Signaling pathways of the F11 receptor (F11R; a.k.a. JAM-1, JAM-A) in human platelets: F11R dimerization, phosphorylation and complex formation with the integrin GPIIIa

The F11 receptor (F11R) (a.k.a. Junctional Adhesion Molecule, JAM) was first identified in human platelets as a 32/35 kDa protein duplex that serves as receptor for a functional monoclonal antibody that activates platelets. We have sequenced and cloned the F11R and determined that it is a member of the immunoglobulin (Ig) superfamily of cell adhesion molecules. The signaling pathways involved in F11R-induced platelet activation were examined in this investigation. The binding of M.Ab.F11 to the platelet F11R resulted in granule secretion and aggregation. These processes were found to be dependent on the crosslinking of F11R with the Fc gammaRII by M.Ab.F11. This crosslinking induced actin filament assembly with the conversion of discoidal platelets to activated shapes, leading to the formation of platelet aggregates. We demonstrate that platelet secretion and aggregation through the F11R involves actin filament assembly that is dependent on phosphoinositide-3 kinase activation, and inhibitable by wortmannin. Furthermore, such activation results in an increase in the level of free intracellular calcium, phosphorylation of the 32 and 35 kDa forms of the F11R, F11R dimerization coincident with a decrease in monomeric F11R, and association of the F11R with the integrin GPIIIa and with CD9. On the other hand, F11R-mediated events resulting from the binding of platelets to an immobilized surface of M.Ab.F11 lead to platelet adhesion and spreading through the development of filopodia and lammelipodia. These adhesive processes are induced directly by interaction of M.Ab.F11 with the platelet F11R and are not dependent on the Fc gammaRII. We also report here that the stimulation of the F11R in the presence of nonaggregating (subthreshold) concentrations of the physiological agonists thrombin and collagen, results in supersensitivity of platelets to natural agonists by a F11R-mediated process independent of the Fc gammaRII. The delineation of the two separate F11R-mediated pathways is anticipated to reveal significant information on the role of this cell adhesion molecule in platelet adhesion, aggregation and secretion, and F11R-dependent potentiation of agonist-induced platelet aggregation. The participation of F11R in the formation and growth of platelet aggregates and plaques in cardiovascular disorders, resulting in enhanced platelet adhesiveness and hyperaggregability, may serve in the generation of novel therapies in the treatment of inflammatory thrombosis, heart attack and stroke, and other cardiovascular disorders.     

Activation of human platelets by a stimulatory monoclonal antibody

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.     

The F2-isoprostane, 8-epi-prostaglandin F2 alpha, a potent agonist of the vascular thromboxane/endoperoxide receptor, is a platelet thromboxane/endoperoxide receptor antagonist.

F2-isoprostanes are a recently discovered series of prostaglandin (PG)F2-like compounds that are produced in vivo in humans by nonenzymatic free radical catalyzed peroxidation of arachidonic acid. One of the compounds that can be produced in abundance by this mechanism is 8-epi-PGF2 alpha. 8-epi-PGF2 alpha is a potent vasoconstrictor in the rat, an effect that has been shown to be mediated via interaction with vascular thromboxane (TxA2)/endoperoxide (PGH2) receptors. In an effort to further understand the biological properties of this prostanoid in relation to its ability to interact with TxA2/PGH2 receptors, we examined its effects on human and rat platelets. At concentrations of 10(-6) M and 10(-5) M, 8-epi-PGF2 alpha induced only a shape change in human platelets and at higher concentrations (10(-4) M) induced reversible but not irreversible aggregation. Both the shape change and reversible aggregation were unaffected by indomethacin but were inhibited by the TxA2/PGH2 receptor antagonist SQ29548. Conversely, 8-epi-PGF2 alpha inhibited platelet aggregation induced by the TxA2/PGH2 receptor agonists U46619 (10(-6) M) and IBOP (3.3 x 10(-7) M) with an IC50 of 1.6 x 10(-6) M and 1.8 x 10(-6) M, respectively. 8-epi-PGF2 alpha also inhibited platelet aggregation induced by arachidonic acid. Similarly, in rat platelets, 8-epi-PGF2 alpha alone induced only modest reversible aggregation but completely inhibited U46619-induced aggregation.     

