Selective activation of the prostaglandin E2 receptor subtype EP2 or EP4 leads to inhibition of platelet aggregation

SummaryThe effect of selective activation of platelet prostaglandin (PG) E2 receptor subtype EP2 or EP4 on platelet aggregation remains to be determined. In platelets prepared from wild-type mice (WT platelets), high concentrations of PGE2 inhibited platelet aggregation induced by U-46619, a thromboxane receptor agonist. However, there was no significant change in the inhibitory effect of PGE2 on platelets lacking EP2 (EP2 –/– platelets) and EP4 (EP4 –/– platelets) compared with the inhibitory effect on WT platelets. On the other hand, AE1–259 and AE1–329, agonists for EP2 and EP4, respectively, potently inhibited U-46619 -induced aggregation with respective IC50 values of 590 ± 14 and 100 ± 4.9 nM in WT platelets, while the inhibition was significantly blunted in EP2 –/– and EP4 –/– platelets. In human platelets, AE1–259 and AE1–329 inhibited U-46619-induced aggregation with respective IC50 values of 640 ± 16 and 2.3 ± 0.3 nM. Notably, the inhibitory potency of AE1–329 in human platelets was much higher than that in murine platelets, while such a difference was not observed in the inhibitory potency of AE1–259. AE1–329 also inhibited adenosine diphosphate-induced platelet aggregation, and the inhibition was almost completely blocked by AE3–208, an EP4 antagonist. In addition, AE1–329 increased intracellular cAMP concentrations in a concentration- and EP4-dependent manner in human platelets. These results indicate that selective activation of EP2 or EP4 can inhibit platelet aggregation and that EP4 agonists are particularly promising as novel anti-platelet agents.

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Opposite Effect of Lysine on Platelet Aggregation Induced by Arachidonate and by Other Aggregants

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657221 ◽

1982 ◽

Vol 48 (01) ◽

pp. 078-083 ◽

Cited By ~ 3

Author(s):

C Ts'ao ◽

S J Hart ◽

D V Krajewski ◽

P G Sorensen

Keyword(s):

Platelet Aggregation ◽

Secondary Phase ◽

Aminocaproic Acid ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Primary Phase ◽

High Doses ◽

The Many ◽

Induced Aggregation

SummaryEarlier, we found that ε-aminocaproic acid (EACA) inhibited human platelet aggregation induced by adenosine diphosphate (ADP) and collagen, but not aggregation by arachidonic acid (AA). Since EACA is structurally similar to lysine, yet these two agents exhibit vast difference in their antifibrinolytic activities, we chose to study the effect of lysine on platelet aggregation. We used L-lysine-HCl in these studies because of its high solubility in aqueous solutions while causing no change in pH when added to human plasma. With lysine, we repeatedly found inhibition of ADP-, collagen- and ristocetin-induced aggregation, but potentiation of AA-induced aggregation. Both the inhibitory and potentiation effects were dose-dependent. Low doses of lysine inhibited the secondary phase of aggregation; high doses of it also inhibited the primary phase of aggregation. Potentiation of AA-induced aggregation was accompanied by increased release of serotonin and formation of malondialdehyde. These effects were not confined to human platelets; rat platelets were similarly affected. Platelets, exposed to lysine and then washed and resuspended in an artificial medium not containing lysine, remained hypersensitive to AA, but no longer showed decreased aggregation by collagen. Comparing the effects of lysine with equimolar concentrations of sucrose, EACA, and α-amino-n-butyric acid, we attribute the potent inhibitory effect of lysine to either the excess positive charge or H+ and C1− ions. The -NH2 group on the α-carbon on lysine appears to be the determining factor for the potentiation effect; the effect seems to be exerted on the cyclooxygenase level of AA metabolism. Lysine and other chemicals with platelet-affecting properties similar to lysine may be used as a tool for the study of the many aspects of a platelet aggregation reaction.

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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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Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C

Blood ◽

10.1182/blood.v79.1.110.bloodjournal791110 ◽

1992 ◽

Vol 79 (1) ◽

pp. 110-116

Author(s):

W Durante ◽

MH Kroll ◽

PM Vanhoutte ◽

AI Schafer

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

Calcium Concentration ◽

Umbilical Vein ◽

Inhibitory Effect ◽

Human Platelets ◽

Relaxing Factor ◽

Kinase C ◽

Endothelium Derived Relaxing Factor ◽

Induced Aggregation

Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin- induced aggregation. These data indicate that EDRF blocks thrombin- induced platelet aggregation by inhibiting the activation of PIP2- specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.

