sunil joseph - Academia.edu (original) (raw)
Papers by sunil joseph
Biochemical Society Transactions, 1985
Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF
Cardiovascular Research, 1992
OBJECTIVE The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to medi... more OBJECTIVE The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2). METHODS Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin. RESULTS U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha. CONCLUSIONS In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.
FEBS Letters, 1986
The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ le... more The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ levels ([Ca2+]J and 5-hydroxy~*~]tryptamine ([%]5HT) secretion was studied. In washed human platelets prelabelled with fYJ5HT and quin 2, OAG (10-50 pg/mI) induced no significant aggregation, [Y]5HT secretion or rise in [Ca*+], in the presence or absence of fibrinogen. However, addition of OAG (IO-50 pg/mi) 10 s to 5 min before or IO-60 s after addition of threshold concentrations of thrombin (co.03 U/ml) resulted in a significant potentiation of aggregation and ['QHT secretion without any effect on the thrombin-induced rise in [Ca2+],. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced ['4C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Caz+]l nor the extent of [Y]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrpmbin-induced [Ca2+], mobilisation but can synergize with low concentrations of thrombin in potentiating [%JSHT secretion even at basal [Ca"],.
• Recent trends in social media and online access to publications have led to new ways of assessi... more • Recent trends in social media and online access to publications have led to new ways of assessing publication impact using article-level metrics (altmetrics). 1 • Altmetrics measure usage, captures/downloads, social media mentions and citations. Two commonly used tools include Impactstory.org and Altmetric.com. o Impactstory.org is a free-to-use, open-source tool that:-Uses both academic metrics (scholars) and altmetrics (public)-Normalizes metrics based on a sample of articles published the same year with results presented as raw data and percentiles compared with other articles (Figure 1a). o Altmetric.com is a proprietary tool adopted by several publishers including Springer, Nature Publishing Group, Scopus (Elsevier) and BioMed Central that:-Focuses on "public" social media, such as blog posts, news pieces, tweets and likes.-Scores each article based on three main factors: number of individuals mentioning the paper, where the mentions occurred and how often the author of each mention talks about scholarly articles (Figure 1b). • As part of a publications gap analysis exercise, Impactstory.org and Altmetric.com were used to measure the influence and reach of publications in a specific therapy area. The present study is a semi-quantitative comparison of the two altmetric tools in assessing publication impact for a compound (drug A) in Phase 3 development. • In addition Topsy (analytics), from Topsy.com, was used to analyze Twitter and assess the social media noise of drug A as shown by real-time tweet volume and sentiment. (Note: at the time of the present analysis, conducted between October and December 2013, access to a free trial of Topsy was available but this service has since been withdrawn). Objective: Recent trends in social media and online access of publications have led to new ways of assessing publication impact, using altmetrics. This study is a semi-quantitative comparison of two commonly used altmetric tools. Research design and methods: As part of a publications gap analysis, altmetrics were assessed using Altmetric.com and Impactstory.org. Here, we focus on publications for one drug in Phase 3 development. Additionally, Twitter activity (tweet volume/sentiment) was assessed using Topsy.com. Results: *Impactstory.org focuses on academic metrics, including academic readers and citations. Highly saved = in the 75 th-100 th percentile of articles added to users' Mendeley libraries vs. all articles published the same year. Highly cited = in the 75 th-100 th percentile of articles cited on PubMed Central or Scopus vs. all articles published the same year. † Altmetric.com focuses on "public" social media, providing results including more investor-related blogs and posts. A weighted score is produced, factoring in relevant impact of each online forum. Articles with a score ≥ 3 are in the top 25% of all articles ranked by attention; a score ≥ 15 puts the article in the top 5% of all articles ranked by attention. Articles classified as highly cited/saved by Impactstory.org could have no or low scores on Altmetric.com and scores did not always correlate across the two tools. Conclusion: Altmetric.com and Impactstory.org report different aspects of publication impact. Further, traditional impact measures, such as journal impact factor, did not equate to Impactstory.org or Altmetric.com scores and calls into question their value as qualitative impact tools. Topsy analysis highlighted that Twitter activity focused around congress presentations and press releases rather than publications (data not shown). Further research is required to assess the utility of altmetrics in tracking publication impact.
XIth International Congress on Thrombosis and Haemostasis, 1987
R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosph... more R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emph...
