Evidence for two mechanisms of thrombin-induced platelet activation: one proteolytic, one receptor mediated

1989 ◽  
Vol 67 (7) ◽  
pp. 332-336 ◽  
Author(s):  
Archibald McNicol ◽  
Jon M. Gerrard ◽  
D. Euan MacIntyre
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Platelet Reactivity ◽  
Platelet Activating Factor ◽  
Receptor Activation ◽  
Acid Formation ◽  
Phosphoinositide Metabolism ◽  
Co Addition ◽  
The Individual ◽  
Dual Mechanism

The possibility that thrombin-induced platelet reactivity could occur via both a receptor-related and a proteolytic process was examined. Thrombin elicited the formation of considerably more [32P)phosphatidic acid (an index of phospholipase C catalysed phosphoinositide metabolism) than did platelet activating factor, 5-hydroxytryptamine, ADP, and the thromboxane A2 analogue EP171, when these agents were added either alone or in combination. Co-addition of thrombin and EP171 did not evoke significantly more [32P]phosphatidic acid than did thrombin alone. The protease inhibitor leupeptin, decreased but did not abolish [32P]phosphatidic acid formation elicited by either thrombin alone or thrombin in combination with EP171. The serine protease, trypsin, stimulated an increase in [32P]phosphatidic acid and this effect was additive with that of EP171. This augmentation by trypsin of EP171-induced [32P]phosphatidic acid formation was inhibited by leupeptin. These results are consistent with the concept that thrombin-induced activation of phospholipase C occurs by two distinct mechanisms: one via proteolysis, which is sensitive to leupeptin, and the other via receptor activation, a process shared by EP171. The individual components of this dual mechanism can be mimicked by the co-addition of a receptor-directed agonist (EP171) and a proteolytic agent (trypsin).Key words: platelet, thrombin, proteolysis, phosphoinositide.

Augmented phosphoinositide metabolism in aortas from genetically hypertensive rats

1990 ◽  
Vol 258 (1) ◽  
pp. H173-H178 ◽  
Author(s):  
M. B. Turla ◽  
R. C. Webb
Keyword(s):  
Phospholipase C ◽  
Vascular Reactivity ◽  
Inositol Phosphates ◽  
Inositol Phosphate ◽  
Second Messengers ◽  
Receptor Activation ◽  
Phosphoinositide Hydrolysis ◽  
Phosphoinositide Metabolism ◽  
Hypertensive Rats ◽  
Wistar Kyoto

Recent studies suggest that serotonergic receptor activation is coupled to phospholipase C-mediated phosphoinositide hydrolysis, which results in the release of intracellular second messengers. The purpose of this study was to determine whether altered phosphoinositide metabolism is the basis for augmented vascular responsiveness to serotonin in genetic hypertension. Thoracic aortic segments isolated from stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto normotensive rats (WKY) were labeled with myo-[3H]inositol and stimulated with serotonin in the presence of LiCl. Accumulation of [3H]inositol phosphates was then quantitated by column chromatography. Basal inositol phosphate accumulation and basal incorporation of myo-[3H]inositol into aortic cell membranes from SHRSP was not significantly different from WKY values. At 2.6 x 10(-7) to 2.6 x 10(-4) M serotonin, phosphoinositide metabolism was significantly augmented in aortae from SHRSP compared with WKY. Depolarization (100 mM KCl) did not increase phosphoinositide hydrolysis above basal levels in SHRSP or WKY. 2-Nitro-4-carboxyphenyl-N,N-diphenyl carbamate (NCDC), an inhibitor of phospholipase C, prevented the serotonin-induced phosphoinositide metabolism. NCDC also partially inhibited phasic contractions (responses in calcium-free solution) to serotonin in aortas from SHRSP and WKY. In conclusion, abnormal phosphoinositide metabolism may be one mechanism responsible for the characteristic increase in vascular reactivity to serotonin in hypertension.

scholarly journals Evidence for the calcium-dependent activation of phospholipase D in thrombin-stimulated human erythroleukaemia cells

1990 ◽  
Vol 267 (2) ◽  
pp. 479-483 ◽  
Author(s):  
S P Halenda ◽  
A G Rehm
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Calf Serum ◽  
Platelet Activating Factor ◽  
Diacylglycerol Kinase ◽  
Sole Source ◽  
Phospholipid Metabolism ◽  
Ionophore A23187 ◽  
Hel Cells

