Leupeptin selectively inhibits human platelet responses induced by thrombin and trypsin; a role for proteolytic activation of phospholipase C

Thrombin and trypsin induce serotonin release and aggregation in human platelets. Both proteases induce activation of phospholipase C as reflected by formation of inositol phosphates and phosphorylation of the resultant 1,2-diacylglycerol to phosphatidic acid. Also, thrombin and trypsin activate protein kinase C and myosin light chain kinase as indicated, respectively, by phosphorylation of the 40,000 and 20,000 dalton proteins. Leupeptin, a known inhibitor of serine proteases, blocks all the observed responses of human platelets to trypsin and thrombin. Leupeptin does not inhibit serotonin release and aggregation induced by other platelet stimuli such as collagen, platelet-activating factor, ionophore A23187, and arachidonic acid. The implication of a proteolytic-mediated pathway in the transmembrane signalling involved in platelet activation is discussed.     

Shape change induced in human platelets by platelet-activating factor. Correlation with the formation of phosphatidic acid and phosphorylation of a 40,000-dalton protein

Washed human platelets that have been separated from plasma in the presence of prostacyclin are activated by the addition of platelet activating factor (PAF). Activation (shape change, serotonin release, and aggregation) correlates closely with the formation of phosphatidic acid and the phosphorylation of a 40,000-dalton protein. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation precede aggregation and are induced at lower concentrations of PAF than those required to induce release of serotonin and platelet aggregation. Platelet shape change, formation of phosphatidic acid, and protein phosphorylation induced by PAF are not affected by trifluoperazine or indomethacin. This indicates that these responses are independent of the liberation of arachidonic acid from platelet phospholipids and the metabolism of arachidonic acid via cyclooxygenase and lipoxygenase. These responses are, however, inhibited by prostacyclin. Platelet shape change is the first measurable physiologic response to platelet agonists and may be associated with the stimulation of phospholipase C, inducing formation of 1,2-diacylglycerol and its phosphorylated product, phosphatidic acid. Transient formation of 1,2-diacylglycerol may also induce the specific activation of the protein kinase C that phosphorylates a 40,000-dalton protein.     

Activation of phospholipase C is dissociated from arachidonate metabolism during platelet shape change induced by thrombin or platelet-activating factor. Epinephrine does not induce phospholipase C activation or platelet shape change

The present study compares the molecular mechanism by which thrombin, platelet-activating factor, and epinephrine induce platelet activation. Thrombin and platelet-activating factor induce an initial activation of phospholipase C, as measured by formation of 1,2-diacylglycerol and phosphatidic acid, during platelet shape change which is independent of and dissociated from metabolism of arachidonic acid. Phospholipase C activation and shape change are independent of extracellular Ca2+ and Mg2+. Formation of cyclooxygenase products occurs subsequent to the initial activation of phospholipase C and those metabolites are associated with platelet aggregation and further activation of phospholipase C. On the other hand, epinephrine is an unique platelet stimulus since it requires extracellular divalent cations and does not induce platelet shape change or activation of phospholipase C. Our results indicate that activation of phospholipase C may be a mechanism by which physiological agonists can activate platelets independently of extracellular divalent cations.     

Dihomogammalinolenic acid, but not eicosapentaenoic acid, activates washed human platelets

Dihomogammalinolenic acid (2.5-20 microM) added to suspensions of washed human platelets induces platelet shape change and the formation of 1,2-diacylglycerol and phosphatidic acid, indicating the activation of phospholipase C. It also stimulates the phosphorylation of a 40 kDa protein, indicating the activation of protein kinase C. Dihomogammalinolenic acid is converted mainly to 12-hydroxyheptadecadienoic acid and to a smaller extent to prostaglandin E1 and thromboxane B1. Small quantities of the lipoxygenase product 12-hydroxyeicosatrienoic acid are also observed. Indomethacin, by blocking platelet cyclooxygenase, prevents the activation of phospholipase C, protein kinase C, and platelet shape change induced by dihomogammalinolenic acid. Compound UK 38485, a specific thromboxane synthetase inhibitor, does not block platelet activation induced by dihomogammalinolenic acid. The results indicate that endoperoxides derived from dihomogammalinolenic acid, such as prostaglandin G1 or prostaglandin H1, may be responsible for the stimulation of phospholipase C and protein kinase C, and for the induction of platelet shape change. Eicosapentaenoic acid does not activate platelets and is poorly metabolized by platelet cyclooxygenase and lipoxygenase. Eicosapentaenoic acid is a better inhibitor of platelet activation induced by various agonists in washed platelets than dihomogammalinolenic acid. Eicosapentaenoic acid and dihomogammalinolenic acid are, however, equally effective in inhibiting aggregation induced by collagen in platelet-rich plasma. We suggest that eicosapentaenoic acid might be a better antithrombotic agent than dihomogammalinolenic acid.     