XIth International Congress on Thrombosis and Haemostasis, 1987
We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate ... more We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate (PMA), both activators of PrkC, on arachidonic acid (AA) release induced by thrombin, collagen and the Ca2+ ionophore, ionomycin, and correlated this with the levels of [Ca2+]i in quin 2-loaded platelets. Experiments were carried out using phenidone (200μM, a combined lipoxygenase and cyclooxygenase inhibitor)-treated washed, human platelets pre-labelled with [3H]-AA or quin 2 and, sub-maximal concentrations of thrombin, collagen and ionomycin. Incubation of platelets with diCg (30 or60μM) or PMA (16nM) over a 15 min period, resulted in no significant release of AA or rise in [Ca2+]i levels compared to that in resting platelets. However, thrombin (0.2U/ml) induced a 6-8% release of [3H]-AA as well as a rise in [Ca2+]i equalling 1μM and, addition of diCg (30-60μM) or PMA (16nM) 10 sec-5 min before or 10-20 sec after thrombin, resulted in a significant reduction (20-40%) of both responses....
XIth International Congress on Thrombosis and Haemostasis, 1987
Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an act... more Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PrkC), has been reported. We have examined the effects of the diacylglycerol (DG) analogues OAG and diCg, both PrkC activators, as well as PMA on intracellular Ca2+ ([Ca2+]i) mobilisation and 5-hydroxytryptamine (5HT) release induced by thrombin (T), collagen (Coll.) and the thromboxane (Tx) mimetic U46619. All studies were performed using washed human platelets pre-labelled with either quin-2 or [14C]-5HT and maximal concentrations of agonists. Neither diCg or PMA elevated [Ca2+]i above resting levels though both agents induced a small (10-15%) secretory response. In response to 0.2U/ml T or 0.6uM U46619, but not 20μg/ml Coll., [Ca2+]i increased from resting levels of lOOnM to 758±108nM and 712±58nM respectively. Addition of diCg (60μM) or PMA (16nM) 10 sec before or after T or U46619 reduced the control responses by 10-15% and 30-80% respectively. In contras...
Effect of the Polyamine-Spermine on Agonist-Induced Human Platelet Activation - Specific Inhibition of "Aggregation-Independent" Events Induced by Thrombin, but not by Collagen, Thromboxane Mimetic, Phorbol Ester or Calcium lonophore
Thrombosis and Haemostasis, 1987
SummarySpermine, a naturally occurring polyamine, has previously been described as an inhibitor o... more SummarySpermine, a naturally occurring polyamine, has previously been described as an inhibitor of purified phospholipase C and protein kinase C in cell-free systems. The present study examines the effect of spermine on platelet aggregation, dense-granule secretion and thromboxane (Tx) B2 synthesis induced by a variety of agonists, which cause the activation of one or both enzymes to different extents. These studies revealed that, while spermine (10 mM) inhibited platelet aggregation in response to all the agonists examined, [14C]-5-hydroxytryptamine (5HT) release and TxB2 synthesis induced by thrombin (0.2 U/ml) and collagen (10-40 μg/ml) alone, were inhibited by spermine, the percentage inhibition being >90% for both responses with thrombin, 30% for 5HT release and 80% for TxB2 synthesis with collagen. The inhibition of collagen-induced [14C]-5HT secretion by spermine was due entirely to the inhibition of aggregation-dependent TxA2 synthesis as addition of a sub-threshold conce...
1,2-Dioctanoylglycerol but not 1-oleoyl-2-acetylglycerol inhibits agonist-induced platelet responses. Dependence of effects on extent of 45-kDa protein phosphorylation and agonist type
European Journal of Biochemistry, 1987
1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro... more 1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro) and 1-oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets. 2. Pre-treatment (10-300 s) with Oco2Gro (15-60 microM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62-125 microM) had no effect on thrombin-induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3. Collagen (20 micrograms/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10-300 s) with Oco2Gro (15-60 microM), PMA (16 nM) or OleAcGro (62 microM) before collagen addition or addition of these agents 30-60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2--2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations (less than 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5-15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 microM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with less than 2-min incubations) at sub-maximal agonist concentrations. 5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while Oco2Gro (60 microM) and PMA (16 nM) caused a 4--5-fold increase in 32P-labelling of this protein over a 5-min incubation period, OleAcGro (62-125 microM) caused a 1.5-fold increase in labelling which was only maintained for a 10--30-s period.(ABSTRACT TRUNCATED AT 400 WORDS)
Synergistic stimulation of the platelet release reaction by ‘weak’ agonist plus thromboxane mimetic: mechanism of ‘weak’ agonist-induced release reaction
Biochemical Society Transactions, 1985
... release reaction. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Rese... more ... release reaction. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK. Abbreviations used ...