Human erythroleukaemia (HEL) cells were exposed to thrombin and other platelet-activating stimuli, and changes in radiolabelled phospholipid metabolism were measured. Thrombin caused a transient fall in PtdInsP and PtdInsP2 levels, accompanied by a rise in diacylglycerol and phosphatidic acid, indicative of a classical phospholipase C/diacylglycerol kinase pathway. However, the rise in phosphatidic acid preceded that of diacylglycerol, which is inconsistent with phospholipase C/diacylglycerol kinase being the sole source of phosphatidic acid. In the presence of ethanol, thrombin and other agonists (platelet-activating factor, adrenaline and ADP, as well as fetal-calf serum) stimulated the appearance of phosphatidylethanol, an indicator of phospholipase D activity. The Ca2+ ionophore A23187 and the protein kinase C activator phorbol myristate acetate (PMA) also elicited phosphatidylethanol formation, although A23187 was at least 5-fold more effective than PMA. Phosphatidylethanol production stimulated by agonists or A23187 was Ca2(+)-dependent, whereas that with PMA was not. These result suggest that phosphatidic acid is generated in agonist-stimulated HEL cells by two routes: phospholipase C/diacylglycerol kinase and phospholipase D. Activation of the HEL-cell phospholipase D in response to agonists may be mediated by a rise in intracellular Ca2+.

Verapamil inhibits phosphatidic acid formation and modifies phosphoinositide metabolism in stimulated platelets

1990 ◽  
Vol 182 (3) ◽  
pp. 457-464 ◽  
Author(s):  
Sheryl T. Homa ◽  
Shah Nawaz Khan ◽  
Dolores M. Conroy ◽  
Andrew E. Speak ◽  
Anthony D. Smith
Keyword(s):  
Phosphatidic Acid ◽  
Acid Formation ◽  
Phosphoinositide Metabolism

scholarly journals Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1676-1683 ◽  
Author(s):  
X Yang ◽  
L Sun ◽  
S Ghosh ◽  
AK Rao
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Ionophore A23187 ◽  
Serotonin Secretion ◽  
Platelet Signaling ◽  
Intracellular Mediators ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Pkc Activation

Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5′-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

scholarly journals Action of guanosine 5′-[β-thio]diphosphate on thrombin-induced activation and Ca2+ mobilization in saponin-permeabilized and intact human platelets

1988 ◽  
Vol 255 (3) ◽  
pp. 885-893 ◽  
Author(s):  
K S Authi ◽  
G H R Rao ◽  
B J Evenden ◽  
N Crawford
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
G Protein ◽  
Protein Function ◽  
Low Dose ◽  
Shape Change ◽  
Acid Formation ◽  
Functional Responses ◽  
Permeabilized Cells ◽  
Fura 2

The non-hydrolysable guanine analogues guanosine 5′-[gamma-thio]triphosphate (GTP[S]) and guanosine 5′-[beta-thio]diphosphate (GDP[S]) have been used extensively (as promoters and inhibitors respectively) to probe the importance of G-protein function. We report on the use of GDP[S] in permeabilized and intact platelets. The stimulatory analogue GTP[S] (9-60 microM) induces shape change, aggregation and 5-hydroxy[14C]-tryptamine secretion when added to saponin (12-14 micrograms/ml)-permeabilized platelets, but not to intact platelets. In line with the activation responses in permeabilized cells, GTP[S] induces an increase in [32P]-phosphatidic acid, which is indicative of phospholipase C activity. GDP[S] (greater than 400 microM) totally inhibits GTP[S] (90 microM)-stimulated phospholipase C activity and functional responses in saponized platelets. GDP[S] (1 mM) was also effective at inhibiting low-dose thrombin (0.1 unit/ml)-induced aggregation and secretion responses (without affecting shape change) in permeabilized platelets with inhibition of [32P]-phosphatidic acid formation. At higher doses of thrombin (greater than 0.5 unit/ml), both functional responses and [32P]phosphatidic acid formation are restored in the presence of GDP[S]. Studies on intact cells revealed that GDP[S] was as effective at inhibiting low-dose thrombin-induced functional responses as in the permeabilized cells, but there was no inhibition of [32P]phosphatidic acid formation, indicating that the agent is nonmembrane-penetrating. This reflected the fact that GDP[S] has additional inhibitory sites on the surface of platelets. In Fura-2-loaded cells GDP[S] inhibited thrombin-induced Ca2+ mobilization, as measured by Fura-2 fluorescence, in a dose-dependent manner. In studies with and without Ca2+ present on the outside, the effect of GDP[S] was to block Ca2+ influx. These studies indicate that, although GDP[S] is a valuable tool in studying G-protein function in permeabilized cells, it also has inhibitory activities on the surface of platelets, and one of these has been identified as an effect on the Ca2+-influx channel after agonist stimulation.