Actin filament content in platelets—A sensitive index of cellular reactivity

Blood platelets have the capacity to participate in a number of physiological as well as pathological processes within the circulation. In order to evaluate their cellular reactivity a number of platelet function tests have been developed. The mainin vitro function tests are assessment of aggregation and adhesion, secretion, arachidonate metabolism, coagulant activities and the characterization of surface membrane glycoproteins (Day and Rao, 1986). Here we measure alterations of the G-/F-actin equilibrium of platelets. High F-actin values of unstimulated platelets indicate a hyperreactivity of the cell as examined in platelets from diabetics. Determination of the actin filament content in platelets can be considered as a new sensitive function test.     

Polymorphonuclear granulocyte expression of CD11a/CD18, CD11b/CD18 and L-selectin in normal individuals

Abstract In this study direct immunofluorescence and flow cytometry with calibration using quantitative bead standards were used to enumerate the cell surface receptors CD11a/CD18, CD11b/CD18 and L-selectin. Holding blood at room temperature and fixation of samples prior to staining induced changes in expression, while immediate staining of polymorphonuclear granulocytes (PMN) in whole blood followed by fixation produced accurate values. The ranges of PMN adhesion molecule expression in 10 normal individuals were CD11a/CD18: 14794–28725, CD11b/CD18: 5300–11939 and L-selectin: 35662–61654 receptors per cell. Differences within individuals over 4 h were also observed. Adhesion molecule expression is used as an index of the adhesive function and state of activation of the cell. The data presented here shows that there is inherent variability in the expression of the PMN adhesion molecules between and within individuals, thus direct comparisons of PMN adhesion molecule expression between patients and “normals” must be interpreted with caution.     

A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation.     

Platelet agonist F11 receptor is a member of the immunoglobulin superfamily and identical with junctional adhesion molecule (JAM): regulation of expression in human endothelial cells and macrophages

The stimulatory mAb F11 binds two platelet membrane proteins of 32 and 35 kDa and causes activation of platelets when cross-linked with the FcgammaRII receptor. We used bioinformatics to identify expressed sequence tags from libraries of cytokine-stimulated human endothelial cell (EC) cDNAs. The protein sequence deduced from full-length F11 cDNA was identical to partial sequences of peptides derived from affinity-purified platelet F11 antigen. F11 mRNA is expressed in human EC, macrophages, and a variety of non-hematopoietic vascular tissues. Expression of F11 mRNA is modulated by cytokines in EC and is up-regulated by oxidized low-density lipoprotein in human macrophages. The F11 receptor contains two immunoglobulin-like domains in its 236-amino-acid-long extracellular region, and has identity to the recently described junctional adhesion molecule. The data indicate that the F11 antigen is a novel receptor or cell adhesion molecule belonging to the immunoglobulin superfamily.     

The role of endogenously formed diacylglycerol in the propagation and termination of platelet activation. A biochemical and functional analysis using the novel diacylglycerol kinase inhibitor, R 59 949

The putative roles for the second messenger, diacylglycerol, were investigated in intact platelets using a novel diacylglycerol kinase inhibitor, R 59 949, or (3-[2-[4-[bis(4-fluorophenyl)methylene]-1-piperidinyl]ethyl]-2,3- dihydro-2-thioxo-4(1H)-quinazolinone). The compound inhibited the diacylglycerol kinase in a concentration-dependent manner (10(-8) to 10(-5) M) in isolated platelet membranes and in intact platelets. When platelets were stimulated with vasopressin in the presence of the compound, protein kinase C activity was markedly increased; the formation of inositol phosphates, the increase in intracellular Ca2+ and shape-change reaction were antagonized while the vasopressin-induced polyphosphoinositide synthesis was amplified, and this in a distinct inositolphospholipid pool. In the presence of R 59 949, vasopressin- as well as collagen-induced release reaction and aggregation was strongly increased, independently of the formation of arachidonate metabolites. It is concluded that diacylglycerol formed after receptor activation, likely by activating the protein kinase C, plays an important role in the propagation of platelet functional responses in casu aggregation and secretion and controls the termination of the primary receptor coupled responses.     