Mepacrine (quinacrine) inhibition of thrombin-induced platelet responses can be overcome by lysophosphatidic acid

Addition of thrombin to human platelets results in production of lysophosphatidic acid. Such synthesis of lysophosphatidic acid can be inhibited by mepacrine, an inhibitor of the phospholipase A2 which attacks phosphatidic acid to give lysophosphatidic acid. In the present study, mepacrine was used at a concentration of 2.5-20 microM, sufficient to block aggregation and lysophosphatidic acid formation induced by 0.1 U/ml thrombin. Mepacrine, at this concentration, also blocked thrombin-induced phosphorylation of platelet myosin light chain and a 47 kDa protein, thrombin-induced secretion and thrombin-induced release of arachidonic acid from platelet phospholipids. However, mepacrine also partly inhibited the formation of phosphatidic acid in response to thrombin, consistent with some simultaneous inhibition of phospholipase C. Lysophosphatidic acid (2.5-22 microM) overcame the mepacrine block in thrombin-stimulated aggregation, protein phosphorylation and secretion without stimulating the release of arachidonic acid from platelet phospholipids or the formation of lysophosphatidic acid, and only slightly increasing phosphatidic acid formation. The results suggest that lysophosphatidic acid primarily acts distal to mepacrine inhibition of phospholipase A2 and phospholipase C and are consistent with the possibility that lysophosphatidic acid might be a mediator of part of the effects of low-dose thrombin on human platelets.     

Stimulation of phosphatidic acid production in platelets precedes the formation of arachidonate and parallels the release of serotonin.

Thrombin rapidly induces the formation of labeled phosphatidic acid from platelets prelabeled with [17C]arachidonate or 32PO34- and specifically decreases by 50--75% the content of phosphatidylinositol. Ionophore A23187 also stimulates phosphatidate labeling, but less effectively than thrombin. This effect on phosphatidic acid is blocked by increasing the levels of cyclic AMP by preincubation with dibutyryl cyclic AMP, cyclic AMP-phosphodiesterase inhibitors or prostacyclin. Indomethacin and eicosatetraynoic acid do not alter the production of phosphatidate, indicating independence from cyclooxygenase or lipoxygenase products. Increased turnover of [14C]- or [32P]phosphatidate occurs within 2--5 s after platelet activation by thrombin and is observed before endogenous, 14C-labeled arachidonate can be detected. The rate of phosphatidate formation parallels the induced rate of serotonin release. Release of [3H]serotonin is not affected by eicosatetraynoic acid. Phosphatidate production reflects the generation of diacylglycerol by C-type phospholipase degradation of phosphatidylinositol. Diacylglycerol and phosphatidic acid may participate in the membrane modification related to the early changes in platelet shape, release reactions or aggregation which occur on stimulation.     

Ca2+ mobilization primes protein kinase C in human platelets. Ca2+ and phorbol esters stimulate platelet aggregation and secretion synergistically through protein kinase C.

