Human platelet secretion and aggregation induced by calcium ionophores. Inhibition by PGE1 and dibutyryl cyclic AMP.

Ca2+, Mg2+-ionophores X537A and A23,187 (10(-7)-10(-6) M) induced the release of adenine nucleotides adenosine diphosphate (ADP, adenosine triphosphate (ATP), serotonin, beta-glucuronidase, Ca2+, and Mg2+ from washed human platelets. Enzymes present in the cytoplasm or mitochondria, and Zn2+ were not released. The rate of ATP and Ca2+ release measured by firefly lantern extract and murexide dye, respectively, was equivalent to that produced by the physiological stimulant thrombin. Ionophore-induced release of ADP, and serotonin was substantially (approximately 60%) but not completely inhibited by EGTA, EDTA, and high extracellular Mg2+, without significant reduction of Ca2+ release. The ionophore-induced release reaction is therefore partly dependent upon uptake of extracellular Ca2+ (demonstrated using 45Ca), but also occurs to a significant extent due to release into the cytoplasm of intracellular Ca2+. The ionophore-induced release reaction and aggregation of platelets could be blocked by prostaglandin E1 (PGE1) or dibutyryl cyclic AMP. The effects of PGE1, and N6, O2-dibutyryl adenosine 3':5'-cyclic monophosphoric acid (dibutyryl cAMP) were synergistically potentiated by the phosphodiesterase inhibitor theophylline. It is proposed that Ca2+ is the physiological trigger for platelet secretion and aggregation and that its intracellular effects are strongly modulated by adenosine 3':5'-cyclic monophosphoric acid (cyclic AMP).

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

10.1055/s-0038-1652801 ◽

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.

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Changes of platelets’ function in preeclampsia

Open Medicine ◽

10.2478/s11536-011-0076-3 ◽

2011 ◽

Vol 6 (6) ◽

pp. 696-700

Author(s):

Goran Babic ◽

Slobodan Novokmet ◽

Slobodan Jankovic

Keyword(s):

Platelet Aggregation ◽

Adenine Nucleotides ◽

Energy Charge ◽

Adenosine Diphosphate ◽

Control Group ◽

Adenylate Energy Charge ◽

Cross Sectional ◽

Blood Samples ◽

Metabolic Pool ◽

Aggregation Of Platelets

AbstractIncreased aggregation of platelets during preeclampsia was shown in several studies, yet several others reported no change. The aim of our study was to investigate platelet aggregation in a group of patients suffering from preeclampsia. In a cross-sectional study blood samples were taken from 89 hospitalized patients in the third trimester of pregnancy: 38 were suffering from mild to moderate preeclampsia and 51 patients were without preeclampsia. From the blood samples platelet aggregation, secretion of adenine nucleotides from platelets, concentration of energy-rich adenine compounds and levels of cyclic adenosine-mono-phosphate and cyclic guanosine mono-phosphate in platelets were measured. In the patients with preeclampsia, the adenosine diphosphate threshold for biphasic aggregation [odds ratio (OR):.75; 95% Confidence Interval (CI): 0.55–1.02; p<0.05], total adenine nucleotides concentration in the metabolic pool of platelets (OR:0.99; CI: 0.62–1.57; p<0.01) and cyclic adenosine-mono-phosphate (OR:0.81; CI: 0.57, 1.14; p<0.05) and cyclic guanosine mono-phosphate (OR:.78; CI: 0.55–1.09; p<0.05) levels in platelets were decreased in comparison with the control group, while adenylate energy charge in the metabolic pool of platelets (OR: >100.00; CI: 0.00->100.00; p<0.05) and secretion of adenosine triphosphate (OR:.13; CI: 0.00–14.26; p<0.05) and adenosine diphosphate (OR:.77; CI: 0.08–36.79; p<0.05) were increased. The results of our study show increased activation and aggregation of platelets in pregnant females with preeclampsia.

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

Blood ◽

10.1182/blood.v56.3.553.553 ◽

1980 ◽

Vol 56 (3) ◽

pp. 553-555 ◽

Cited By ~ 46

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.