Biochemical Journal, 1986
Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdo... more Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubati...
Stimulation of U937 human monocytic cells by thrombin results in inositol trisphosphate formation and the mobilisation of intracellular Ca2+but not the synthesis of thromboxane B2
Biochemical Society Transactions, 1991
Regulation of thrombin-induced arachidonic acid metabolism by extracellular Ca 2+
Biochemical Society Transactions, 1985
... extracellular Ca 2+. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis R... more ... extracellular Ca 2+. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK. Abbreviations used ...
Selective desensitization of protein-kinase-C-mediated platelet granule secretion with prolonged incubation of protein kinase C activators despite sustained 45 kDa phosphorylation
Biochemical Society Transactions, 1988
... SUSHILA KRISHNAMURTHI, YATIN PATEL, SUNIL JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Uni... more ... SUSHILA KRISHNAMURTHI, YATIN PATEL, SUNIL JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Rayne Insitute, 123 Coldharbour Lane, London SE5 9NU, UK. ...
Pharmacological manipulation of diacylglycerol-dependent protein kinase C
Trends in Pharmacological Sciences, 1989
1. Trends Pharmacol Sci. 1989 Oct;10(10):396. Pharmacological manipulation of diacylglycerol-depe... more 1. Trends Pharmacol Sci. 1989 Oct;10(10):396. Pharmacological manipulation of diacylglycerol-dependent protein kinase C. Joseph S, Krishnamurthi S. PMID: 2617665 [PubMed - indexed for MEDLINE]. Publication Types: Letter. MeSH Terms: ...
The identity of IgE receptors (FcεRII) That mediate cellular activation of human macrophages: Evidence against a role for CD23
Molecular Immunology, 1992
Abstract There is now compelling evidence that macrophages bind IgE, and are involved in several ... more Abstract There is now compelling evidence that macrophages bind IgE, and are involved in several IgE-dependent responses. The CD23 antigen mediates a mitogenic response in “primed” B-lymphocytes, although its expression is not confined to B cells, and CD23 is inducably expressed in many cells including macrophages. CD23 is also known to bind IgE, a property that leads to inhibition of the mitogenic response in B cells. In the present review, the possibility that CD23 mediates IgE-dependent responses in macrophages has been re-examined, and it is proposed that the functional receptor for IgE on macrophages may be quite separate from the CD23 antigen.
European Journal of Pharmacology: Molecular Pharmacology, 1991
The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic eff... more The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have u: the U937 human monocytic cell line Io explore the signal transduction mechanisms utilised by thrombin ia these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP 3) and the mobilisatmn of intracellutar Ca 2+ ([Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B 2 synthesis in the differentiated ce~ls. In contrast, tile chemotactic peptide N-formy-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxBz synthesis under conditions where it evoked increases in IP 3 formation and [Ca 2~ ]~ mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thmmbin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. Th,;se results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP 3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB 2 synthesis or cause Gi-mediated inhibition of adcnylate cycla,,e in U937 cells contrasts with its effects in human :;latelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin recc~tor or receptor-eflector coupling mechanism(s) present in other thrombin-re~;ponsive cells.
British Journal of Pharmacology, 1992
Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (P... more Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; K., = 0.7 nM) in a concentrationdependent manner when exposed to bradykinin. 2 The bradykinin-dependent release of PG12 (but not EDRF) was inhibited by 1 1AM isoprenaline or 5 &M forskolin, and the inhibitory effect of isoprenaline could be reversed by the P2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PG12 release stimulated by exogenous arachidonic acid. 3 Exposure of cells to bradykinin increased the cytosolic concentration of Ca2`ions ([Ca2+]i; Kad = 4.8 nM), and the effect was inhibited by both 1 LM isoprenaline and 5 AM forskolin. 4 In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of that required for PG12 biosynthesis and that these corresponded to a [Ca2f], threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5 These differences in Ca2' ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2t]j.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased 32P incorporation into phos... more Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased 32P incorporation into phospholipids namely phosphatadyl-inositol (PI); phosphatidyl-inositol-4-phosphate (PIP); phosphatidyl-inositol-4,5-bis-phosphate (PIP2); phosphatidyl-acid (PA); phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE) particularly at the 20-30th min after treatment. The ratio of members of the phosphoinositol system, especially PIP and PI, related to the total phospholipid content was increased. PMA (2 x lo-' M) was the most effective of the three concentrations tested. The results call attention to the presence of a working phosphoinositol system in Protozoa.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1988
R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the co... more 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-dioctanoylglyceroi (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 DiC 8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC 8 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.
