Regulation of Arachidonic Acid-Dependent Ca++ Influx in Human Platelets

1988 ◽  
Vol 59 (01) ◽  
pp. 086-092 ◽  
Author(s):  
Amalia Bosia ◽  
Wolfgang Losche ◽  
Antonella Pannocchia ◽  
Silvia Treves ◽  
Dario Ghigo ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Reduced Glutathione ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Selective Blockade ◽  
Adp Receptor ◽  
Cyclic Endoperoxide

SummaryQuin2 was used to study the rise in cytoplasmic free calcium ([Ca++]i) and the role of prostaglandin (PG) endoperoxides/thromboxane A2 (TxA2), reduced glutathione (GSH), ADP and the glycoprotein (GP) Ilb IIIa complex in mediating [Ca++]i rise during àiachidonic acid(AA)induced platelet aggregation. Ca++mobilization, mostly due to an influx across the plasma membrane, is completely inhibited by aspirin and persists after selective blockade of TxA2 synthase by dazoxiben. GSH total depletion causes a complete aggregation block and 90% inhibition of the transient: U-46619, a stable analog of cyclic endoperoxide PGH2, stimulates [Ca++]i transient in aspirintreated or in GSH depleted platelets. ADPscavengers, ATP (which competes for the ADP receptor), and monoclonal antibodies against the GP Ilb IIIa complex reduce AAinduced Ca++ influx. Therefore, PG endoperoxides alone or a PGH2/TxA2 mimetic stimulate Ca++ influx. Synthesis of PGH2 and TxA2 depends on the availability of GSH, which acts as the reducing cofactor for the PG peroxidase activity. ADP and GP II b ill a are regulating factors of AA mediated Ca++ influx during platelet activation.

scholarly journals The role of cytoplasmic free calcium in the responses of quin2-loaded human platelets to vasopressin

1984 ◽  
Vol 221 (3) ◽  
pp. 897-901 ◽  
Author(s):  
T J Hallam ◽  
N T Thompson ◽  
M C Scrutton ◽  
T J Rink
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Free Calcium ◽  
Similar Time ◽  
Transient Rise ◽  
Thromboxane Production

Responses to vasopressin were studied in human platelets loaded with the fluorescent Ca2+ indicator, quin2. In the presence of 1 mM external Ca2+, vasopressin caused a transient rise in [Ca2+]i from the basal level near 100nM to about 700 nM; peak [Ca2+]i was reached in a few seconds and the level then declined towards resting over several minutes. In the absence of external Ca2+ there was a much smaller rise of similar time-course, suggesting that vasopressin increases [Ca2+]i mainly by stimulated-influx across the plasma membrane but also by partly releasing internal Ca2+. Inhibition of thromboxane A2 formation somewhat reduced the peak [Ca2+]i in the presence of external Ca2+, but had no effect on the response attributed to release of internal Ca2+. With external Ca2+, vasopressin stimulated shape-change, secretion and aggregation. Secretion and aggregation were decreased by about half following blockage of thromboxane production. The ability of vasopressin to induce shape-change and secretion even at near basal [Ca2+]i suggests that activators other than Ca2+ are involved.

ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)

1987 ◽  
Author(s):  
Y Patel ◽  
S Krishnamurthi ◽  
V V Kakkar
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Synergistic Effects ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Presence And Absence ◽  
Pre Treatment ◽  
Arachidonate Release

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

THE ROLE OF IONISED INTRACELLULAR FREE CALCIUM ([Ca++]i) IN THROMBIN-INDUCED DENSE GRANULE SECRETION

1987 ◽  
Author(s):  
C T Poll ◽  
J Westwick
Keyword(s):  
Human Platelets ◽  
Dense Granule ◽  
Free Calcium ◽  
Fluorescent Properties ◽  
Fura 2 ◽  
Stimulus Response ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion

Fura 2 is one of a recently-introduced family of Ca++ indicators with improved fluorescent properties compared to quin 2 (Grynkiewicz et al 1985). This study has examined the role of [Ca++]i in thrombin-induced dense granule release using prostacyclin-washed human platelets loaded with either thedense granule marker 14C-5HT (5HT) alone or with 5HT together with quin 2 ([quin2]i = 0.8mM) or fura 2 ([fura 2]i 20-30µM). In the presence of ImM extracellular calcium concentration ([Ca++]i) the [Ca++]e in quin 2 and fura 2 loaded platelets was 93±2 (n=10 experiments) and 133±0.3nM (n=12 experiments) respectively. In either quin 2 or fura 2 loaded platelets suspended in the presence of ImM [Ca++]e, thrombin (0.23-23.InM) promoted a rapid (in secs)concentration-dependent elevation of [Ca++]i from basal values to levels l-2µM, together with a parallel release of dense granules almost identical to that obtained with thrombin in non dye loaded platelets. In fura 2 loaded cells, removal of [Ca++]e inhibited the elevation of [Ca++]i induced by a sub-maximal concentration of thrombin (0.77nM) by 43+5% (n=4) but interestingly had no significant effect (p<0.05) on the rise in [Ca++]i elicited by low thrombin doses (0.231nM). Neither did lowering [Ca++]e inhibit the release of 5HT evoked by thrombin ( 0.231-23.InM) from either fura 2 loaded or non dye loaded platelets. In contrast, in quin 2 loaded platelets, removal of [Ca++]e inhibited the thrombin (0.231-23.InM) stimulated rise in [Ca++]i-by 90% and the 5HT release response to either low (0.231nM), sub-maximal (0.77nM) or maximal (23.InM) thrombin by 100% (n=4), 87+2°/o (n=6)and 2+l°/o (n=4) respectively. Fura 2 but not quin 2 loaded cells suspended in ImM [Ca++]e exhibited a Ca++ response to thrombin concentrations >2.31nM which could be separated into a rapid phasic component and a more sustained 'tonic' like component inhibitable by removal of [Ca++]e or by addition of ImM Ni++ . These data suggest the use of fura 2 rather than quin 2 for investigating stimulus response coupling in platelets, particularly when [Ca++]e is less than physiological. We thank the British Heart Foundation and Ciba-Geigy USA for financial support.

