Thromboxane-induced phosphatidate formation in human platelets. Relationship to receptor occupancy and to changes in cytosolic free calcium

The inter-relationships between receptor occupancy, inositol phospholipid metabolism and elevation of cytosolic free Ca2+ in thromboxane A2-induced human platelet activation were investigated by using the stable thromboxane A2 mimetic, 9,11-epoxymethanoprostaglandin H2, and the thromboxane A2 receptor antagonist, EPO45. 9,11-Epoxymethanoprostaglandin H2 stimulated platelet phosphatidylinositol metabolism as indicated by the rapid accumulation of [32P]phosphatidate and later accumulation of [32P]phosphatidylinositol in platelets pre-labelled with [32P]Pi. These effects of 9,11-epoxymethanoprostaglandin H2 were concentration-dependent and half-maximal [32P]phosphatidate formation occurred at an agonist concentration of 54 +/- 8 nM. With platelets labelled with the fluorescent Ca2+ indicator quin 2, resting cytosolic free Ca2+ was 86 +/- 12 nM. 9,11-Epoxymethanoprostaglandin H2 induced a rapid, concentration-dependent elevation of cytosolic free Ca2+ to a maximum of 300-700 nM. Half-maximal stimulation was observed at an agonist concentration of 80 +/- 23 nM. The thromboxane A2 receptor antagonist EPO45 selectively inhibited 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and elevation of cytosolic free Ca2+, indicating that both events are sequelae of receptor occupancy. Human platelets contain a single class of stereospecific, saturable, high affinity (KD = 70 +/- 13 nM) binding sites for 9,11-epoxymethano[3H]prostaglandin H2. The concentration-response curve for receptor occupancy (9,11-epoxymethano-[3H]prostaglandin H2 binding) is similar to that for 9,11-epoxymethanoprostaglandin H2-induced [32P]phosphatidate formation and for elevation of cytosolic free Ca2+. These observations indicate that human platelet thromboxane A2 receptor occupation is closely linked to inositol phospholipid metabolism and to elevation of cytosolic free Ca2+. Both such events may be necessary for thromboxane A2-induced human platelet activation.

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Neomycin does not interfere with the inositol phospholipid metabolism, but blocks binding of α-thrombin to intact human platelets

Biochemical Journal ◽

10.1042/bj2730241 ◽

1991 ◽

Vol 273 (1) ◽

pp. 241-243 ◽

Cited By ~ 5

Author(s):

O B Tysnes ◽

E Johanessen ◽

V M Steen

Keyword(s):

Platelet Activation ◽

Phospholipid Metabolism ◽

Human Platelets ◽

Cellular Receptor ◽

Inositol Phospholipid ◽

Induced Changes

Neomycin was demonstrated to inhibit the binding of thrombin to intact human platelets. The effects of neomycin on both thrombin binding and thrombin-induced changes in inositol phospholipid metabolism could be reproduced by the thrombin antagonist hirudin. We propose that neomycin inhibits thrombin-induced platelet activation by interference with the cellular receptor.

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The Endocannabinoid 2-Arachidonoylglycerol Regulates Platelet Function.

Blood ◽

10.1182/blood.v108.11.3904.3904 ◽

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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The Potent Inhibition of Vapiprost, a Novel Thromboxane A2 Receptor Antagonist, on the Secondary Aggregation and ATP Release of Human Platelets.

Biological and Pharmaceutical Bulletin ◽

10.1248/bpb.20.625 ◽

1997 ◽

Vol 20 (6) ◽

pp. 625-631 ◽

Cited By ~ 3

Author(s):

Shuichi HORIE ◽

Mayumi YAMADA ◽

Megumi SATOH ◽

Shinobu NORITAKE ◽

Sayuri HIRAISHI ◽

...

Keyword(s):

Receptor Antagonist ◽

Atp Release ◽

Thromboxane A2 ◽

Human Platelets ◽

Thromboxane A2 Receptor ◽

Potent Inhibition

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Agonist-Induced Inositol Phospholipid Metabolism and Ca++ Flux in Human Platelet Activation

Advances in Experimental Medicine and Biology - Mechanisms of Stimulus—Response Coupling in Platelets ◽

10.1007/978-1-4615-9442-0_10 ◽

1985 ◽

pp. 127-144 ◽

Cited By ~ 28

Author(s):

D. Euan MacIntyre ◽

W. Kenneth Pollock ◽

Angus M. Shaw ◽

Mark Bushfield ◽

Linda J. MacMillan ◽

...

