Is Phospholipase A2 (PLA2) Involved In PAF-Acether Formation By Platelets

1981 ◽  
Author(s):  
M Chignard ◽  
B B Vargaftig ◽  
J P Le Couedic ◽  
J Benveniste
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Chelating Agents ◽  
Creatine Phosphokinase ◽  
Platelet Activating Factor ◽  
Calcium Ionophore ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Dibutyryl Cyclic Amp ◽  
A 23187

PAF-acether (platelet-activating factor) has been recently identified as l-O-alkyl-2-acetyl-sn-glyceryl-phosphorylcholine, and later chemically synthetized. Platelets form PAF-acether upon stimulation with the calcium ionophore A 23187 or with more physiological stimuli such as thrombin or collagen. By contrast, arachidonic acid (AA) and adenosine diphosphate (ADP) do not trigger formation of PAF-acether. Since 1) PAF-acether is a phospholipid derivative and 2) aggregating agents which trigger PAF-acether formation are potent platelet PLA2 stimulators, we speculated that PLA2 could be implicated in its formation.Rabbit washed platelets were incubated at 37°C in the presence of thrombin (2.5 U/ml) or of ionophore A 23187 (2.5 uM) for 7 min and ethanol (80 % final) was added. After centrifugation, the supernatant was evaporated and concentrated. The extract was tested for its aggregating property on rabbit washed platelets preincubated with a cyclo-oxygenase inhibitor (aspirin) and an ADP scavenging system (creatine phosphate and creatine phosphokinase).In the presence of calcium chelating agents such as EDTA (5 mM) and EGTA (5 mM) most of the synthesis of PAF-acether was suppressed (93 % and 100 % of inhibition respectively). Dibutyryl cyclic AMP (5 mM) also suppressed PAF-acether formation from platelets challenged by thrombin or by the ionophore A 23187 (100 % and 62 % inhibition respectively). Bromophenacyl bromide (0.1 mM) and compound CB 874 (0.1 mM) proved also to be very potent inhibitors of PAF-acether synthesis (100 % inhibition both). All these drugs are well-known platelet PLA2 inhibitors. Upon stimulation platelets also form a deacetylated PAF-acether (lyso- PAF-acether) which could be the direct precursor of PAF-acether. The release of lyso-PAF-acether and the blockade of PAF-acether formation by various molecules having in common a PLA- inhibitory activity lead us to conclude that a PLA2 may be implicated in PAF-acether formation from platelets. Alternative explanations include the possibility that the various inhibitors act on other membrane-related sites.

Effects of an ATP analogue (α,β-methylene-adenosine-5′-triphosphate) on cyclic AMP and cyclic GMP levels, 45Ca efflux, and protein secretion from rat pancreas

1976 ◽  
Vol 54 (5) ◽  
pp. 692-697 ◽  
Author(s):  
Seymour Heisler
Keyword(s):  
Cyclic Amp ◽  
Cyclic Gmp ◽  
Calcium Ionophore ◽  
Competitive Inhibitor ◽  
The Other ◽  
Dibutyryl Cyclic Amp ◽  
Onset Of Action ◽  
Rat Pancreas ◽  
A 23187

The effects of the α,β-methylene analogue of ATP (Ap(CH2)pp), described as a competitive inhibitor of adenylate cyclase (EC 4.6.1.1), were studied in the rat pancreas in vitro. The analogue did not alter basal cyclic AMP production and basal or carbachol-stimulated efflux of 45Ca from isotope-preloaded glands. On the other hand, Ap(CH2)pp reduced the secretory responses to carbachol, carbachol in the presence of dibutyryl cyclic AMP, pancreozymin (PZ), and the calcium ionophore, A-23187. Release of pancreatic protein in response to dibutyryl cyclic AMP itself was unaffected by the ATP analogue, suggesting that the other secretagogues tested share a common, Ap(CH2)pp-inhibitable pathway in their respective stimulatory actions. Though carbachol, PZ, and A-23187 all stimulated a rapid production (30 s) of pancreatic cyclic GMP, these responses were not affected by Ap(CH2)pp. Additional studies with the analogue indicated that it has a slow onset of action (30–45 min), i.e., its effect on secretion is preceded by secretagogue-induced changes in nucleotide levels and calcium efflux. Nonetheless, the analogue may affect cellular calcium homeostasis, if not during the initial events triggering secretion, then during those events which maintain continued secretory output in the presence of a stimulus.

