyphosphoinositides TPI and DPI, has been raised [4,5]. Therefore, it is of importance to understand the basic biochemical mechanisms which control the breakdown of polyphosphoinositides. This can occur via phosphodiesterase and/or phosphomonoesterase route(s). The phospholipase C-like, phosphodiesterase cleavage of polyphosphoinositides has been reported in a variety of tissues [4,6-lo]. This enzyme activity is strictly Ca*+ -activated and attacks both DPI and TPI thus inducing the release of inositol bisor triphosphate and of diacylglycerol [IO]. The existence of polyphosphoinositide phosphomonoesterase(s) has been reported in brain and in kidney [ 1 l-141. A Mg*+ -activated phosphatase specilically directed against TPI has been reported to be present in human erythrocyte cytosol [ 151; its existence within erythrocyte membranes has only been hypothesized in swine and rat [ 16-181. Here, we report on the Mg*+ -activated hydrolysis of TPI by a membrane-bound phosphomonoesterase. This was achieved by measurement of the redistribution of the radioactivities incorporated into DPI and TPI

Drug-Induced Modifications of Phosphoinositide Metabolism

The isolated red cell ghost was used as a biomembrane model to investigate the influence of aminoglycosides upon phosphoinositide metabolism by measuring 32P incorporation into the polyphosphoinositides (PIP and PI P2). A rapid and dose-dependent decrease in [32P]PIP2 and an increase in [32P]PIP were observed. Studies with neomycin revealed that these changes could likely be ascribed to an inhibition of the PIP-kinase activity by the drug. The order of potency of various aminoglycosides for the impairment of phosphoinositide metabolism varied as a function of the cationicity of the drugs. A possible relationship between the aminoglycoside-induced nephrotoxicity and the drug-induced alteration of phosphoinositide turnover is discussed.