Biochemical Society Transactions, 1985
Activation of bovine endothelial thromboxane receptors triggers release of prostacyclin but not EDRF
Cardiovascular Research, 1992
OBJECTIVE The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to medi... more OBJECTIVE The aim was to examine the capacity of U46619 (a stable thromboxane A2 mimetic) to mediate release of endothelium derived relaxing factor (EDRF) from bovine aortic endothelial cells, and compare the response to the U46619 dependent release of prostacyclin (PGI2). METHODS Bovine aortic endothelial cells (AG4762) were cultured in vitro on microcarrier beads, which were then loaded onto a column and perfused. The cells were challenged with U46619, bradykinin, or the Ca2+ ionophore ionomycin in the perfusate, and measurements made of the release of 6-oxo-PGF1 alpha (the stable hydrolysis product of PGI2, measured by radioimmunoassay) and EDRF (bioassay). Cells were also cultured on glass cover slips, loaded with Fura 2-AM, and measurements made of the rise in intracellular Ca2+ after challenge with U46619, bradykinin or ionomycin. RESULTS U46619 triggered release of 6-oxo-PGF1 alpha but not EDRF from AG4762 cells, contrasting with bradykinin which released both 6-oxo-PGF1 alpha and EDRF. Ionomycin had little or no capacity to mimic and trigger release of 6-oxo-PGF1 alpha, although ionomycin mediated large increases in intracellular Ca2+. In contrast, staurosporine (a putative inhibitor of protein kinase C) substantially inhibited the U46619 and bradykinin dependent release of 6-oxo-PGF1 alpha. CONCLUSIONS In contrast to bradykinin linked receptors on AG4762 endothelial cells, which are coupled to the release of both prostacyclin and EDRF, activation of thromboxane A2 receptors on these cells selectively triggers release of PGI2 but not EDRF. Further, based on the distinct effects of ionomycin and staurosporine, it appears that agonist stimulated PGI2 release from these cells is mediated predominantly by protein kinase C, rather than by rises in intracellular Ca2+. This observation contrasts with previously described mechanisms of PGI2 release from endothelium obtained from other sources.
FEBS Letters, 1986
The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ le... more The effect of I-oleoyl-2-acetylgIy~ro1 (OAG) on the thrombin-endued rise in intracellular Ca2+ levels ([Ca2+]J and 5-hydroxy~*~]tryptamine ([%]5HT) secretion was studied. In washed human platelets prelabelled with fYJ5HT and quin 2, OAG (10-50 pg/mI) induced no significant aggregation, [Y]5HT secretion or rise in [Ca*+], in the presence or absence of fibrinogen. However, addition of OAG (IO-50 pg/mi) 10 s to 5 min before or IO-60 s after addition of threshold concentrations of thrombin (co.03 U/ml) resulted in a significant potentiation of aggregation and ['QHT secretion without any effect on the thrombin-induced rise in [Ca2+],. Both EGTA, which abolished the latter and creatine phosphate/creatine phosphokinase, the ADP scavenger, totally inhibited the aggregation but only partially reduced ['4C]5HT secretion in response to thrombin plus OAG. At higher concentrations of thrombin, neither the rise in [Caz+]l nor the extent of [Y]5HT secretion was significantly altered by OAG addition. The results demonstrate that, unlike phorbol esters, OAG has no inhibitory effect on thrpmbin-induced [Ca2+], mobilisation but can synergize with low concentrations of thrombin in potentiating [%JSHT secretion even at basal [Ca"],.