Keyword(s):

Platelet Activation ◽

Human Platelet ◽

Phospholipid Metabolism ◽

Inositol Phospholipid

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Desensitization and antagonism of vasopressin-induced phosphoinositide metabolism and elevation of cytosolic free calcium concentration in human platelets

Biochemical Journal ◽

10.1042/bj2340067 ◽

1986 ◽

Vol 234 (1) ◽

pp. 67-73 ◽

Cited By ~ 28

Author(s):

W K Pollock ◽

D E MacIntyre

Keyword(s):

Platelet Activation ◽

Human Platelet ◽

Receptor Occupancy ◽

Human Platelets ◽

Free Calcium ◽

Phosphoinositide Metabolism ◽

Subsequent Challenge ◽

Free Calcium Concentration ◽

20 Nm ◽

Stimulation Of

The receptor mechanisms underlying vasopressin-induced human platelet activation were investigated with respect to stimulation of phosphoinositide metabolism and changes in the cytosolic free Ca2+ concentration ([Ca2+]i). Vasopressin stimulated phosphoinositide metabolism, as indicated by the early formation of [32P]phosphatidic acid ([32P]PtdA) and later accumulation of [32P]phosphatidylinositol ([32P]PtdIns). In addition, vasopressin elicited a transient depletion of [glycerol-3H]PtdIns and accumulation of [glycerol-3H]PtdA. The effects of vasopressin on phosphoinositide metabolism were concentration-dependent, with half maximal [32P]PtdA formation occurring at 30 +/- 15 nM-vasopressin. In the presence of 1 mM extracellular free Ca2+, vasopressin induced a rapid, concentration-dependent elevation of [Ca2+]i in quin2-loaded platelets: half-maximal stimulation was observed at 53 +/- 20 nM-vasopressin. The V1-receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyl)tyrosine,8-arginine]-vasopressin selectively inhibited vasopressin (100 nM)-induced [32P]PtdA formation [I50 (concn. giving 50% inhibition) = 5.7 +/- 2.4 nM] and elevation of [Ca2+]i (I50 = 3 +/- 1.5 nM). Prior exposure of platelets to vasopressin rendered them unresponsive, in terms of [32P]PtdA formation and elevation of [Ca2+]i, to a subsequent challenge with vasopressin, but responsive to a subsequent challenge with U44069, a thromboxane-A2 mimetic. These results indicate that vasopressin-induced human platelet activation is initiated by combination with specific V1 receptors on the platelet, and that the sequelae of receptor occupancy (stimulation of phosphoinositide metabolism and elevation of [Ca2+]i) are equally susceptible to inhibition by receptor antagonists and by receptor desensitization.

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Intrinsic activity of the non-prostanoid thromboxane A2 receptor antagonist, daltroban (BM 13,505), in human platelets in vitro and in the rat vasculature in vivo

British Journal of Pharmacology ◽

10.1111/j.1476-5381.1995.tb16341.x ◽

1995 ◽

Vol 115 (1) ◽

pp. 210-216 ◽

Cited By ~ 6

Author(s):

Frédéric Bertolino ◽

Jean-Pierre Valentin ◽

Myriam Maffre ◽

Françoise Grelac ◽

Anne-Marie Bessac ◽

...

Keyword(s):

Receptor Antagonist ◽

Thromboxane A2 ◽

Human Platelets ◽

Intrinsic Activity ◽

Thromboxane A2 Receptor

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Effects of SQ 27,427, a thromboxane A2 receptor antagonist, in the human platelet and isolated smooth muscle

European Journal of Pharmacology ◽

10.1016/0014-2999(84)90183-3 ◽

1984 ◽

Vol 103 (1-2) ◽

pp. 9-18 ◽

Cited By ~ 13

Author(s):

Don N. Harris ◽

Roland Greenberg ◽

Marie B. Phillips ◽

Inge M. Michel ◽

Harold J. Goldenberg ◽

...

Keyword(s):

Smooth Muscle ◽

Receptor Antagonist ◽

Human Platelet ◽

Thromboxane A2 ◽

Thromboxane A2 Receptor

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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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