Aluminum inhibits phosphatidic acid formation by blocking the phospholipase C pathway

Planta ◽  
2006 ◽  
Vol 225 (2) ◽  
pp. 393-401 ◽  
Author(s):  
Ana Ramos-Díaz ◽  
Ligia Brito-Argáez ◽  
Teun Munnik ◽  
S. M. Teresa Hernández-Sotomayor
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Acid Formation

scholarly journals Human platelet signaling defect characterized by impaired production of inositol-1,4,5-triphosphate and phosphatidic acid and diminished Pleckstrin phosphorylation: evidence for defective phospholipase C activation

Blood ◽  
1996 ◽  
Vol 88 (5) ◽  
pp. 1676-1683 ◽  
Author(s):  
X Yang ◽  
L Sun ◽  
S Ghosh ◽  
AK Rao
Keyword(s):  
Phosphatidic Acid ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Ionophore A23187 ◽  
Serotonin Secretion ◽  
Platelet Signaling ◽  
Intracellular Mediators ◽  
Gamma S ◽  
Hydrolysis Of ◽  
Pkc Activation

Abstract Signal transduction on platelet activation involves phosphoinositide- specific phospholipase C (PLC)-mediated hydrolysis of phosphatidylinositides and formation of inositol-1,4,5-triphosphate [I(1,4,5)P3], which mediates Ca2+ mobilization, and diacylglycerol (DG), which activates protein kinase C (PKC) to phosphorylate a 47-kD protein (Pleckstrin). We studied these events in two related patients previously reported (Blood 74:664, 1989) to have abnormal aggregation and 14C-serotonin secretion, and impaired intracellular Ca2+ mobilization in response to several agonists. Thrombin-induced I(1,4,5)P3 and phosphatidic acid formation were diminished. Pleckstrin phosphorylation was impaired on activation with thrombin, platelet- activating factor, and ionophore A23187, but was normal with PKC activator 1,2-dioctonyl-sn-glycerol (DiC8). Ca2+ mobilization induced by guanosine triphosphate (GTP) analog guanosine 5′-0-(3 thiotriphosphate) (GTP gamma S) was diminished. Pretreatment with either A23187 or DiC8 did not correct the impaired adenine diphosphate- induced secretion; however, upon stimulation with A23187 plus DiC8, pleckstrin phosphorylation and secretion were normal, indicating that both PKC activation and Ca2+ mobilization are essential for normal secretion. We conclude that these patients have a unique inherited platelet defect in formation of two key intracellular mediators [I(1,4,5)P3 and DG] and in the responses mediated by them due to a defect in postreceptor mechanisms of PLC activation.

scholarly journals A comparative study of endothelin- and platelet-activating-factor-mediated signal transduction and prostaglandin synthesis in rat Kupffer cells

1992 ◽  
Vol 281 (2) ◽  
pp. 485-492 ◽  
Author(s):  
C R Gandhi ◽  
K Stephenson ◽  
M S Olson
Keyword(s):  
Prostaglandin E2 ◽  
Phospholipase C ◽  
Kupffer Cells ◽  
Inositol Phosphates ◽  
Platelet Activating Factor ◽  
Phosphoinositide Metabolism ◽  
Guanine Nucleotide Binding ◽  
Nucleotide Binding Proteins ◽  
Hydrolysis Of ◽  
Stimulation Of

Endothelin-3 (ET-3) stimulated phosphoinositide metabolism and synthesis of prostaglandins in cultured rat Kupffer cells. ET-3-induced hydrolysis of phosphoinositides was characterized by the production of various inositol phosphates and of glycerophosphoinositol. The mechanism of ET-3-stimulated metabolism of phosphoinositides and synthesis of prostaglandins appeared to be distinct from the effect of platelet-activating factor (PAF) on these processes described previously [Gandhi, Hanahan & Olson (1990) J. Biol. Chem. 265, 18234-18241]. On a molar basis ET-3 was significantly more potent than PAF in stimulating phosphoinositide metabolism, e.g. ET-3-induced hydrolysis of phosphoinositides occurred at 1 pM, whereas PAF was ineffective at concentrations less than 1 nM. Upon challenging Kupffer cells with both ET-3 and PAF, an additive stimulation of phosphoinositide metabolism was observed, suggesting that the actions of these factors may be exerted on separate phosphoinositide pools. Treatment of Kupffer cells with pertussis toxin resulted in an inhibition of ET-3-induced phospholipase C activation; in contrast, cholera toxin treatment caused potentiation of ET-3-stimulated phospholipase C activity. Both toxins, however, inhibited PAF-stimulated phospholipase C activity. The present results suggest that the stimulatory effects of ET-3 and PAF on the phosphodiesteric metabolism of phosphoinositides in Kupffer cells require different guanine-nucleotide-binding proteins. Furthermore, the effects of bacterial toxins on ET-3- and PAF-induced phosphoinositide metabolism were not mediated by cyclic AMP. ET-3-induced metabolism of phosphoinositides was inhibited completely in Kupffer cells pretreated with ET-3, suggesting homologous ligand-induced desensitization of the ET-3 receptors. In contrast, similar experiments using PAF showed only a partial desensitization of subsequent PAF-induced phosphoinositide metabolism. In contrast to the increased production of prostaglandins E2 and D2 observed upon stimulation of Kupffer cells with PAF, ET-3 stimulated the biosynthesis of prostaglandin E2 only. Consistent with their additive effects on phosphoinositide metabolism, PAF and ET-3 exhibited an additive stimulation of the synthesis of prostaglandin E2.

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