SummaryThe stimulating effect of adrenaline on human platelet phospholipase C (PLC) activation and responses in vitro (shape change, aggregation and dense granule secretion) was investigated with respect to its dependence on exogenously added agonists. All experiments were performed with human gel-filtered platelets pretreated with acetylsalicylic acid to prevent endogenous stimulation by the arachidonate pathway. (1) Preliminary experiments demonstrated the presence of trace amounts of extracellular ADP (0.05-0.58 μM) in non-stimulated platelet suspensions; ADP was effectively converted to ATP by the enzyme system creatine phosphate (CP)/creatine phosphokinase (CPK). (2) The adrenaline-induced optical aggregation and single particle (platelet) disappearance in the presence of trace amounts of ADP were almost abolished by the ADP-scavanger system CP/ CPK. (3) The response of CP/CPK-treated thrombin- or platelet-activating factor (PAF)-stimulated platelets was markedly increased by a subsequent addition of adrenaline. When hirudin or BN 50726 was added just prior to adrenaline to terminate the activation by thrombin or PAF, respectively, the stimulating effect of adrenaline was also abolished. (4) CP/CPK-treated, PAF-stimulated platelets rapidly developed decreased responsiveness to a subsequent addition of PAF. When adrenaline was added instead of a second addition of PAF, the stimulating effect of adrenaline was gradually decreased and prevented in parallel with the homologous desensitization of PAF. (5) The weak platelet agonist serotonin by itself induced only shape change in CP/CPK-treated platelets. Adrenaline failed to enhance the extent of this serotonin-induced platelet activation. (6) These results strongly suggest that adrenaline per se is not a platelet agonist in vitro but acts to enhance the stimulation induced by true agonists.

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The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by shortterm labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP.1. Under short-term labelling conditions, the 4-and 5-phosphate groups of phosphatidylinositol4-phosphate (PtdIns4P) and phosphatidylinositol4,5-bisphosphate [PtdIns(4,5)Pz] had the same specific 32P radioactivity as the y-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4 -13% compared to that of the ATP-y-phosphate.2. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics.3. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)Pz, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)Pz in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)Pz are present as a single metabolic pool in human platelets. Turnover of the 4-and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the y-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.The polyphosphoinositol lipids constitute a minor fraction of total cell phospholipids. Nonetheless, important functions are assigned to them, such as receptor-mediated transmembrane signalling (reviewed in [l]), facilitation of exocytotic processes [2, 31, the anchorage of membrane-associated proteins [4 -81 and the regulation of ion transport [9]. The polyphosphoinositol lipids are highly dynamic and undergo continuous phosphorylation and dephosphorylation. PtdIns is converted in two separate ATP-dependent reactions to PtdIns4P and PtdIns(4,5)P2. The latter are dephosphorylated again, thereby constituting 'futile' cycles in which two moles of ATP are consumed for each mole of recycled PtdIns. This cycling was estimated by Maretzki et al. [lo] to account for as much as 20% of basal ATP consumption in mature erythrocytes, but in more thorough studies Muller et al. [ll] and Dale [I21 found much lower values of 2-5% and 0.5-1 %, respectively. The relative importance of phosphoinositol lipid cycling for total ATP consumption in other cell types has not yet been quantified.In platelets, the polyphosphoinositol lipids are thought to be crucial in signal processing. Upon stimulation the steady-

On the basis of differences in specific radioactivity (SA), separate pools of phosphoinositides have recently been proposed in platelets. Human platelets were labelled for 60 min with [32p]p- and subsequently transferred to a phosphate- and Ca2+free Tyrode’s solution by gel-filtration. Thereafter, the platelets were either incubated at 37°C for 120 min, a condition which induces increase in specific labelling of the diester phosphate of phosphatitylinositol (PI), or stimulated with 0.5 U/ml of thrombin. The changes in SA of both diester and monoester phosphates of the phosphoinositides were detrmined. Immediately after the gel filtration, the SA of the diester phosphate of phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2) were both similar to that of PI and amounted to 4% or the SA of the monoester groups of PIP and PIP2- Whereas the SA of the monoester phosphate essentially remained constant and the same for PIP and PIP2 during the entire incubation, the SA of their diester phosphates increased gradually in parallel to that of PI, and reached 20% of the monoster groups after 120 min. The effect of thrombin was studied at 15, 60 and 180 sec after the addition. The absolute radioactivity of both diester and monoester phosphates of all phosphoinositides increased conciderably after an initial decrease. However, for the monoester groups, the changes in radioactivity were parallelled by the changes in mass for both PIP and PIP2. Thrombin therefore induced no changes in SA of the monoester phosphates. In contrast, the SA of the diester phosphates increased 5-fold and remained similar for all three phosphoinositides during the 180 sec of stimulation.In conclusion, our results demonstrate close metabolic equilibrium between all three phosphoinositides.Thrombin-induced changes in SA of PIP and PIP2 are purely secondary to changes in specific labelling of the diester phosphate.

The specific radioactivity of the phosphodiester and phosphomonoester moieties of the phosphoinositides was determined in resting and thrombin-stimulated platelets that were prelabelled with [32p] Pi. In the unstimulated cells, the specific radioactivity of the monoester phosphates of PIP and PIP2 was similar. Both prolonged incubation at 37°C and upon stimulation with 0.5 U/ml of thrombin, the specific radioactivity of the monoester phosphates decreased 10-15 % in both polypbosphoinositides. In the unstimulated cells, the specific radioactivity of the diester phosphates was similar in PI, PIP and PIP2 but amounted only to 3 % of the activity of the monoester groups. Prolonged incubation of the unstimulated cells as well as stimulation with thrombin induced a similar 5-6 fold increase in specific radioactivity of the diester phosphate of PI, PIP and PIP2. The results indicate that the phosphoinositides in both resting and thrombin-stimulated platelets exist in a metabolically homogeneous pool.Phospholipid metabolism; Inositol phospholipid; Thrombin; (Human platelet)