Sphingosine-1-phosphate modulates PAR1-mediated human platelet activation in a concentration-dependent biphasic manner

AbstractSphingosine 1-phosphate (S1P) is a bioactive signalling sphingolipid that is increased in diseases such as obesity and diabetes. S1P can modulate platelet function, however the direction of effect and S1P receptors (S1PRs) involved are controversial. Here we describe the role of S1P in regulating human platelet function and identify the receptor subtypes responsible for S1P priming. Human platelets were treated with protease-activated receptor 1 (PAR-1)-activating peptide in the presence or absence of S1P, S1PR agonists or antagonists, and sphingosine kinases inhibitors. S1P alone did not induce platelet aggregation but at low concentrations S1P enhanced PAR1-mediated platelet responses, whereas PAR1 responses were inhibited by high concentrations of S1P. This biphasic effect was mimicked by pan-S1PR agonists. Specific agonists revealed that S1PR1 receptor activation has a positive priming effect, S1PR2 and S1PR3 have no effect on platelet function, whereas S1PR4 and S1PR5 receptor activation have an inhibitory effect on PAR-1 mediated platelet function. Although platelets express both sphingosine kinase 1/2, enzymes which phosphorylate sphingosine to produce S1P, only dual and SphK2 inhibition reduced platelet function. These results support a role for SphK2-mediated S1P generation in concentration-dependent positive and negative priming of platelet function, through S1PR1 and S1PR4/5 receptors, respectively.

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Cigarette Smoke Extract Inhibits Platelet Aggregation by Suppressing Cyclooxygenase Activity

TH Open ◽

10.1055/s-0037-1607979 ◽

2017 ◽

Vol 01 (02) ◽

pp. e122-e129

Author(s):

Hitoshi Kashiwagi ◽

Koh-ichi Yuhki ◽

Yoshitaka Imamichi ◽

Fumiaki Kojima ◽

Shima Kumei ◽

...

Keyword(s):

Platelet Aggregation ◽

Cigarette Smoke ◽

Platelet Function ◽

Inhibitory Effect ◽

Cigarette Smoke Extract ◽

Human Platelets ◽

Receptor Subtypes ◽

Ic50 Value ◽

Induced Aggregation ◽

Cox 1

AbstractThe results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A2 receptor agonist, and that induced by collagen with respective IC50 values of 1.05 ± 0.14% and 1.34 ± 0.19%. A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid–induced TXA2 production in murine platelets with an IC50 value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA2 receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I2 receptor and PGE2 receptor subtypes EP2 and EP4, were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA2 production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC50 value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA2 production in platelets, leading to inhibition of platelet aggregation.

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NAADP regulates human platelet function

Biochemical Journal ◽

10.1042/bj20111175 ◽

2011 ◽

Vol 441 (1) ◽

pp. 435-442 ◽

Cited By ~ 12

Author(s):

Carmen H. Coxon ◽

Alexander M. Lewis ◽

Amanda J. Sadler ◽

Sridhar R. Vasudevan ◽

Andrew Thomas ◽

...

Keyword(s):

Receptor Antagonist ◽

Binding Site ◽

Platelet Activation ◽

Platelet Function ◽

Cell Activation ◽

Radioligand Binding ◽

Human Platelet ◽

Inhibitory Effect ◽

Human Platelets ◽

Glycoprotein Vi

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid–adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.

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Effects of Halothane on Platelet Function

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650105 ◽

1980 ◽

Vol 44 (03) ◽

pp. 143-145 ◽

Cited By ~ 19

Author(s):

J Dalsgaard-Nielsen ◽

J Gormsen

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Halothane Anaesthesia ◽

Inhibition Of Platelet Aggregation ◽

Transient Impairment ◽

High Concentrations ◽

Uptake And Release ◽

Platelet Functions

SummaryHuman platelets in platelet rich plasma (PRP) incubated at 37° C with 0.3–2% halothane for 5–10 min lost the ability to aggregate with ADP, epinephrine and collagen.At the same time uptake and release of 14C-serotonin was inhibited. When halothane supply was removed, platelet functions rapidly returned to normal. However, after high concentrations of halothane, the inhibition of platelet aggregation was irreversible or only partially reversible.The results suggest that halothane anaesthesia produces a transient impairment of platelet function.

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Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657151 ◽

1982 ◽

Vol 47 (02) ◽

pp. 150-153 ◽

Cited By ~ 13

Author(s):

P Han ◽

C Boatwright ◽

N G Ardlie

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

Antiarrhythmic Drugs ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Second Phase ◽

Ionophore A23187 ◽

High Concentrations ◽

Artery Disease

SummaryVarious cardiovascular drugs such as nitrates and propranolol, used in the treatment of coronary artery disease have been shown to have an antiplatelet effect. We have studied the in vitro effects of two antiarrhythmic drugs, verapamil and disopyramide, and have shown their inhibitory effect on platelet function. Verapamil, a calcium channel blocker, inhibited the second phase of platelet aggregation induced by adenosine diphosphate (ADP) and inhibited aggregation induced by collagen. Disopyramide similarly inhibited the second phase of platelet aggregation caused by ADP and aggregation induced by collagen. Either drug in synergism with propranolol inhibited ADP or collagen-induced platelet aggregation. Disopyramide at high concentrations inhibited arachidonic add whereas verapamil was without effect. Verapamil, but not disopyramide, inhibited aggregation induced by the ionophore A23187.