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Mechanism of platelet plug formation and role of adenosine diphosphate

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1964.206.6.1267 ◽

1964 ◽

Vol 206 (6) ◽

pp. 1267-1274 ◽

Cited By ~ 148

Author(s):

Theodore H. Spaet ◽

Marjorie B. Zucker

Keyword(s):

Connective Tissue ◽

Divalent Cation ◽

Platelet Rich Plasma ◽

Adenine Nucleotides ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Suitable Model ◽

Plug Formation ◽

Rat Platelets

Traumatized rat omentum was used to demonstrate the development of "platelet plugs" following agitation in platelet-rich plasma. In the absence of divalent cation there was only platelet adhesion to connective tissue fibers; in the presence of divalent cation masses of platelets formed (cohesion) even in plasma adequately anticoagulated with heparin. Exposure of these platelet masses to thrombin produced greater compactness and stability. Human and rat platelets behaved alike with the traumatized rat omentum; platelets from two patients with von Willebrand's disease gave normal reactions whereas platelets from a patient with thrombasthenia showed adhesion only. Exposure of human platelets to washed connective-tissue fragments or to thrombin elicited clumping accompanied by release of serotonin and of adenine nucleotides (AN) of which about one-third was adenosine diphosphate. Intermediate concentrations of connective tissue and thrombin also caused clumping but no liberation of AN or serotonin. ADP caused intense clumping but failed to liberate serotonin or additional ADP. It is suggested that cohesion reaction is mediated by release of ADP. The traumatized omentum appears to be a suitable model for studying the hemostatic process.

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Evidence for the involvement of cyclic AMP in the pheromonal modulation of barnacle settlement

Journal of Experimental Biology ◽

10.1242/jeb.198.3.655 ◽

1995 ◽

Vol 198 (3) ◽

pp. 655-664 ◽

Cited By ~ 4

Author(s):

A Clare ◽

R Thomas ◽

D Rittschof

Keyword(s):

Signal Transduction ◽

Cyclic Amp ◽

Adenylate Cyclase ◽

Cyclic Gmp ◽

Phosphodiesterase Inhibitor ◽

Balanus Amphitrite ◽

Dibutyryl Cyclic Amp ◽

Physico Chemical ◽

Miconazole Nitrate

The involvement of cyclic AMP in the settlement of the cypris larva of Balanus amphitrite amphitrite Darwin has been examined through the use of compounds that affect intracellular cyclic AMP levels. The activation of adenylate cyclase with forskolin, and the inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine, caffeine and theophylline, significantly increased the settlement of cyprids. Although the analogue dibutyryl cyclic AMP appeared to increase settlement, the effect was not significant. No marked increase in settlement resulted from the incubation of cyprids with dibutyryl cyclic GMP, 8-(4-chlorophenylthio) (CPT) cyclic AMP or papaverine (a phosphodiesterase inhibitor). Miconazole nitrate, an adenylate cyclase inhibitor, prevented settlement, but this effect appeared to be physico-chemical rather than pharmacological. Radioimmunoassay did not clearly show whether cyclic AMP levels changed following exposure of cyprids to a pulse of crude barnacle extract. However, exposure to forskolin significantly increased the cyclic AMP titre of cyprids. We conclude that compounds that alter intracellular cyclic AMP levels alter normal patterns of cyprid settlement. Whether this is because of an alteration in signal transduction is unclear.

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Short communication: possible association of newly absorbed serotonin with nonmetabolic, granule-located adenine nucleotides in human blood platelets

Blood ◽

10.1182/blood.v45.3.413.bloodjournal453413 ◽

1975 ◽

Vol 45 (3) ◽

pp. 413-416

Author(s):

H Holmsen ◽

CA Setkowsky ◽

HJ Day

Keyword(s):

Guinea Pig ◽

Human Blood ◽

Short Communication ◽

Adenine Nucleotides ◽

Blood Platelets ◽

Human Platelets ◽

Serotonin Release ◽

Release Reaction ◽

Human Blood Platelets

[3H]-adenine-labeled human platelets in plasma were incubated with or without nonradioactive serotonin. Release reaction was then induced by ADP, epinephrine, collagen, or thrombin. Platelets that had been incubated with serotonin released four times as much serotonin as platelets incubated without serotonin. The specific radioactivities of the ATP and ADP released to plasma during release reaction induced with all four inducers were the same in both systems. This shows that when serotonin is taken up by human platelets, it enters the compartment containing nonmetabolic, granula-stored ATP, and not the compartment with metabolic extragranular ATP. These results suggest that the mechanism of serotonin storage in human platelets is similar to that in other species investigated, i.e., rabbit, guinea pig, and pig.