• Recent trends in social media and online access to publications have led to new ways of assessi... more • Recent trends in social media and online access to publications have led to new ways of assessing publication impact using article-level metrics (altmetrics). 1 • Altmetrics measure usage, captures/downloads, social media mentions and citations. Two commonly used tools include Impactstory.org and Altmetric.com. o Impactstory.org is a free-to-use, open-source tool that:-Uses both academic metrics (scholars) and altmetrics (public)-Normalizes metrics based on a sample of articles published the same year with results presented as raw data and percentiles compared with other articles (Figure 1a). o Altmetric.com is a proprietary tool adopted by several publishers including Springer, Nature Publishing Group, Scopus (Elsevier) and BioMed Central that:-Focuses on "public" social media, such as blog posts, news pieces, tweets and likes.-Scores each article based on three main factors: number of individuals mentioning the paper, where the mentions occurred and how often the author of each mention talks about scholarly articles (Figure 1b). • As part of a publications gap analysis exercise, Impactstory.org and Altmetric.com were used to measure the influence and reach of publications in a specific therapy area. The present study is a semi-quantitative comparison of the two altmetric tools in assessing publication impact for a compound (drug A) in Phase 3 development. • In addition Topsy (analytics), from Topsy.com, was used to analyze Twitter and assess the social media noise of drug A as shown by real-time tweet volume and sentiment. (Note: at the time of the present analysis, conducted between October and December 2013, access to a free trial of Topsy was available but this service has since been withdrawn). Objective: Recent trends in social media and online access of publications have led to new ways of assessing publication impact, using altmetrics. This study is a semi-quantitative comparison of two commonly used altmetric tools. Research design and methods: As part of a publications gap analysis, altmetrics were assessed using Altmetric.com and Impactstory.org. Here, we focus on publications for one drug in Phase 3 development. Additionally, Twitter activity (tweet volume/sentiment) was assessed using Topsy.com. Results: *Impactstory.org focuses on academic metrics, including academic readers and citations. Highly saved = in the 75 th-100 th percentile of articles added to users' Mendeley libraries vs. all articles published the same year. Highly cited = in the 75 th-100 th percentile of articles cited on PubMed Central or Scopus vs. all articles published the same year. † Altmetric.com focuses on "public" social media, providing results including more investor-related blogs and posts. A weighted score is produced, factoring in relevant impact of each online forum. Articles with a score ≥ 3 are in the top 25% of all articles ranked by attention; a score ≥ 15 puts the article in the top 5% of all articles ranked by attention. Articles classified as highly cited/saved by Impactstory.org could have no or low scores on Altmetric.com and scores did not always correlate across the two tools. Conclusion: Altmetric.com and Impactstory.org report different aspects of publication impact. Further, traditional impact measures, such as journal impact factor, did not equate to Impactstory.org or Altmetric.com scores and calls into question their value as qualitative impact tools. Topsy analysis highlighted that Twitter activity focused around congress presentations and press releases rather than publications (data not shown). Further research is required to assess the utility of altmetrics in tracking publication impact.
XIth International Congress on Thrombosis and Haemostasis, 1987
R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosph... more R59022 is a recently described inhibitor of the enzyme DG kinase [1], which converts DG to phosphatidic acid. While R59002 inhibits DG conversion in platelets resulting in enhanced protein kinase C (PrkC) activation [1], little is known on its effect on other platelet responses. In this study, we have examined the effect of R59022 on agonist-induced platelet aggregation and [14C]-5-hydroxytryptamine (5HT) release using washed human platelets. With a sub-maximal concentration of thrombin (T, 0.05U/ml) R59022 (10-30μM) significantly potentiated T-induced platelet aggregation and [14C]-5HT release eg % [14C]-5HT release:- 0.05U/ml T-52±5,30μM R59022+T-76±8. Removal of external Ca2+ (ImM) using EGTA (5mM) reduced T-induced 5HT release but not the potentiation of it by R59022 eg EGTA+ 0.05U/ml T-36±6%, EGTA+R59022+T- 72±5%. These results show that in the presence of EGTA and R59022 the increased DG levels can compensate for the diminished rise in T-induced Ca/2+ mobilisation thus re-emph...
XIth International Congress on Thrombosis and Haemostasis, 1987
We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate ... more We have examined the effect of 1,2 dioctanoylglycerol (diCg) and phorbol 12-myristate 13-acetate (PMA), both activators of PrkC, on arachidonic acid (AA) release induced by thrombin, collagen and the Ca2+ ionophore, ionomycin, and correlated this with the levels of [Ca2+]i in quin 2-loaded platelets. Experiments were carried out using phenidone (200μM, a combined lipoxygenase and cyclooxygenase inhibitor)-treated washed, human platelets pre-labelled with [3H]-AA or quin 2 and, sub-maximal concentrations of thrombin, collagen and ionomycin. Incubation of platelets with diCg (30 or60μM) or PMA (16nM) over a 15 min period, resulted in no significant release of AA or rise in [Ca2+]i levels compared to that in resting platelets. However, thrombin (0.2U/ml) induced a 6-8% release of [3H]-AA as well as a rise in [Ca2+]i equalling 1μM and, addition of diCg (30-60μM) or PMA (16nM) 10 sec-5 min before or 10-20 sec after thrombin, resulted in a significant reduction (20-40%) of both responses....