Diabetic Retinopathy And Thromboxane Like Substance

1981 ◽  
Author(s):  
M A Lazzari ◽  
M Gimeno ◽  
N M Sutton ◽  
J R Lopez
Keyword(s):  
Diabetes Mellitus ◽  
Arachidonic Acid ◽  
Diabetic Retinopathy ◽  
Risk Factor ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Initial Step ◽  
Platelet Activity

Diabetes Mellitus (DM) is a risk factor in the development of vasculopathies and its complications. It produces also its own microangiopathy. Evidence was reported of increased platelet activity in DM in different assays. Platelets aggregation and the arachidonic cycle could play a key role in the increased tendency to thrombosis. A disorder of ratio TXA2/PGI2, two opposing prostaglandin derivatives, could be the initial step. We intended to evaluate a thromboxane like substance (TLS) produced from platelet rich plasma (PRP) and to compare between normals and diabetic retinopathy (DR) patients. TLS was measured in 16 controls and 16 patients. Assay was done with the aggregating activity developed in PRP (considered TLS) after addition of arachidonic acid (f.c. 2 mM). The supernatant of the PRP (100 μl) was taken 40 sec. after the aggregation started and were added to a normal PRP treated with aspirin (f.c. 40 μl/ml) adjusted to 250.000 - 300.000 pl/μl and the degree of platelet aggregation measured in a Chrono Log Aggregometer. TLS was inactivated after its incubation during 2 min. at 37°C. This finding suggests this activity is due to TXA2.The results obtained (expressed in % of platelet aggregation) were: controls x 16.37% ± 6.28 and DR x 36.00% ± 9.72.The increase detected in the DR group supports previous experimental reports suggesting the role of the thromboxane A2 in vaso occlusive complication of diabetes mellitus.

Aspirin Does Not Affect the Flow Cytometric Detection of Fibrinogen Binding to, or Release of α-Granules or Lysosomes from, Human Platelets

1994 ◽  
Vol 87 (5) ◽  
pp. 575-580 ◽  
Author(s):  
Nicolas A. F. Chronos ◽  
Darren J. Wilson ◽  
Sarah L. Janes ◽  
Ronald A. Hutton ◽  
Nigel P. Buller ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Arachidonic Acid ◽  
Thromboxane A2 ◽  
Normal Subjects ◽  
Human Platelets ◽  
Aspirin Therapy ◽  
Flow Cytometric Assay ◽  
Blood Samples ◽  
Flow Cytometric ◽  
Fibrinogen Binding

1. Aspirin inhibits the conversion of arachidonic acid to thromboxane A2 which reinforces the effects of weak agonists such as ADP in platelets. 2. In this study the effect of aspirin (300 mg/day) on platelet agonist response was measured by whole blood flow cytometry of unfixed blood samples from normal subjects (n = 10), an assay that investigates aggregation-independent changes in the platelet. 3. Fibrinogen binding to unstimulated platelets or to platelets stimulated with ADP or thrombin was unaffected by aspirin. 4. Under the conditions of this assay, platelets undergo a partial degranulation of α-granules and lysosomes (evidenced by expression of P-selectin and CD63, respectively) in response to ADP, and full degranulation in response to thrombin. P-selectin expression was paralleled by release of β-thromboglobulin. None of these events was affected by aspirin. 5. Thromboxane formation was totally prevented by the aspirin treatment, as shown by Born aggregometry in which the platelet aggregatory response to arachidonic acid was abolished and secondary aggregation by ADP was inhibited. 6. The flow cytometric assay can therefore be used to investigate platelets in patients, regardless of aspirin therapy. 7. These findings suggest that platelet fibrinogen binding and the release of platelet α-granule and lysosomal contents, in response to stimulation with physiological agonists, can continue in patients despite aspirin therapy. This may help to explain why aspirin is only partially effective in preventing thrombotic events.