Low concentrations of Ca2+-mobilizing agonists such as vasopressin, platelet-activating factor, ADP, the endoperoxide analogue U44069 and the Ca2+ ionophore A23187 enhance the binding of [3H]phorbol 12,13-dibutyrate (PdBu) to intact human platelets. This effect is prevented by preincubation of platelets with prostacyclin (except for A23187). Adrenaline, which does not increase Ca2+ in the platelet cytosol, does not enhance the binding of [3H]PdBu to platelets. In addition, all platelet agonists except adrenaline potentiate the phosphorylation of the substrate of protein kinase C (40 kDa protein) induced by PdBu. Potentiation of protein kinase C activation is associated with increased platelet aggregation and secretion. Stimulus-induced myosin light-chain phosphorylation and shape change are not significantly affected, but formation of phosphatidic acid is decreased in the presence of PdBu. The results may indicate that low concentrations of agonists induce in intact platelets the translocation of protein kinase C to the plasma membrane by eliciting mobilization of Ca2+, and thereby place the enzyme in a strategic position for activation by phorbol ester. Such activation enhances platelet aggregation and secretion, but at the same time suppresses activation of phospholipase C. Therefore, at least part of the synergism evoked by Ca2+ and phorbol ester is mediated through a single pathway which involves protein kinase C. It is likely that the priming of protein kinase C by prior Ca2+ mobilization occurs physiologically in activated platelets.     

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.     

Differential effects of the diacylglycerol kinase inhibitor R59022 on thrombin versus collagen-induced human platelet secretion

R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the conversion of DAG to phosphatidic acid, causes an increase in endogenous DAG levels and the activity of the DAG-dependent enzyme protein kinase C. This property of the drug was utilized in the present study to assess the role of DAG, i.e., its relative importance as a potentiatory versus inhibitory mediator, in agonist-induced platelet activation. The phosphorylation of the 40-47-kDa protein by protein kinase C was monitored as an indicator of endogenous DAG levels and correlated with other agonist-induced platelet responses such as platelet aggregation, 5-hydroxytryptamine (5HT) secretion and arachidonate release, the agonists used being those that induce DAG formation, e.g., thrombin and collagen. Pretreatment of platelets with R59022 before agonist addition resulted in the potentiation of 5HT secretion as well as 45 kDa protein phosphorylation induced by thrombin and the DAG analogue, 1,2-dioctanoylglycerol (DiC8). However, collagen-induced 5HT secretion was significantly inhibited (70%) in the presence of R59022, which also had strong inhibitory effects on aggregation induced by collagen, as well as by thrombin and DiC8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC8 on the same, suggesting that even although DAG acts as a potentiatory signal in this system, the inhibitory effects of R59022 on collagen-induced aggregation can mask any effects of endogenous DAG. This inhibitory effect of R59022 on agonist-induced platelet aggregation makes it unsuitable as a tool in studying the role of DAG in platelet activation induced by agonists such as collagen as well as the 'weak' agonists (ADP, adrenaline and platelet-activating factor), where aggregation mediates other responses such as arachidonate release and secretion. Furthermore, potentiatory effects of R59022 on 5HT secretion induced by phorbol 12-myristate 13-acetate and ionomycin, which are effects unlikely to be related to inhibition of DAG kinase was observed, and these effects further underline the non-specificity in the actions of R59022 and its limitations as a tool in studying platelet stimulus-response coupling.     

Potential involvement of vicinal sulfhydryls in stimulus-induced rabbit platelet activation

The potential involvement of vicinal dithiols in the expression of platelet-activating factor (AGEPC)- and A23187-induced alterations in rabbit platelets was explored through the use of phenylarsine oxide (PhAsO) and certain analogous derivatives. PhAsO (As3+) but not phenylarsonic acid (As5+) inhibited markedly at 1 microM concentration the release of arachidonic acid initiated by AGEPC and the ionophore A23187. In contrast, AGEPC-induced phosphatidic acid formation, phosphorylation of 40- and 20-kDa proteins, and Ca2+ uptake from external medium were not inhibited substantially by 1 microM PhAsO. However, these latter metabolic responses to AGEPC were inhibited by PhAsO at higher doses (10 microM). AGEPC- and thrombin-induced platelet aggregation and serotonin secretion also were prevented by PhAsO. The IC50 value of PhAsO was 2.7 +/- 1.2 microM toward AGEPC (5 X 10(-10) M)-induced serotonin release. Further, ATP and cAMP levels in PhAsO-treated platelets were not changed from controls. Interestingly, addition of Ca2+ to platelet sonicates (prepared in EDTA) caused diacylglycerol production and free arachidonic acid formation, even in the presence of 133 microM PhAsO. This would suggest that in the intact platelets PhAsO acted indirectly on phospholipase A2 and/or phospholipase C activities. Finally, a dithiol compound, 2,3-dimercaptopropanol, reversed the inhibition of platelet aggregation and arachidonic acid release effected by PhAsO. On the other hand, a monothiol compound, 2-mercaptoethanol, was not effective in preventing or in reversing the action of PhAsO. These observations suggest that vicinal sulfhydryl residues may be involved in stimulus-induced platelet activation.     