XIth International Congress on Thrombosis and Haemostasis, 1987
Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an act... more Inhibition of agonist-induced platelet responses by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PrkC), has been reported. We have examined the effects of the diacylglycerol (DG) analogues OAG and diCg, both PrkC activators, as well as PMA on intracellular Ca2+ ([Ca2+]i) mobilisation and 5-hydroxytryptamine (5HT) release induced by thrombin (T), collagen (Coll.) and the thromboxane (Tx) mimetic U46619. All studies were performed using washed human platelets pre-labelled with either quin-2 or [14C]-5HT and maximal concentrations of agonists. Neither diCg or PMA elevated [Ca2+]i above resting levels though both agents induced a small (10-15%) secretory response. In response to 0.2U/ml T or 0.6uM U46619, but not 20μg/ml Coll., [Ca2+]i increased from resting levels of lOOnM to 758±108nM and 712±58nM respectively. Addition of diCg (60μM) or PMA (16nM) 10 sec before or after T or U46619 reduced the control responses by 10-15% and 30-80% respectively. In contras...
Effect of the Polyamine-Spermine on Agonist-Induced Human Platelet Activation - Specific Inhibition of "Aggregation-Independent" Events Induced by Thrombin, but not by Collagen, Thromboxane Mimetic, Phorbol Ester or Calcium lonophore
Thrombosis and Haemostasis, 1987
SummarySpermine, a naturally occurring polyamine, has previously been described as an inhibitor o... more SummarySpermine, a naturally occurring polyamine, has previously been described as an inhibitor of purified phospholipase C and protein kinase C in cell-free systems. The present study examines the effect of spermine on platelet aggregation, dense-granule secretion and thromboxane (Tx) B2 synthesis induced by a variety of agonists, which cause the activation of one or both enzymes to different extents. These studies revealed that, while spermine (10 mM) inhibited platelet aggregation in response to all the agonists examined, [14C]-5-hydroxytryptamine (5HT) release and TxB2 synthesis induced by thrombin (0.2 U/ml) and collagen (10-40 μg/ml) alone, were inhibited by spermine, the percentage inhibition being >90% for both responses with thrombin, 30% for 5HT release and 80% for TxB2 synthesis with collagen. The inhibition of collagen-induced [14C]-5HT secretion by spermine was due entirely to the inhibition of aggregation-dependent TxA2 synthesis as addition of a sub-threshold conce...
1,2-Dioctanoylglycerol but not 1-oleoyl-2-acetylglycerol inhibits agonist-induced platelet responses. Dependence of effects on extent of 45-kDa protein phosphorylation and agonist type
European Journal of Biochemistry, 1987
1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro... more 1. The effect of the membrane-permeable diacylglycerol analogues, 1,2-dioctanoylglycerol (Oco2Gro) and 1-oleoyl-2-acetyl-glycerol (OleAcGro) on agonist-induced platelet activation processes were compared with those of the phorbol ester, phorbol 12-myristate 13-acetate (PMA), using appropriately labelled washed human platelets. 2. Pre-treatment (10-300 s) with Oco2Gro (15-60 microM) or PMA (16 nM) before addition of thrombin (0.2 U/ml) or, addition of these agents 10-20 s after thrombin, resulted in a significant reduction (20-80%) in the extent of thrombin-induced intracellular Ca2+ ([Ca2+]i) mobilisation and arachidonate/thromboxane B2 release. OleAcGro (62-125 microM) had no effect on thrombin-induced [Ca2+]i elevations but had a slight (15%) inhibitory effect on thrombin-induced arachidonate release with a 5-min pre-incubation. Addition of Oco2Gro, PMA or OleAcGro on their own caused no rise in [Ca2+]i levels or arachidonate release. 3. Collagen (20 micrograms/ml) induced substantial arachidonate release without a detectable rise in [Ca2+]i. Pretreatment (10-300 s) with Oco2Gro (15-60 microM), PMA (16 nM) or OleAcGro (62 microM) before collagen addition or addition of these agents 30-60 s after collagen addition resulted in a significant potentiation of arachidonate release (1.2--2-fold over control), even though thromboxane B2 formation in response to collagen was inhibited in the presence of Oco2Gro or PMA. 4. Both Oco2Gro and PMA had dual effects on 5-hydroxytryptamine secretion induced by thrombin or collagen. Short pre-incubations (less than 2 min) with these agents caused a potentiation of sub-maximal agonist-induced secretion, while not affecting secretion induced by maximal agonist concentrations. With longer pre-incubation times (5-15 min) however, a significant reduction in the level of agonist-induced secretion in the presence of Oco2Gro or PMA was observed. Inhibition of secretion was also observed in platelets treated with indomethacin (10 microM), suggesting that inhibition of thromboxane B2 formation alone does not account for inhibition of 5-hydroxytryptamine secretion. OleAcGro had no inhibitory effects on agonist-induced secretion even though it potentiated it (with less than 2-min incubations) at sub-maximal agonist concentrations. 5. Time courses of phosphorylation of a 45-kDa protein, a marker of protein kinase C activation, in 32P-labelled platelets showed that while Oco2Gro (60 microM) and PMA (16 nM) caused a 4--5-fold increase in 32P-labelling of this protein over a 5-min incubation period, OleAcGro (62-125 microM) caused a 1.5-fold increase in labelling which was only maintained for a 10--30-s period.(ABSTRACT TRUNCATED AT 400 WORDS)
Synergistic stimulation of the platelet release reaction by ‘weak’ agonist plus thromboxane mimetic: mechanism of ‘weak’ agonist-induced release reaction
Biochemical Society Transactions, 1985
... release reaction. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Rese... more ... release reaction. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK. Abbreviations used ...
