D. de Chaffoy de Courcelles

12-O-Tetradecanoylphorbol 13-acetate stimulates inositol lipid phosphorylation in intact human platelets

Potyphosphoinositide Phorbol ester Protein kinase C

Stimulation by serotonin of 40 kDa and 20 kDa protein phosphorylation in human platelets

In human platelets, serotonin is known to induce a shape change followed by (reversible) aggregation. Recently, it was found that the amine triggers the elevation of cytosolic free calcium and activates phospholipase C. On stimulation of human platelets with serotonin we found an immediate increase in protein kinase C activity, phosphorylating its 40 kDa substrate protein. A 20 kDa protein, most likely the myosin light chain, was phosphorylated to the same extent. Ketanserin, a highly selective serotonin‐S2 antagonist inhibited both phosphorylation processes at subnanomolar concentrations.

Agents that elevate platelet cAMP stimulate the formation of phosphatidylinositol 4-phosphate in intact human platelets.

The present study investigates the effect of compounds that are known to elevate cAMP on the phospholipid metabolism of platelets. Prostaglandin E1, forskolin and isobutylmethylxanthine induce an increase in [32P]‐phosphatidylinositol 4‐phosphate (PIP) in platelets prelabelled with [32P]orthophosphate. Possible roles of this phenomenon are discussed in view of the inhibitory effect of cAMP elevation on platelet activation.

1-Oleoyl-2-acetyl-glycerol (OAG) stimulates the formation of phosphatydylinositol 4-phosphate in intact human platelets

Diacylglycerol (DAG) is one of the primary products formed upon activation of platelets with stimuli that induce inositol lipid turnover. Its synthetic analog, 1-oleoyl-2-acetyl-glycerol (OAG) is often used as a tool for studying the involvement of the lipid in platelet activation. We found that OAG induces a concomitant increase in [32P]-incorporation in phosphatidylinositol 4-phosphate (PIP) and in the 40K protein, the endogenous substrate for protein kinase C in human platelets. It is hypothesized that in receptor mediated platelet activation a metabolic link might exist between both processes.

The synergistic effect of serotonin and epinephrine on the human platelet at the level of signal transduction

Simultaneous addition to platelets of submaximal amounts of excitatory agonists acts synergistically in provoking secretory and aggregatory responses. By measuring changes in intracellular free Ca2+ concentration, inositol phospholipid metabolism and protein phosphorylation, we verified whether synergism could be evidenced at the level of signal transduction. Challenging platelets with epinephrine only induced minor changes on the measured parameters. However, when added together with serotonin, epinephrine amplified mobilisation of intracellular C2+, PA formation, PIP formation, protein kinase C and myosin light chain kinase activity as compared to the alterations induced by serotonin alone. It is concluded that synergistic effects on simultaneous addition of serotonin and epinephrine might originate at the level of signal transduction.

Ouabain increases the calcium concentration in intracellular stores involved in stimulus-response coupling in human platelets.

The effect of ouabain on Ca2+ homeostasis in human platelets was studied using both quin 2 and chlorotetracycline to monitor changes in cytosolic Ca2+ as well as changes in the amount of Ca2+ accumulated in intracellular storage sites. In resting platelets, ouabain induces a concentration- and time-dependent increase in cytosolic Ca2+ concentration and a marked elevation of Ca2+ in the intracellular stores. The amount of Ca2+ mobilized from these stores upon stimulation with thrombin, as well as thrombin-induced secretion of platelet 5-hydroxytryptamine, was increased after preincubation with the glycoside (3 x 10(-6) M). These data show that ouabain induces an elevation of intracellular Ca2+ levels, most likely mediated via Na(+)-Ca2+ exchange, and that this incremental amount of Ca2+ is accumulated in an intracellular store involved in stimulus-response coupling. This may explain the enhanced functional responses of platelets to agonists in the presence of ouabain and suggests a role for Na(+)-Ca2+ exchange in Ca2+ homeostasis of the human platelet.

Amplification Mechanisms in Platelet Activation

Platelets, as they contribute to the formation of an hemostatic plug or an arterial thrombus, are exposed to a multitude of agonists, acting with different strength in concert on the same target cells to amplify each other effects. Such amplification, rather then the potency of the agonists singly, determines the true impact of a so-called weak agonist as serotonin on platelet reactions in pathology.

The signal transducing system coupled to serotonin-S2 receptors

The signal transducing system coupled to the serotonin-S2 receptor on platelets involves metabolism of inositolcontaining phospholipid, elevation of intracellular free Ca2+ and activation of protein kinase C. Evidence for coupling of the serotonin-S2 receptor to the same signal transducing system in brain and smooth muscle tissue is reviewed.

Cyclic AMP-phosphodiesterase IIIA1 inhibitors decrease cytosolic Ca2+ concentration and increase the Ca2+ content of intracellular storage sites in human platelets

The effect of cyclic AMP-phosphodiesterase (cAMP-PDE) inhibitors on Ca2+ homeostasis in human platelets was studied using both quin-2 (2-(bis-(acetylamino)-5-methyl-phenoxy)methyl-6-methoxy-8-bis-(acetylami no) quinoline) and chlorotetracycline (CTC) to measure changes in cytosolic Ca2+ as well as changes in the amount of Ca2+ accumulated in intracellular storage sites. At therapeutic concentrations (1 microM) milrinone and R 80 122 but not enoximone decreased the cytosolic Ca2+ concentration in the resting platelet while the Ca2+ content in intracellular stores was increased. These observations are in accord with the proposed mechanism of action of cAMP-PDE inhibitors on cardiomyocites and highlight the particular role of cAMP in regulation of Ca2+ homeostasis.

Amplifying Effects of 5-Hydroxytryptamine on Human Platelets and Signal Transduction

The receptors involved in the 5-hydroxytryptaminergic activation process of human platelets resemble the 5-HT2 type. The signal transducing system, linked to the 5-HT2 receptors involves the phosphodiesteratic breakdown of phospholipids, the activation of protein kinase C and the increase of intracellular free Ca2+. 5-HT amplifies the functional responses of human platelets to several excitatory agonists. To gain deeper insights into the molecular mechanism of this amplification, we investigated its occurrence at the level of signal transduction, using 5-HT and epinephrine as a pair of platelet agonists. We found that the amplifying effect originated at the levels of the signal transducing system coupled to the 5-HT2-receptor.