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Inhibition of ADP-Induced Responses in Human Platelets by Agents Elevating the Cyclic AMP Level: Comparison of Aggregation and Shape Change

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661208 ◽

1984 ◽

Vol 52 (03) ◽

pp. 333-335 ◽

Cited By ~ 4

Author(s):

Vider M Steen ◽

Holm Holmsen

Keyword(s):

Inhibitory Action ◽

Human Platelet ◽

Platelet Rich Plasma ◽

Shape Change ◽

Inhibitory Effect ◽

Human Platelets ◽

Inhibitory Effects ◽

Early Step ◽

Stimulus Response ◽

Sequential Relationship

SummaryThe inhibitory effect of cAMP-elevating agents on shape change and aggregation in human platelets was studied to improve the understanding of the sequential relationship between these two responses.Human platelet-rich plasma was preincubated for 2 min at 37° C with prostaglandin E1 or adenosine, agents known to elevate the intracellular level of cAMP. Their inhibitory effects on ADP-induced shape change and aggregation were determined both separately and simultaneously. The dose-inhibition patterns for shape change and aggregation were similar for both PGE1 and adenosine. There was no distinct difference between the inhibitory action of these two inhibitors.These observations suggest that elevation of the intracellular concentration of cAMP interferes with an early step in the stimulus-response coupling that is common for aggregation and shape change.

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Effect Of Solvents On Indium-111 Oxine Transport In Human Platelets And On Platelet Function In Vitro

10.1055/s-0038-1653277 ◽

1981 ◽

Author(s):

T Tsukada

Keyword(s):

Platelet Function ◽

Kinetic Constant ◽

Inhibitory Effect ◽

Human Platelets ◽

Polysorbate 80 ◽

Autologous Plasma ◽

Indium 111 ◽

Induced Aggregation ◽

Water Space

Mechanism of Indium-111 oxine(In) transport in human platelets in buffered saline and the effect of In-labeling on platelet function were studied using In dissolved in 25% of ethanol in saline (In-ES) or 0.01% of polysorbate 80 in HEPES buffer(In-PH). Increase in temperature up to 37° C progressively enhanced the transport of In-ES, while transport of In-PH reached to plateau at 15°C. A states of equilibrium was not reached during 2 hr incubation at 22°C in In-ES. Uptake of In-PH reached to plateau after only 15 min of incubation. Distribution of In taken up by platelets in InES was 57% in cytosol and 27% in stroma, while in In-PH 69% in stroma and 22% in cytosol. 88% of In in cytosol was bound to lipids(46% in cholesterol and 27% in PS+PI). 82% of In in stroma was found in PS+PI fraction.The fact that the ratio of free In between the platelet water space and the outside medium after 30 min of incubation at up to 0.1 uM of In exceeded unity, suggests satura- , ble component of In transport prevails at this concentration in In-ES and In-PH. Kinetic constant could be calculated, Kt= 2nM, Vmax= 2.5 pmol/min/ml in In-ES, and Kt= InM, Vmax=0.7 pmol/min/ml in In-PH.Elution of In from radiolableled platelets in autologous plasma incubated at 37°C for 5 hr was less than 10% in the case of In-ES and 56% in the case of In-PH. Less than 3% of labeled-In was eluated from platelets in collagen-induced aggregation and 4-7% of In was eluated in thrombin-induced aggregation.Although 0.3% of ethanol and/or 6nM of oxine have no inhibitory effect of platelet aggregation, collagen-induced aggregation and release reaction of In-labeled platelet was impaired. 0.003% of polysorbate 80 itself abolished completely the aggregability of platelets by collagen or thrombin.It is concluded In-PH is unsuitable for platelet labeling. In-111 oxine also seems to have problems which Cr-51 has, i.e. inhomogenous distribution of In in a platelet population, elution of In from labeled platelets in circulation.

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Activation of human platelets by immune complexes prepared with cationized human IgG

Blood ◽

10.1182/blood.v82.10.3045.bloodjournal82103045 ◽

1993 ◽

Vol 82 (10) ◽

pp. 3045-3051

Author(s):

M Schattner ◽

M Lazzari ◽

AS Trevani ◽

E Malchiodi ◽

AC Kempfer ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Platelet Rich Plasma ◽