Role of phosphoinositide 3-kinase β in platelet aggregation and thromboxane A2 generation mediated by Gi signalling pathways

2010 ◽  
Vol 429 (2) ◽  
pp. 369-377 ◽  
Author(s):  
Analia Garcia ◽  
Soochong Kim ◽  
Kamala Bhavaraju ◽  
Simone M. Schoenwaelder ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Functional Responses ◽  
Induce Platelet Aggregation ◽  
Erk Phosphorylation

PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms α, β, γ and δ in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kβ-selective inhibitor, but not by PIK75 (a PI3Kα inhibitor), AS252424 (a PI3Kγ inhibitor) or IC87114 (a PI3Kδ inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1−/− mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kβ in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kβ plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kβ mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.

Marked Temperature Dependence of the Platelet Calcium Signal Induced by Human von Willebrand Factor

Blood ◽  
1999 ◽  
Vol 94 (1) ◽  
pp. 199-207 ◽  
Author(s):  
John C. Kermode ◽  
Qi Zheng ◽  
Elizabeth P. Milner
Keyword(s):  
Arachidonic Acid ◽  
Temperature Dependence ◽  
Von Willebrand Factor ◽  
Thromboxane A2 ◽  
Underlying Mechanism ◽  
Calcium Signal ◽  
Free Calcium ◽  
Strong Temperature Dependence ◽  
Von Willebrand ◽  
Willebrand Factor

Interaction of von Willebrand factor (vWF) with the platelet is essential to hemostasis when vascular injury occurs. This interaction elevates the intracellular free calcium concentration ([Ca2+]i) and promotes platelet activation. The present study investigated the temperature dependence of vWF-induced [Ca2+]i signaling in human platelets. The influence of temperature can provide invaluable insight into the underlying mechanism. Platelet [Ca2+]i was monitored with Fura-PE3. Ristocetin-mediated binding of vWF induced a transient platelet [Ca2+]i increase at 37°C, but no response at lower temperatures (20°C to 25°C). This temperature dependence could not be attributed to a reduction in vWF binding, as ristocetin-mediated platelet aggregation and agglutination were essentially unaffected by temperature. Most other platelet agonists (U-46619, -thrombin, and adenosine 5′-diphosphate [ADP]) induced a [Ca2+]isignal whose amplitude did not diminish at lower temperatures. The [Ca2+]i signal in response to arachidonic acid, however, showed similar temperature dependence to that seen with vWF. Assessment of thromboxane A2 production showed a strong temperature dependence for metabolism of arachidonic acid by the cyclo-oxygenase pathway. vWF induced thromboxane A2production in the platelet. Aspirin treatment abolished the vWF-induced [Ca2+]i signal. These observations suggest that release of arachidonic acid and its conversion to thromboxane A2 play a central role in vWF-mediated [Ca2+]i signaling in the platelet at physiological temperatures.

The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3904-3904
Author(s):  
Samantha Baldassarri ◽  
Alessandra Bertoni ◽  
Paolo Lova ◽  
Stefania Reineri ◽  
Chiara Sarasso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Cannabinoid Receptors ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Dependent Manner ◽  
Cb2 Receptor ◽  
Thromboxane A2 Receptor

Abstract 2-Arachidonoylglycerol (2-AG) is a naturally occurring monoglyceride that activates cannabinoid receptors and meets several key requisites of an endogenous cannabinoid substance. It is present in the brain and hematopoietic cells, including macrophages, lymphocytes and platelets. 2-AG is released from cells in a stimulus-dependent manner and is rapidly eliminated by uptake into cells and enzymatic hydrolysis in arachidonic acid and glycerol. 2-AG might exert a very fine control on platelet function either through mechanisms intertwining with the signal transduction pathways used by platelet agonists or through mechanisms modulating specific receptors. The aim of this study was to define the role of 2-AG in human platelets and characterize the mechanisms by which it performs its action. Platelets from healthy donors were isolated from plasma by differential centrifugations and gel-filtration on Sepharose 2B. The samples were incubated with 2-AG (10–100 μM) under constant stirring in the presence or absence of various inhibitors. Platelet aggregation was measured by Born technique. We have found that stimulation of human platelets with 2-AG induced irreversible aggregation, which was significantly enhanced by co-stimulation with ADP (1–10 μM). Furthermore, 2-AG-dependent platelet aggregation was completely inhibited by ADP scavengers, aspirin, and Rho kinase inhibitor, as well as by antagonists of the 2-AG receptor (CB2), of the ADP P2Y12 receptor, and of the thromboxane A2 receptor. We further investigated the role of endocannabinoids on calcium mobilization. Intracellular [Ca2+] was measured using FURA-2-loaded platelets prewarmed at 37°C under gentle stirring in a spectrofluorimeter. 2-AG induced rapid increase of cytosolic [Ca2+] in a dose-dependent manner. This effect was partially blocked by ADP scavengers and CB2 receptor antagonists. Furthermore, 2-AG-induced [Ca2+] mobilization was totally suppressed by aspirin or the thromboxane A2 receptor antagonist. These results suggest that 2-AG is able to trigger platelet activation, and that this action is partially mediated by CB2 receptor and ADP. Furthmore, 2-AG-dependent platelet activation is totally dependent on thromboxane A2 generation.

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