Prostacyclin inhibition of phosphatidic acid synthesis in human platelets is not mediated by protein kinase C

The activation of protein kinase C in human platelets by phorbol-12, 13- dibutyrate (PDBu) results in the phosphorylation of a 40,000 dalton protein. This phosphorylation is time- and concentration-dependent. Maximal phosphorylation is rapid and is not affected by indomethacin or prostacyclin. PDBu does not promote activation of the phosphodiesteratic cleavage (phospholipase C) of the inositol phospholipids and the subsequent formation of 1,2-diacylglycerol or its phosphorylated product, phosphatidic acid. If platelets exposed to PDBu are subsequently stimulated with thrombin, this stimulus does not initiate further 40,000 dalton protein phosphorylation but will promote the formation of phosphatidic acid and also the phosphorylation of a 20,000 dalton protein (myosin light chain). However, prostacyclin will prevent the subsequent stimulation of phosphatidic acid synthesis by thrombin in a concentration-dependent manner. The fact that prostacyclin can affect the response to thrombin, even in the presence of phorbol ester, supports the idea that the enzymes related to the formation of phosphatidic acid or inhibition of its synthesis are not related to the phosphorylated 40K protein.     

Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP.

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.     

Phosphatidic acid with medium-length fatty acyl chains synergistically stimulates phospholipase C with Ca2+ in rabbit platelets.

The mode of phospholipase C activation in platelet cells induced by didecanoyl (C10)-phosphatidic acid (PA) was investigated with washed rabbit platelets. The C10-PA dose-dependently induced aggregation and serotonin secretion, as well as increases in cytoplasmic free Ca2+ concentration and 1,2-diacylglycerol formation. None of these responses was evoked unless Ca2+ had been added to the platelet suspension. Furthermore, under the conditions of various intracellular Ca2+ concentrations which were set by treatment of the cells with ionomycin and Ca2+, C10-PA promoted 1,2-diacylglycerol formation only at the Ca2+ concentration of 300 nM or higher, whereas thrombin induced the formation even at 100 nM Ca2+. These results suggest that PA activates platelet phospholipase C in cooperation with Ca2+ and contributes to platelet activation through such an effect.     

Decanoyl lysophosphatidic acid induces platelet aggregation through an extracellular action. Evidence against a second messenger role for lysophosphatidic acid.

Platelets rapidly convert 1,2-didecanoyl-sn-glycerol into its corresponding phosphatidic acid and lysophosphatidic acid derivatives, thereby providing a means of introducing these two compounds into platelets. 1-Decanoyl-2-lyso-3-sn-phosphatidic acid, when added directly to platelets, induced platelet aggregation and raised intracellular Ca2+ levels at concentrations of 0.3 microM upwards, but was without effect when formed intracellularly from 1,2-didecanoylglycerol at an estimated concentration of approx. 47 microM. This indicates that the site of platelet activation by lysophosphatidic acid is extracellular. A concentration of thrombin (0.2 unit/ml), which produced maximal platelet aggregation, caused an estimated intracellular formation of 20 microM-lysophosphatidic acid in the presence of 2 mM-Ca2+; however, there was no detectable release of lysophosphatidic acid into the bathing medium. Lysophosphatidic acid, therefore, may not be an intracellular second messenger involved in platelet aggregation by thrombin.     

Neomycin cannot be used as a selective inhibitor of inositol phospholipid hydrolysis in intact or semi-permeabilized human platelets. Aminoglycosides activate semi-permeabilized platelets.