Effects of Sodium Azide on Platelet Function

1977 ◽  
Vol 38 (04) ◽  
pp. 1042-1053 ◽  
Author(s):  
Jeanne Stibbe ◽  
Holm Holmsen
Keyword(s):  
Arachidonic Acid ◽  
Platelet Function ◽  
Sodium Azide ◽  
Creatine Phosphokinase ◽  
Shape Change ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Guanosine Monophosphate ◽  
A 23187 ◽  
Serotonin Secretion

SummarySodium azide in low concentrations (0.1-10 μM) was found to have inhibitory effects on human platelet function. Primary aggregation induced by ADP, epinephrine, thrombin and the ionophore A 23187 was decreased. To evaluate the effect of azide apart from secondary processes, the platelets were treated with indomethacin to prevent prostaglandin/thromboxane synthesis for all inducers; in addition, effects of secreted ADP, in the case of thrombin and A 23187, was prevented by the presence of creatine phosphate plus creatine phosphokinase. ADP, epinephrine and A 23187, but not thrombin-induced primary aggregates, dispersed immediately upon addition of azide. Azide powerfully inhibited dense granule secretion induced by collagen, ADP and epinephrine as measured both by 14C-serotonin secretion and as judged by secondary aggregation. Shape change induced by ADP, thrombin or A 23187 was not affected. Azide had no effect on energy metabolism. Since the aggregation experiments were performed in the presence of indomethacin, and malondialdehyde formation from arachidonic acid was not affected by azide, it seemed unlikely that the inhibition by azide of platelet function was related to inhibition of synthesis of prostaglandins and thromboxanes. It is concluded that azide exerts its effects directly on the common pathway for platelet responses.Abbreviations used: AA: arachidonic acid; PG-TX: PGG2, PGH2 and thromboxane A2; ETYA: 5, 8, 11, 14-eicosa-tetraynoic acid; MDA: malondialdehyde; PGE1 prostaglandin E1; db-c-GMP: dibutyryl 3’, 5’-cyclic guanosine monophosphate; ADP: adenosine diphosphate; c-AMP: 3’, 5’-cyclic adenosine monophosphate; CP: creatine phosphate; CPK: creatine phosphokinase; PRP: píatelet-rich plasma; PPP: platelet-poor plasma; GFP: gel filtered platelets.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Information transfer during embryonic induction in amphibians

Development ◽  
1985 ◽  
Vol 89 (Supplement) ◽  
pp. 349-364
Author(s):  
Horst Grunz
Keyword(s):  
Cyclic Amp ◽  
Concanavalin A ◽  
Information Transfer ◽  
Cell Communication ◽  
Calcium Ionophore ◽  
Neural Induction ◽  
Target Cells ◽  
Binding Studies ◽  
A 23187 ◽  
Intracellular Calcium Level

Neural induction and differentiation has been studied using Concanavalin A, cyclic AMP, tunicamycin and calcium ionophore A 23187. Competent ectoderm of Xenopus laevis treated with Concanavalin A differentiates into neural (archencephalic) structures. Binding studies with gold-labelled ConA indicate that the superficial ectodermal layer contains fewer ConA-sensitive sites (α-D-mannoside and α-D-glucoside residues of glycoproteins) than the inner ectodermal layer. The small number of ConA-sensitive sites can be correlated with the fact that the isolated superficial ectoderm layer, in contrast to the inner layer, does not differentiate into neural structures. The gold-ConA particles bound to inner ectoderm are quickly (within 30 minutes) internalized, presumably by receptor-mediated endocytosis. However, endocytosis is not a prerequisite for neural induction. On the contrary ConA apparently must be bound to the plasma membrane for a certain period to initiate neural induction. The rapid internalization of ConA could explain why neural inductions are evoked only if ectoderm is incubated in ConA-containing medium for longer than 30 minutes. On the other hand cyclic AMP or calcium ionophore A 23187 does not elicit neural inductions. On the contrary calcium ionophore A 23187 apparently, inhibits neural and mesodermal differentiation. This effect could be correlated with an increase of intracellular calcium level of the ectodermal target cells, which could influence the permeability of gap junctions resulting in a loss of cell communication, followed by a change of differentiation and pattern formation.

scholarly journals Prostacyclin and beta-adrenergic catecholamines inhibit arachidonate release and PGI2 synthesis by vascular endothelium