Platelet adhesion to collagen-coated wells: analysis of this complex process and a comparison with the adhesion to matrigel-coated wells.

The mechanisms of platelet adhesion to collagen type III-coated wells and Matrigel-coated wells were analyzed. The adhesion of 51Cr-labeled platelets to collagen-coated wells showed a biphasic pattern. The early stage of adhesion was inhibited by antibodies against platelet glycoprotein(GP)s Ia/IIa and VI. The later stage of platelet adhesion was inhibited by an antibody against the GPIIb/IIIa complex and a concomitant release of 14C-labeled serotonin was observed. The percentage of adhered platelets was increased when a higher platelet concentration was added in the reaction medium. These results indicated that the adhesion assay of platelets to collagen-coated wells was composed of two reactions: the first one is the platelet-collagen interaction that depends on GPIa/IIa and GPVI on the platelet surface; and the second reaction is the platelet-platelet interaction, platelet aggregation, which depends on GPIIb/IIIa. The adhesion of platelets to Matrigel-coated wells was indicated to involve platelet-Matrigel interactions that were partly dependent on the laminin in the Matrigel solution.     

FcεR and FcγR Expressed on Murine IgE-Specific Suppressor T Hybridomas

The expression of Fc receptors specific for IgE (Fc_εR) and those for IgG (Fc_γR) on murine IgE-specific suppressor T hybridomas was studied. While parental T lymphoma cells (BW5147) failed to bind IgE-sensitized red cells (mIgE-TNP-ORBC), the majority of T hybridoma cell lines having IgE-specific suppressor activity contained rosette-forming cells (RFC) binding mIgE-TNP-ORBC (2 to 13% of the total cells). The expression of Fc_εR was poor (2% or less) in T hybridoma cell lines without IgE-specific suppressor activity. In addition to Fc_εR, IgE-specific suppressor T hybridomas also expressed Fc_γR as detected by ORBC sensitized with IgG antibodies (EA_oxγ). β-Interferon (IFN) (1,000 to 2,000 U/ml) augmented the expression of Fc_γR, but not of Fc_εR. On the other hand, preculture of the cells with 40 μg of mIgE per ml enhanced the expression of Fc_εR without augmenting Fc_γR expression. IgE-specific suppressor activity in the culture supernatants of T hybridomas was also augmented by preculture with mIgE. However, a dissociation between the expression of Fc_εR and IgE-suppressor activity was observed. In some of the subclones, mIgE augmented the suppressor activity without inducing the expression of Fc_εR. Furthermore, when treated with 5 μg of melittin, a phospholipase A₂ activator, per ml, IgE-specific suppressor activity of the hybridomas was completely abrogated, whereas IgE-induced expression of Fc_εR was enhanced by melittin.     

Characterization of the Fc gamma receptor on human platelets

IgG-containing immune complexes may play a role in the immune destruction of human platelets by interacting with an Fc gamma receptor on the platelet surface. We studied the platelet Fc gamma receptor and characterized its interaction with IgG ligand and anti-Fc gamma receptor monoclonal antibodies. Oligomers of IgG, but not monomeric IgG, bound to platelets and the number of binding sites was significantly increased at low ionic strength. Ligand-binding studies indicated that normal human platelets express a single Fc gamma receptor (Fc gamma RII) with 8559 +/- 852 sites per cell, Kd = 12.5 +/- 1.7 X 10(-8) M using trimeric IgG. Results of studies with bivalent and Fab monoclonal anti-Fc gamma RII were consistent with each Fc gamma receptor expressing two epitopes recognized by the antibody. The number of Fc gamma binding sites and affinity of binding were unchanged by the presence of 2.0 mM Mg2+ or 10 micrograms/ml cytochalasin B. Platelet stimulation with thrombin or ADP in the presence of fibrinogen also did not alter the number of Fc gamma binding sites or the affinity of binding. However, platelets preincubated with 5 microM dexamethasone expressed a decreased number of Fc gamma binding sites as well as decreased IgG-dependent platelet aggregation. Platelets from patients with Glanzmann's thrombasthenia and from patients with the Bernard Soulier syndrome expressed a normal number and affinity of Fc gamma binding sites. The data suggest that platelet Fc gamma RII binding of trimeric IgG occurs independent of actin filament interaction, Mg2+, ADP, or thrombin and does not require GPIIb/IIIa or GPIIb/IIIa-fibrinogen interaction. Furthermore, this receptor appears to be normally expressed on GPIb-deficient platelets and susceptible to modulation by glucocorticoids. Finally, the Fc gamma-binding protein was isolated from whole platelets as a 220-kDa protein which upon reduction dissociates into 50,000 Mr subunits.     