Biochemical Journal, 1986
Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdo... more Previous studies have demonstrated an inhibition of agonist-induced inositol phospholipid breakdown and intracellular Ca2+ ([Ca2+]i) mobilization by phorbol esters in platelets. In this study, we have examined the effect of phorbol 12-myristate 13-acetate (PMA) on agonist-induced granule secretion and correlated it with agonist-induced [Ca2+]i mobilization, arachidonate and thromboxane (Tx) release in human platelets. With increasing times of incubation with PMA (10 s-5 min), the rise in [Ca2+]i induced by thrombin and the TxA2 mimetic, U46619, was increasingly inhibited (90-100% with 5 min incubation) and, correlating with this, thrombin-induced [3H]arachidonate, TxB2 and beta-thromboglobulin (beta TG) release were also inhibited. In addition, the conversion of exogenously added arachidonate to TxB2 was inhibited (50-80%) by a 10 s-5 min pretreatment with PMA. However, secretion of 5-hydroxy[14C]tryptamine (5HT) induced by thrombin or U46619 was not inhibited by 10 s-2 min incubati...
Stimulation of U937 human monocytic cells by thrombin results in inositol trisphosphate formation and the mobilisation of intracellular Ca2+but not the synthesis of thromboxane B2
Biochemical Society Transactions, 1991
Regulation of thrombin-induced arachidonic acid metabolism by extracellular Ca 2+
Biochemical Society Transactions, 1985
... extracellular Ca 2+. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis R... more ... extracellular Ca 2+. SUSHILA KRISHNAMURTHI, SUNIL K. JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Denmark Hill, London SE5 8RX, UK. Abbreviations used ...
Selective desensitization of protein-kinase-C-mediated platelet granule secretion with prolonged incubation of protein kinase C activators despite sustained 45 kDa phosphorylation
Biochemical Society Transactions, 1988
... SUSHILA KRISHNAMURTHI, YATIN PATEL, SUNIL JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Uni... more ... SUSHILA KRISHNAMURTHI, YATIN PATEL, SUNIL JOSEPH and VIJAY V. KAKKAR. Thrombosis Research Unit, King's College School of Medicine and Dentistry, Rayne Insitute, 123 Coldharbour Lane, London SE5 9NU, UK. ...
Pharmacological manipulation of diacylglycerol-dependent protein kinase C
Trends in Pharmacological Sciences, 1989
1. Trends Pharmacol Sci. 1989 Oct;10(10):396. Pharmacological manipulation of diacylglycerol-depe... more 1. Trends Pharmacol Sci. 1989 Oct;10(10):396. Pharmacological manipulation of diacylglycerol-dependent protein kinase C. Joseph S, Krishnamurthi S. PMID: 2617665 [PubMed - indexed for MEDLINE]. Publication Types: Letter. MeSH Terms: ...
The identity of IgE receptors (FcεRII) That mediate cellular activation of human macrophages: Evidence against a role for CD23
Molecular Immunology, 1992
Abstract There is now compelling evidence that macrophages bind IgE, and are involved in several ... more Abstract There is now compelling evidence that macrophages bind IgE, and are involved in several IgE-dependent responses. The CD23 antigen mediates a mitogenic response in “primed” B-lymphocytes, although its expression is not confined to B cells, and CD23 is inducably expressed in many cells including macrophages. CD23 is also known to bind IgE, a property that leads to inhibition of the mitogenic response in B cells. In the present review, the possibility that CD23 mediates IgE-dependent responses in macrophages has been re-examined, and it is proposed that the functional receptor for IgE on macrophages may be quite separate from the CD23 antigen.