Human Platelets ◽

Human Igg ◽

Experimental Conditions ◽

Induce Platelet Aggregation ◽

Soluble Immune Complexes ◽

High Concentrations

The present study shows that the ability of soluble immune complexes (IC), prepared with human IgG and rabbit IgG antibodies against human IgG, to trigger platelet activation was markedly higher for IC prepared with cationized human IgG (catIC) compared with those prepared with untreated human IgG (cIC). CatIC induced platelet aggregation and adenosine triphosphate release in washed platelets (WP), gel-filtered platelets (GFP), or platelet-rich plasma (PRP) at physiologic concentrations of platelets (3 x 10(8)/mL) and at low concentrations of catIC (1 to 30 micrograms/mL). On the contrary, under similar experimental conditions, cIC did not induce aggregation in PRP, WP, or GFP. Low aggregation responses were only observed using high concentrations of both WP (9 x 10(8)/mL) and cIC (500 micrograms/mL). Interestingly, catIC were also able to induce platelet activation under nonaggregating conditions, as evidenced by P-selectin expression. Cationized human IgG alone did not induce platelet aggregation in PRP but triggered either WP or GFP aggregation. However, the concentration needed to induce these responses, was about eightfold higher than those required for catIC. The responses induced either by catIC or cationized human IgG were completely inhibited by treatment with heparin, dextran sulphate, EDTA, prostaglandin E1, or IV3, a monoclonal antibody against the receptor II for the Fc portion of IgG (Fc gamma RII). The data presented in this study suggest that IgG charge constitutes a critical property that conditions the ability of IC to trigger platelet activation.

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Selective Inhibition of Platelet Agglutination and Aggregation by Aromatic Amidino Compounds

10.1055/s-0039-1680564 ◽

1977 ◽

Author(s):

J. D. Geratz ◽

R. R. Tidwell ◽

K. M. Brinkhous ◽

S. F. Mohammad

Keyword(s):

Selective Inhibition ◽

Inhibitory Effect ◽

Human Platelets ◽

The Other ◽

Future Studies ◽

Bovine Plasma ◽

Inhibitory Activities ◽

High Concentrations ◽

Ristocetin Cofactor ◽

Specific Inhibitors

A series of aromatic amidino compounds were investigated for their inhibitory effect on platelet agglutination and platelet aggregation. Agglutination of fresh or fixed human platelets was produced by bovine plasma or by human plasma in combination with ristocetin, while aggregation of fresh platelets was induced by ADP, thrombin or collagen. Highly effective inhibitors were found for both types of platelet clumping, but there was no parallelism between the inhibitory activities in the two test systems.5-(5-Amidino-2-benzimidazolyl)-2-(4-hydroxybenzene)benzimidazole suppressed agglutination exclusively. Pentamidine, on the other hand, strongly blocked the aggregation reactions, but did not interfere with agglutination, even at high concentrations. Compounds which inhibited aggregation also prevented the liberation of serotonin from the platelets.This investigation has led to the identification of new specific inhibitors of platelet agglutination and aggregation which can serve an important role in future studies of the two processes. The exact mode of interaction between ami dines and platelets is still being explored.. In the case of agglutination, inhibition most likely occurred at the level of binding of ristocetin cofactor to the platelet membrane. In the case of aggregation, however, interference could have taken place at the membranes or in the cytoplasm and could have been enzymatic or non-enzymatic in nature.

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

Biochemical Journal ◽

10.1042/bj20100166 ◽

2010 ◽

Vol 429 (2) ◽

pp. 369-377 ◽

Cited By ~ 64

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.

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Alteration of Platelet Cyclic AMP (cAMP) by Ethanol

10.1055/s-0039-1680810 ◽

1977 ◽

Author(s):

D.H. Cowan ◽

M. Kikta ◽

D. Baunach

Keyword(s):

Cyclic Amp ◽

Platelet Function ◽

Inhibitory Effect ◽

Human Platelets ◽

Platelet Dysfunction ◽

Camp Metabolism ◽

Subnormal Level ◽

Camp Levels ◽

Stimulation Of

Studies of cAMP in human platelets exposed to ethanol were done to assess one possible mechanism for ethanol-related platelet dysfunction. Ingestion of ethanol by 3 subjects produced blood ethanol levels from 65-76 mM. Thrombocytopenia occurred in 1 subject and impaired platelet function occurred in all. Platelet cAMP decreased 36,51, and 59% below control levels. Infusion of ethanol to 2 normals produced blood ethanol levels of 43 mM and decreased platelet cAMP by 15% and 22%. Incubation of normal platelets with 86 mM ethanol in vitro decreased cAMP from 13.8 ± 2.9 (1 SD) to 9.4 ± 3.5 (p<0.02). By contrast, ethanol did not impair the increase in cAMP that occurred with 1.3 μM PGE1. Further, ethanol enhanced the increase in cAMP produced by 2.0 mM papaverine (Pap) by 160-220% and that produced by Pap + PGE1 by 58%. Dopamine, 0.1 mM, caused a 23% decrease in the basal level of cAMP, a 31% decrease below the subnormal level of cAMP seen with ethanol alone, and a 41% reduction in the increased level of cAMP produced by Pap + ethanol. The effect of ethanol on platelet cAMP metabolism is complex. Ethanol reduces basal levels of cAMP, does not decrease elevated levels that result from PGE1 stimulation of adenylate cyclase, and augments the inhibitory effect of Pap on platelet phosphodiesterase (PDE). Despite causing a decrease in basal cAMP levels, ethanol may impair platelet function by potentiating the effect of agents or other conditions which increase cAMP. The effect of ethanol on Pap-stimulated PDE activity may be blocked by dopamine, a neuropharmacologic agent that is actively accumulated by platelets.

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