High concentrations of neomycin (2-10 mM) inhibited aggregation, but not shape change, of intact platelets by collagen, ADP and the Ca2+ ionophore, A23187, the last two studies being carried out in the presence of the cyclo-oxygenase inhibitor indomethacin. In contrast, over the same range of concentrations neomycin inhibited both aggregation and shape change induced by thrombin. Under these conditions activation of platelets by collagen and by thrombin, but not by A23187 or by ADP, is believed to be dependent on the hydrolysis of membrane inositol phospholipids. These data therefore suggest that the inhibitory action of neomycin on intact platelets is not related to its previously reported inhibitory effect on phosphoinositide metabolism. The selective inhibition of thrombin-induced shape change indicates a second site of action of neomycin on intact platelets. On platelets rendered semi-permeable with saponin, neomycin and a second aminoglycoside antibiotic, streptomycin (each 0.06-2 mM), stimulated secretion and aggregation responses. These effects were inhibited by indomethacin and by EGTA. Activation of semi-permeabilized platelets by neomycin is associated with the formation of inositol phosphates and phosphatidic acid, indicating activation by phospholipase C. This effect is also inhibited by indomethacin, implying that it is secondary to the formation of prostaglandins and endoperoxides. These results are discussed in the context of the use of neomycin as a selective inhibitor of polyphosphoinositide metabolism.     

Ethanol interferes with collagen-induced platelet activation by inhibition of arachidonic acid mobilization

The effect of ethanol on signal generation in collagen-stimulated human platelets was evaluated. Incubation of washed human platelets with physiologically relevant concentrations of ethanol (25-150 mM) resulted in a dose-dependent inhibition of aggregation and secretion in response to collagen (0.5-10 micrograms/ml), but did not inhibit shape change. In platelets labeled with [3H]arachidonic acid, ethanol significantly inhibited the release of arachidonic acid from phospholipids, in both the presence and the absence of indomethacin. Thromboxane B2 formation was also inhibited in proportion to the reduction in free arachidonic acid. There was a close correlation between the extent of inhibition of arachidonic acid release and secretion. The inhibition of platelet aggregation and secretion by ethanol was partially overcome by the addition of exogenous arachidonic acid. In the presence of indomethacin, ethanol had no effect on the activation of phospholipase C by collagen as determined by the formation of inositol phosphates and phosphatidic acid. Moreover, ethanol had no effect on the mobilization of intracellular calcium by collagen and only minimally inhibited the early phases of the phosphorylation of myosin light chain (20 kDa) and a 47-kDa protein, a known substrate for protein kinase C. Arachidonic acid formation was also inhibited by ethanol in response to ionomycin under conditions where phospholipase C activation was prevented. The results suggest that the functional effects of ethanol on collagen-stimulated platelets are due, at least in part, to an inhibition of phospholipase A2.     

Membrane microenvironmental changes during activation of human blood platelets by thrombin. A study with a fluorescent probe.

Membrane microenvironmental changes associated with thrombin-induced platelet activation were followed by fluorescence intensity and polarization studies of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labeled human platelets. The labeling of washed platelets with DPH did not alter platelet intactness and morphology. In response to thrombin, DPH-labeled platelets exhibited reduced serotonin release, yet aggregation was barely inhibited. Shape change induced by thrombin or ADP was indistinguishable in control and in DPH-labeled platelets. During platelet aggregation induced by thrombin, fluorescence intensity increased by about 14%, which may indicate a more hydrophobic exposure of the probe. However, no change in fluorescence was detected during platelet shape change, induced either by thrombin in presence of EDTA or by ADP. Thrombin-activated platelets exhibited an increase in values of fluorescence polarization (P) during the stages of shape change and secretion, which further increased during aggregation. A similar pattern of increase in P values characterized platelet shape changes, caused either by thrombin in the presence of EDTA or by ADP. Changes in individual platelets are discernible from the alterations of the aggregating cells. These results may indicate that platelet activation is accompanied by an increase in rigidity of the membrane lipids. Functionally, the elevated "microviscosity" may reflect a primary role of membrane lipids in modulating the process of platelet activation or secondary transitions in lipids due to membrane events mediated by proteins.     