Characterization of a novel focal adhesion kinase inhibitor in human platelets

Focal adhesion kinase (FAK) is activated in human platelets downstream of integrins, e.g. α_IIbβ₃, and other adhesion receptors e.g. GPVI. Mice in which platelets lack FAK have been shown to exhibit extended bleeding times and their platelets have been shown to display decreased spreading on fibrinogen-coated surfaces. Recently, a novel FAK inhibitor (PF-573,228) has become available, its selectivity for FAK shown in vitro and in cell lines. We determined the effect of this inhibitor on platelet function and signaling pathways. Like murine platelets lacking FAK, we found that PF-573,228 was effective at blocking human platelet spreading on fibrinogen-coated surfaces but did not affect the initial adhesion. We also found a reduced spreading on CRP-coated surfaces. Further analysis of the morphology of platelets adhered to these surfaces showed the defect in spreading occurred at the transition from filopodia to lamellipodia. Similar to that seen with murine neutrophils lacking FAK, we also observed an unexpected defect in intracellular calcium release in human platelets pre-treated with PF-573,228 which correlated with impaired dense granule secretion and aggregation. The aggregation defect could be partially rescued by addition of ADP, normally secreted from dense granules, suggesting that PF-573,228 has effects on FAK downstream of α_IIbβ₃ and elsewhere. Our data show that PF-573,228 is a useful tool for analysis of FAK function in cells and reveal that in human platelets FAK may regulate a rise in cell calcium and platelet spreading.     

Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Helicobacter pylori urease activates blood platelets through a lipoxygenase‐mediated pathway

The bacterium Helicobacter pylori causes peptic ulcers and gastric cancer in human beings by mechanisms yet not fully understood. H. pylori produces urease which neutralizes the acidic medium permitting its survival in the stomach. We have previously shown that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring platelet secretion, activation of calcium channels and lipoxygenase‐derived eicosanoids. We investigated whether H. pylori urease displays platelet‐activating properties and defined biochemical pathways involved in this phenomenon. For that the effects of purified recombinant H. pylori urease (HPU) added to rabbit platelets were assessed turbidimetrically. ATP secretion and production of lipoxygenase metabolites by activated platelets were measured. Fluorescein‐labelled HPU bound to platelets but not to erythrocytes. HPU induced aggregation of rabbit platelets (ED₅₀ 0.28 μM) accompanied by ATP secretion. No correlation was found between platelet activation and ureolytic activity of HPU. Platelet aggregation was blocked by esculetin (12‐lipoxygenase inhibitor) and enhanced ～3‐fold by indomethacin (cyclooxygenase inhibitor). A metabolite of 12‐lipoxygenase was produced by platelets exposed to HPU. Platelet responses to HPU did not involve platelet‐activating factor, but required activation of verapamil‐inhibitable calcium channels. Our data show that purified H. pylori urease activates blood platelets at submicromolar concentrations. This property seems to be common to ureases regardless of their source (plant or bacteria) or quaternary structure (single, di‐ or tri‐chain proteins). These properties of HPU could play an important role in pathogenesis of gastrointestinal and associated cardiovascular diseases caused by H. pylori.     