European Journal of Pharmacology: Molecular Pharmacology, 1991
The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic eff... more The blood coagulation factor, human thrombin has been shown to have chemotactic and mitogenic effects on mononuclear phagocytic inflammatory cells. In the present study, we have u: the U937 human monocytic cell line Io explore the signal transduction mechanisms utilised by thrombin ia these cells. In U937 cells differentiated into a macrophage-like phenotype, thrombin stimulated the formation of inositol trisphosphate (IP 3) and the mobilisatmn of intracellutar Ca 2+ ([Ca2+]i) via a mechanism which was partially sensitive to pertussis toxin. Thrombin failed, however, to evoke thromboxane (Tx) B 2 synthesis in the differentiated ce~ls. In contrast, tile chemotactic peptide N-formy-L-methionylleucyl-L-phenylalanine (FMLP) stimulated TxBz synthesis under conditions where it evoked increases in IP 3 formation and [Ca 2~ ]~ mobilisation, via a pertussis toxin-sensitive mechanism, comparable in extent to those mediated by thrombin. Thmmbin also failed to cause inhibitory guanine nucleotide binding protein (Gi)-mediated inhibition of adenylate cyclase activity in U937 cell membranes. Th,;se results indicate that U937 cells express receptors for thrombin which are in part coupled via a pertussis toxin-sensitive guanine nucleotide binding protein to phospholipase C activation, the formation of IP 3 and the mobilisation of [Ca2+]i. However, the failure of thrombin to stimulate TxB 2 synthesis or cause Gi-mediated inhibition of adcnylate cycla,,e in U937 cells contrasts with its effects in human :;latelets and other thrombin-responsive cells. These results suggest that the thrombin receptor or receptor-effector coupling mechanism(s) in mononuclear cells is functionally distinct from the thrombin recc~tor or receptor-eflector coupling mechanism(s) present in other thrombin-re~;ponsive cells.
British Journal of Pharmacology, 1992
Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (P... more Bovine aortic endothelial cells were cultured in vitro, and shown to release both prostacyclin (PGI2; Kact = 24.1 nM) and endothelium-derived relaxing factor (EDRF, NO; K., = 0.7 nM) in a concentrationdependent manner when exposed to bradykinin. 2 The bradykinin-dependent release of PG12 (but not EDRF) was inhibited by 1 1AM isoprenaline or 5 &M forskolin, and the inhibitory effect of isoprenaline could be reversed by the P2-adrenoceptor antagonist, ICI 118551. In contrast, isoprenaline had no capacity to inhibit PG12 release stimulated by exogenous arachidonic acid. 3 Exposure of cells to bradykinin increased the cytosolic concentration of Ca2`ions ([Ca2+]i; Kad = 4.8 nM), and the effect was inhibited by both 1 LM isoprenaline and 5 AM forskolin. 4 In similar experiments, exposure of cells to ionomycin also increased [Ca2+]i and the values of [Ca2+]i were calibrated in terms of the ionomycin concentration. In subsequent experiments involving exposure of endothelial cells to selected concentrations of ionomycin, it was possible to show that the biosynthesis of NO was triggered at ionomycin concentrations about one tenth of that required for PG12 biosynthesis and that these corresponded to a [Ca2f], threshold of 350 nM for PGI2 release while that for EDRF release was less than 200 nM. 5 These differences in Ca2' ion sensitivity explain the selective inhibition of bradykinin-stimulated PGI2 biosynthesis (to the exclusion of NO biosynthesis) by isoprenaline or forskolin, both of which attenuate bradykinin-dependent increases in [Ca2t]j.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1987
Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased 32P incorporation into phos... more Phorbol 12-myristate 13-acetate (PMA) treatment elicited an increased 32P incorporation into phospholipids namely phosphatadyl-inositol (PI); phosphatidyl-inositol-4-phosphate (PIP); phosphatidyl-inositol-4,5-bis-phosphate (PIP2); phosphatidyl-acid (PA); phosphatidyl-choline (PC) and phosphatidyl-ethanolamine (PE) particularly at the 20-30th min after treatment. The ratio of members of the phosphoinositol system, especially PIP and PI, related to the total phospholipid content was increased. PMA (2 x lo-' M) was the most effective of the three concentrations tested. The results call attention to the presence of a working phosphoinositol system in Protozoa.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1988
R59022 is an inhibitor of the enzyme 1,2-diacylglycerol (DAG) kinase, which, by inhibiting the co... more 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-dioctanoylglyceroi (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 DiC 8. The inhibition of collagen-induced secretion by R59022 was in contrast to the potentiatory effects of DiC 8 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.