Blood ◽  
1981 ◽  
Vol 58 (3) ◽  
pp. 514-517 ◽  
Author(s):  
B Adler ◽  
MA Jr Gimbrone ◽  
AI Schafer ◽  
RI Handin
Keyword(s):  
Endothelial Cells ◽  
Cyclic Amp ◽  
Vascular Endothelium ◽  
Calcium Ionophore ◽  
Phosphodiesterase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Membrane Phospholipids ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Arachidonate Release

Abstract We have investigated the mechanism by which cyclic AMP inhibits PGI2 synthesis in cultured bovine aortic endothelial cells. Inhibition of cyclic AMP phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) blocks calcium ionophore-induced PGI2 production by 62%. The addition of 3 mM dibutyryl cyclic AMP, alone with IBMX, increases the inhibition to 96%. Release o 3H-arachidonate from membrane phospholipids was inhibited 25% by dibutyryl cyclic AMP, 48% by IBMX, and 76% by isoproterenol plus IBMX. Inhibition by isoproterenol was reversed by 10 micro M propranolol. Release of 3H-arachidonate was also reduced 75% by a combination of 10 micro M PGI2 and 3 mM IBMX. We conclude that hormones like isoproterenol and PGI2 may regulate endothelial cell PGI2 biosynthesis by increasing intracellular cyclic AMP, which then inhibits release of endogenous arachidonate from membrane phospholipids.

scholarly journals Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 914-921 ◽  
Author(s):  
AK Rao ◽  
K Koike ◽  
J Willis ◽  
JL Daniel ◽  
C Beckett ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Myosin Light Chain Phosphorylation ◽  
Serotonin Secretion ◽  
Intracellular Ca ◽  
Thromboxane Production ◽  
Platelet Secretion

Abstract We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.

scholarly journals Participation of ADP in the binding of fibrinogen to thrombin- stimulated platelets

Blood ◽  
1980 ◽  
Vol 56 (3) ◽  
pp. 553-555 ◽  
Author(s):  
EF Plow ◽  
GA Marguerie
Keyword(s):  
Creatine Phosphokinase ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Human Platelets ◽  
Serotonin Release ◽  
Fibrinogen Binding ◽  
Platelet Secretion

Abstract Thrombin and adenosine diphosphate (ADP) supported the binding of 125I- fibrinogen to washed human platelets with similar kinetics and affinity. Platelet secretion, as measured by 14C-serotonin release, and fibrinogen binding exhibited an identical dependence on thrombin concentration. Enzymatic removal of ADP with apyrase or creatine phosphate/creatine phosphokinase (CP/CPK) from thrombin-stimulated platelets markedly inhibited 125I-fibrinogen binding, but pretreatment of platelets with CP/CPK prior to thrombin stimulation was without effect. Thus, ADP, released from the platelet, participates in the binding of fibrinogen to thrombin-stimulated platelets.

scholarly journals Platelet secretion defect associated with impaired liberation of arachidonic acid and normal myosin light chain phosphorylation

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 914-921
Author(s):  
AK Rao ◽  
K Koike ◽  
J Willis ◽  
JL Daniel ◽  
C Beckett ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Platelet Activating Factor ◽  
Adenosine Diphosphate ◽  
Myosin Light Chain Phosphorylation ◽  
Serotonin Secretion ◽  
Intracellular Ca ◽  
Thromboxane Production ◽  
Platelet Secretion

We describe four patients with impaired platelet aggregation and 14C- serotonin secretion during stimulation with adenosine diphosphate (ADP), epinephrine, collagen, and platelet-activating factor. The response to arachidonic acid was normal in all patients with regard to aggregation and in three of the four with regard to 14C-serotonin secretion. The total platelet adenosine triphosphate (ATP) and ADP content and the ATP to ADP ratio was normal in all patients, thereby excluding storage pool deficiency as the cause of the secretion defect. Studies with 3H-arachidonic acid-labeled platelets revealed that the thrombin-induced liberation of arachidonic acid from membrane-bound phospholipids was impaired in these patients. Further, platelet thromboxane B2 production, measured using a radioimmunoassay, was diminished during stimulation with ADP and thrombin, but was normal with arachidonic acid, indicating that the oxygenation of arachidonic acid was normal and that the diminished thromboxane production was due to a defect in the liberation of arachidonic acid. Release of arachidonic acid is mediated by phospholipases that are Ca++ dependent. To examine whether these patients may have a defect in making intracellular Ca++ available, another Ca++-dependent process, myosin light chain phosphorylation, was studied during thrombin stimulation. Platelets from three of the patients were found to behave the same as normal ones, suggesting that the deficiency in phospholipase activity may not be due to impaired Ca++ mobilization. Our studies demonstrate a novel group of patients with platelet secretion defects associated with impaired liberation of arachidonic acid from phospholipids. These patients exemplify a congenital defect, other than deficiencies of cyclooxygenase and thromboxane synthetase, by which thromboxane production may be impaired in platelets.