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Effects of Lead Acetate on Platelet Aggregation, Ultrastructure and Serotonin Release

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653698 ◽

1971 ◽

Vol 26 (03) ◽

pp. 455-466 ◽

Cited By ~ 1

Author(s):

R. B Davis ◽

G. C Holtz

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Lead Acetate ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

Blood Platelet ◽

Human Platelets ◽

Serotonin Release ◽

Aggregation Of Platelets

SummaryThe effects of lead on blood platelet function and ultrastructure have been investigated. Lead acetate was injected intravenously in 27 rats and was added to rat and human platelet rich plasma in vitro. In vitro studies showed that concentrations of 2.5 × 10-3 M lead acetate reduced or blocked aggregation of rat and human platelets by adenosine diphosphate, collagen, and thrombin. Radioactive serotonin release from human platelets was inhibited by 10-4 M lead acetate. One hour after the injection of lead, platelet aggregation by thrombin was reduced, but platelet aggregation by adenosine diphosphate and collagen showed little change. Three days after lead, aggregation of platelets by collagen and thrombin was blocked and aggregation by adenosine diphosphate reduced. Thrombocytopenia was present 4 days after intravenous lead acetate. Electron micrographs of platelets showed that the mean number of mitochondria per platelet was increased, whereas alpha granules were reduced. Dense bodies were not significantly changed. Lead acetate affects platelet function in concentrations reported in human bone marrow in lead poisoning, and may relate to the binding of free sulfhydryl groups by lead.

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The Effects of Citrate and Extracellular Calcium Ions on the Platelet Release Reaction Induced by Adenosine Diphosphate and Collagen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666916 ◽

1979 ◽

Vol 42 (02) ◽

pp. 778-793 ◽

Cited By ~ 32

Author(s):

Stanley Hepinstall ◽

Patricia M Taylor

Keyword(s):

Calcium Ions ◽

Sodium Citrate ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

Human Platelets ◽

Extracellular Calcium ◽

Release Reaction ◽

Calcium Dependent ◽

Platelet Release ◽

Dependent Mechanism

SummaryThe ADP-induced release of 3H-serotonin from human platelets in heparinized platelet rich plasma is markedly stimulated by the addition of sodium citrate. The aggregation and release that is induced by collagen is less affected by citrate. Data is presented that supports the view that the effects of citrate on both ADP-and collagen-induced release are largely via alteration of the concentration of ionized calcium in plasma.Collagen can induce release of 3H-serotonin via extracellular calcium-independent and -dependent mechanisms. The possibility that the calcium-dependent mechanism is aggregation-dependent and that the calcium is required for platelet aggregation rather than directly involved in the release reaction is discussed.

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Regulation of the synthesis of lutropin-induced protein in rat testis Leydig cells

Biochemical Journal ◽

10.1042/bj1700009 ◽

1978 ◽

Vol 170 (1) ◽

pp. 9-15 ◽

Cited By ~ 13

Author(s):

Felix H. A. Janszen ◽

Brian A. Cooke ◽

Maria J. A. Van Driel ◽

Henk J. Van Der Molen

Keyword(s):