Involvement of RPL11 in the enhancement of P53 stability by a podophyllum derivative,a topoisomerase II inhibitor

In previous work, we presented experimental and theoretical evidence that D‐3F or 4‐N‐(2‐Amino‐3‐fluoropyridine)‐4‐deoxidation‐4′‐demethylepipofophyllotoxin induced G₂/M phase arrest and apoptosis, purportedly by increasing the expression of P53. However, the precise mechanism of D‐3F action is currently unknown. Here, we investigated the mechanism by which D‐3F treatment induces increased expression of P53. This study showed that D‐3F definitively inhibited the activity of topoisomerase II in a dose‐dependent manner and resulted in DNA damage. The results were in overall agreement with modeling and docking studies performed on D‐3F . In addition, D‐3F increased the levels of P53 and P21 in HeLa cells in a dose‐dependent manner, this in turn prolonged the half‐life of P53. Taken together, these data suggested that D‐3F ‐mediated transient enhancement of P53 stabilization may be critical for the P53/P21 signalling pathway leading to G₂/M phase arrest on HeLa cells. Furthermore, D‐3F downregulated the phosphorylation of E3 ubiquitin‐protein ligase murine double minute 2 (Mdm2) at Ser166, inhibited Mdm2‐mediated ubiquitination of P53, and released 60S ribosomal protein L11 (RPL11) from the nucleolus into the nucleoplasm. To conclude, the topoisomerase II inhibitor D‐3F causes P53 to accumulate in HeLa cell lines by enhancing its stability as a result of DNA‐damage induced RPL11 relocalization and subsequent blocking of the P53‐Mdm2 feedback loop.     

Deletion of junctional adhesion molecule A from platelets increases early‐stage neointima formation after wire injury in hyperlipidemic mice

Platelets play an important role in the pathogenesis of vascular remodelling after injury. Junctional adhesion molecule A (JAM‐A) was recently described to regulate platelet activation. Specific deletion of JAM‐A from platelets resulted in increased reactivity and in accelerated progression of atherosclerosis. The aim of this study was to investigate the specific contribution of platelet‐derived JAM‐A to neointima formation after vascular injury. Mice with or without platelet‐specific (tr)JAM‐A‐deficiency in an apolipoprotein e (apoe^?/?) background underwent wire‐induced injury of the common carotid artery. Ex vivo imaging by two‐photon microscopy revealed increased platelet coverage at the site of injury in trJAM‐A‐deficient mice. Cell recruitment assays showed increased adhesion of monocytic cells to activated JAM‐A‐deficient platelets than to control platelets. Inhibition of α_Mβ₂ or GPIbα, but not of CD62P, suppressed those differences. Up to 4 weeks after wire injury, intimal neoplasia and neointimal cellular content were analysed. Neointimal lesion area was increased in trJAM‐A^?/? apoe^?/? mice and the lesions showed an increased macrophage accumulation and proliferating smooth muscle cells compared with trJAM‐A^+/+ apoe^?/? littermates 2 weeks, but not 4 weeks after injury. Re‐endothelialization was decreased in trJAM‐A^?/? apoe^?/? mice compared with controls 2 weeks after injury, yet it was complete in both groups after 4 weeks. A platelet gain of function by deletion of JAM‐A accelerates neointima formation only during earlier phases after vascular injury, through an increased recruitment of mononuclear cells. Thus, the contribution of platelets might become less important when neointima formation progresses to later stages.     

Clinical isolates of Enterococcus faecalis aggregate human platelets

Many endocarditis pathogens activate human platelets and this has been proposed to contribute to virulence. Here we report for the first time that many clinical isolates of Enterococcus faecalis, a common pathogen in infective endocarditis, aggregate human platelets. 84 isolates from human blood and urine were screened for their ability to aggregate platelets from four different donors. Platelet aggregation occurred for between 11 and 65% of isolates depending on the donor. In one donor, a significantly larger proportion of isolates from blood than from urine caused platelet aggregation. Median time to aggregation was 11 min and had a tendency to be shorter for blood isolates as compared to urine isolates. Immunoglobulin G (IgG) was shown to be essential in mediating activation and aggregation. Platelet aggregation could be abolished by an IgG-specific proteinase (IdeS), by an antibody blocking FcRγIIa on platelets, or by preabsorption of plasma with an E. faecalis isolate. Fibrinogen binding to bacteria or platelets does not contribute to platelet activation or aggregation under our experimental conditions. These results indicate that platelet activation and aggregation by E. faecalis is dependent on both host and bacterial factors and that it may be involved in the pathogenesis of invasive disease with this organism.     

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.