Epinephrine potentiates calcium mobilization and activation of protein kinases in platelets stimulated by ADP through a mechanism unrelated to phospholipase C

ADP, added to suspensions of aspirinized 32P-prelabelled washed platelets, induced reversible platelet aggregation, the rapid elevation of cytosolic Ca2+ (maximum at 2 s), 20 kDa myosin light chain phosphorylation (maximum faster than 3 s), 40 kDa protein phosphorylation (maximum at 3-10 s) and phosphatidic acid formation (maximum at 30 s). Prior addition of epinephrine potentiated platelet aggregation, cytosolic Ca2(+)-elevation, 20 and 40 kDa protein phosphorylation evoked by ADP, but it did not enhance phosphatidic acid formation induced by ADP. The potentiating effect of epinephrine on aggregation, cytosolic Ca2(+)-increase and 20 and 40 kDa protein phosphorylation induced by ADP was also observed in the presence of EGTA. Ethylisopropylamiloride, an inhibitor of Na+/H(+)-exchange, did not affect the potentiation of ADP-induced platelet aggregation by epinephrine. We conclude that epinephrine primes platelets to increase Ca2(+)-influx and Ca2(+)-mobilization in response to ADP. The potentiation of cytosolic Ca2(+)-elevation by epinephrine leads to further stimulation of myosin light chain phosphorylation and protein kinase C activation and ultimately to enhanced platelet aggregation. These effects of epinephrine do not seem to take place at the level of phospholipase C.     

1,2-Diacylglycerols do not potentiate the action of phospholipases A2 and C in human platelets

1,2-Diacylglycerol has recently been reported to potentiate the ability of phospholipases A and C to hydrolyze phospholipids in a cell-free system. The present study has been undertaken to investigate whether 1,2-diacylglycerol can also perform this function in intact cells using the platelet as a test system. Exogenous 1-oleoyl-2-acetyl-glycerol ( OAG ) and 1,2- didecanoylglycerol , at concentrations sufficient to produce maximal phosphorylation of a 40,000 dalton protein, caused no significant formation of [3H]inositol phosphates and [32P]phosphatidic acid (products of phospholipase C activation) or [14C]arachidonic acid metabolites and lysophosphatidyl[3H]inositol (products of phospholipase A2 activation). These data therefore imply that 1,2-diacylglycerols do not potentiate the actions of phospholipases A2 and C in intact platelets at concentrations that are physiologically relevant.     

The energetics of early platelet responses. Energy consumption during shape change and aggregation with special reference to protein phosphorylation and the polyphosphoinositide cycle.

Among the different platelet responses, secretion requires the greatest amount of metabolic energy. The velocities of dense, alpha- and acid hydrolase granule secretion vary in parallel with the increase in energy consumption seen in thrombin-stimulated cells. This covariance is preceded by a phase in which energy consumption is increased without the extracellular appearance of secretion markers. By treating the platelets with thrombin and hirudin we have stimulated the platelets for short intervals and succeeded in separating shape change, single platelet disappearance and secretion to a great extent. In this report we show that the early increase in energy consumption reflects the energy requirement of aggregation but not of shape change. The cost of 100% of single platelet disappearance is 2.8 mumol of ATPeq. X (10(11) platelets)-1. Concurrent analysis of phosphorylation of Mr 20 000 and 47 000 proteins and of 32P-labelled phosphatidylinositol metabolites led to the following observations. Firstly, shape change is neither accompanied by an increase in protein phosphorylation nor by changes in the steady state levels of 32P-labelled phosphatidylinositol metabolites. Secondly, when aggregation occurs both proteins are phosphorylated, but the phosphatidylinositol metabolites do not change. Thirdly, when secretion follows, more phosphorylation of the Mr 47 000 protein occurs and initially only phosphatidic acid accumulates. At a later stage of the secretion responses, more protein phosphorylation and phosphatidic acid accumulation become evident, and are now accompanied by alterations in the steady state levels of 32P-labelled (poly)phosphoinositides. Hence, the early increase in energy consumption coincides with protein phosphorylation and, at a later stage, with alterations in (poly)phosphoinositides metabolites. This demonstrates that metabolic energy is directly involved in stimulus-response coupling in aggregating platelets.