scholarly journals Prostacyclin and beta-adrenergic catecholamines inhibit arachidonate release and PGI2 synthesis by vascular endothelium

Blood ◽  
1981 ◽  
Vol 58 (3) ◽  
pp. 514-517
Author(s):  
B Adler ◽  
MA Jr Gimbrone ◽  
AI Schafer ◽  
RI Handin
Keyword(s):  
Endothelial Cells ◽  
Cyclic Amp ◽  
Vascular Endothelium ◽  
Calcium Ionophore ◽  
Phosphodiesterase Activity ◽  
Dibutyryl Cyclic Amp ◽  
Membrane Phospholipids ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Arachidonate Release

We have investigated the mechanism by which cyclic AMP inhibits PGI2 synthesis in cultured bovine aortic endothelial cells. Inhibition of cyclic AMP phosphodiesterase activity by 3-isobutyl-1-methylxanthine (IBMX) blocks calcium ionophore-induced PGI2 production by 62%. The addition of 3 mM dibutyryl cyclic AMP, alone with IBMX, increases the inhibition to 96%. Release o 3H-arachidonate from membrane phospholipids was inhibited 25% by dibutyryl cyclic AMP, 48% by IBMX, and 76% by isoproterenol plus IBMX. Inhibition by isoproterenol was reversed by 10 micro M propranolol. Release of 3H-arachidonate was also reduced 75% by a combination of 10 micro M PGI2 and 3 mM IBMX. We conclude that hormones like isoproterenol and PGI2 may regulate endothelial cell PGI2 biosynthesis by increasing intracellular cyclic AMP, which then inhibits release of endogenous arachidonate from membrane phospholipids.

scholarly journals Platelet agonist synergism by the acute phase reactant C-reactive protein

Blood ◽  
1985 ◽  
Vol 65 (2) ◽  
pp. 264-269
Author(s):  
BA Fiedel
Keyword(s):  
Acute Phase ◽  
Creatine Phosphokinase ◽  
Platelet Rich Plasma ◽  
Adenosine Diphosphate ◽  
Creatine Phosphate ◽  
Acute Phase Reactant ◽  
C Reactive Protein ◽  
Reactive Protein ◽  
Phase Reactant ◽  
Irreversible Aggregation

C-reactive protein is the prototypic acute phase reactant. A self- complexed form (H-CRP) can induce isolated platelets to undergo aggregation, secretion of dense and alpha-granule constituents, and generation of thromboxane A2, but fails to function in platelet-rich plasma (PRP) as a direct agonist. In contrast, when PRP was activated with an amount of adenosine diphosphate (ADP) that produced only reversible platelet aggregation, the presence of H-CRP resulted in irreversible aggregation and the secretion of adenosine triphosphate (ATP). Following a maximum stimulus with ADP alone, where platelet secretion occurred late during the aggregation response, the presence of H-CRP shifted and increased the secretory burst to a time simultaneous with the onset of aggregation. This hypersecretion required H-CRP to be present prior to platelet stimulation or to be added within 15 to 30 seconds following the addition of ADP. H-CRP also potentiated platelet activation stimulated with epinephrine, thrombin, and collagen. When the synergism generated in PRP by H-CRP in the presence of ADP or epinephrine was compared to the synergism similarly produced by aggregated human IgG, collagen, or thrombin, it more closely resembled that of collagen, as reflected by the kinetics and characteristics of synergism and sensitivity to creatine phosphate/creatine phosphokinase or 5,8,11,14-eicosatetraynoic acid. These data provide a philosophically ideal niche for the acute phase (and C-reactive protein) in that a platelet-directed activity associated with this acute phase reactant is not utilized unless platelets are otherwise challenged.

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