Protein Synthesis ◽

Cyclic Amp ◽

Leydig Cells ◽

Actinomycin D ◽

Polyacrylamide Gel Electrophoresis ◽

Phosphodiesterase Inhibitor ◽

Dibutyryl Cyclic Amp ◽

Rat Testis ◽

Stimulation Of

The mechanism of action of lutropin on the stimulation of the synthesis of a specific lutropin-induced protein in rat testis Leydig cells was investigated. Lutropin-induced protein has a mol.wt. of approx. 21000 and is detected by labelling the Leydig-cell proteins with [35S]methionine, followed by separation by polyacrylamide-gel electrophoresis and radioautography of the dried gel. The incorporation of35S into lutropin-induced protein was used as an estimate for the synthesis of the protein. Incubation of Leydig cells with dibutyryl cyclic AMP or cholera toxin also resulted in the stimulation of synthesis of the protein. Synthesis of lutropin-induced protein, when maximally stimulated with 100ng of lutropin/ml, could not be stimulated further by addition of dibutyryl cyclic AMP. Addition of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, further increased synthesis of the protein in the presence of a submaximal dose of lutropin (10ng/ml) but not in the absence of lutropin or with maximal amounts of lutropin (100 and 1000ng/ml). Actinomycin D prevented the effect of lutropin on the stimulation of lutropin-induced protein synthesis when added immediately or 1h after the start of the incubation, but not when added after 5–6h. This is interpreted as reflecting that, after induction of mRNA coding for lutropin-induced protein, lutropin had no influence on the synthesis of the protein in the presence of actinomycin D. Synthesis of the protein was also stimulated in vivo by injection of choriogonadotropin into rats 1 day after hypophysectomy, and the time course of this stimulation of lutropin-induced protein synthesis in vivo was similar to that obtained by incubating Leydig cells in vitro with lutropin. From these results it is concluded that stimulation of lutropin-induced protein synthesis by lutropin is most probably mediated by cyclic AMP and involves synthesis of mRNA.

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Effects of collagen, ionophore A23187 and prostaglandin E1 on the phosphorylation of specific proteins in blood platelets

Biochemical Journal ◽

10.1042/bj1780397 ◽

1979 ◽

Vol 178 (2) ◽

pp. 397-406 ◽

Cited By ~ 118

Author(s):

Richard J. Haslam ◽

James A. Lynham ◽

Joan E. B. Fox

Keyword(s):

Cyclic Amp ◽

Prostaglandin E1 ◽

Blood Platelets ◽

Short Circuit ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Ionophore A23187 ◽

Release Reaction ◽

Specific Proteins

Human platelets that had been preincubated with 5-hydroxy[3H]tryptamine and [32P]Pi were stirred with various agents; the secretion of 5-hydroxy[3H]tryptamine from platelet granules and the radioactivity of platelet [32P]phosphopolypeptides separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis were then measured. Exposure of the platelets to collagen fibres or ionophore A23187 selectively increased the phosphorylation of polypeptides with apparent mol.wts. of 47000 (P47) and 20000 (P20) by approx. 3-fold, in association with the release of 5-hydroxy[3H]tryptamine. The 47000-mol.wt. phosphopolypeptide (P47) was clearly separated from platelet actin by the electrophoresis system used. Prostaglandin E1, which inhibits platelet function by increasing platelet cyclic AMP, decreased the phosphorylation of polypeptides caused by collagen as well as the release of 5-hydroxy[3H]tryptamine. Prostaglandin E1 also selectively increased the phosphorylation of distinct polypeptides with apparent mol.wts. of 24000 (P24) and 22000 (P22) by approx. 2-fold. As the phosphorylation reactions caused by collagen are probably mediated by an increase in Ca2+ concentration in the platelet cytosol and may have a role in the release reaction [Haslam & Lynham (1977) Biochem. Biophys. Res. Commun.77, 714–722; (1978) Thromb. Res.12, 619–628], we suggest that a cyclic AMP-dependent phosphorylation of the 24000- and/or 22000-mol.wt. polypeptides caused by prostaglandin E1 may initiate processes that decrease the Ca2+ concentration in the cytosol, so inhibiting both the Ca2+-dependent phosphorylation reactions and the release reaction. Treatment of platelets with prostaglandin E1 did not inhibit the increased phosphorylation of polypeptides with apparent mol.wts. of 47000 and 20000 (P47 and P20) caused by ionophore A23187, which may therefore short-circuit cyclic AMP-dependent mechanisms that decrease the Ca2+ concentration in the platelet cytosol. As prostaglandin E1 did inhibit the release of 5-hydroxy[3H]tryptamine by ionophore A23187, cyclic AMP may also inhibit the release reaction by additional mechanisms.

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