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Endothelium-derived relaxing factor inhibits thrombin-induced platelet aggregation by inhibiting platelet phospholipase C

Blood ◽

10.1182/blood.v79.1.110.110 ◽

1992 ◽

Vol 79 (1) ◽

pp. 110-116 ◽

Cited By ~ 45

Author(s):

W Durante ◽

MH Kroll ◽

PM Vanhoutte ◽

AI Schafer

Keyword(s):

Platelet Aggregation ◽

Phospholipase C ◽

Calcium Concentration ◽

Umbilical Vein ◽

Inhibitory Effect ◽

Human Platelets ◽

Relaxing Factor ◽

Kinase C ◽

Endothelium Derived Relaxing Factor ◽

Induced Aggregation

Abstract Endothelium-derived relaxing factor (EDRF) inhibits platelet function, but the mechanism underlying this inhibitory effect is not known. To examine this, cultured acetylsalicylic acid (ASA)-treated endothelial cells (EC) from bovine aorta (BAEC) or from human umbilical vein (HUVEC) were incubated with washed, ASA-treated human platelets. Incubation of platelets with either BAEC or HUVEC resulted in inhibition of thrombin-induced platelet aggregation that was dependent on the number of EC added. This effect was potentiated by superoxide dismutase and reversed by treating EC with NG-nitro-L-arginine or by treating platelets with methylene blue, indicating that the inhibition of platelet aggregation was due to the release of EDRF by EC. EC significantly blocked the thrombin stimulated breakdown of phosphatidylinositol-4,5-bisphosphate (PIP2) and the production of phosphatidic acid in [32P]orthophosphate-labeled platelets and of inositol trisphosphate in [3H]myoinositol-labeled platelets. In addition, the thrombin-mediated activation of protein kinase C (PKC) and phosphorylation of myosin light chain were inhibited in the presence of EC. Finally, thrombin stimulated an increase in cytosolic ionized calcium concentration ([Ca2+]i) in fura2-loaded platelets that was abolished by concentrations of EC which also blocked thrombin- induced aggregation. These data indicate that EDRF blocks thrombin- induced platelet aggregation by inhibiting the activation of PIP2- specific phospholipase C and thereby suppressing the consequent activation of PKC and the mobilization of [Ca2+]i.

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Synthesis of intracellular histamine in platelets is associated with activation of protein kinase C, but not with mobilization of Ca2+

Biochemical Journal ◽

10.1042/bj2730405 ◽

1991 ◽

Vol 273 (2) ◽

pp. 405-408 ◽

Cited By ~ 6

Author(s):

S P Saxena ◽

C Robertson ◽

A B Becker ◽

J M Gerrard

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Human Platelets ◽

Ionophore A23187 ◽

Dependent Manner ◽

Kinase C ◽

Histamine Synthesis ◽

Ic50 Values ◽

Induced Aggregation ◽

Pkc Activation

In previous reports, we have provided evidence indicating that newly formed histamine is an intracellular messenger in human platelets. The involvement of protein kinase C (PKC) and intracellular calcium (Ca2+i) in the synthesis of histamine was investigated. Human platelets were stimulated by phorbol 12-myristate 13-acetate (PMA), collagen and the Ca2+ ionophore A23187, with or without the PKC inhibitor staurosporine. Aggregation, histamine synthesis and phosphorylation of pleckstrin (47 kDa; P47) and myosin light chain (20 kDa; P20) proteins were monitored. Staurosporine inhibited PMA- and collagen-induced aggregation, histamine synthesis and phosphorylation of 47 kDa and 20 kDa proteins in a dose-dependent manner. For PMA, median inhibitory concentrations (IC50 values) for staurosporine inhibition of aggregation, histamine synthesis and phosphorylation were similar, suggesting that histamine synthesis induced by this agonist may be a consequence of PKC activation. Conversely, collagen-stimulated histamine synthesis was inhibited by staurosporine at concentrations significantly higher than those required to inhibit aggregation (P less than 0.005) or pleckstrin phosphorylation (P less than 0.01), indicating the possible involvement of non-PKC mechanism(s) in the synthesis of histamine induced by this agonist. A23187 failed to induce the synthesis of intracellular histamine in platelets, whereas staurosporine blocked A23187-induced aggregation and phosphorylation of the 20 kDa protein at significantly higher concentrations than those needed to inhibit PKC. When platelets were stimulated with a combination of A23187 and PMA, the increase in platelet histamine was less than that with PMA alone. The results provide evidence that the synthesis of intracellular histamine in platelets occurs as a consequence of PKC activation and may be down-regulated under conditions where there is a substantial rise in [Ca2+]i.

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The Gas6 Receptor Axl Enhances Platelet Activation Responses through Stimulation of PI3kinase- and PLC-Dependent Signaling Pathways.

Blood ◽

10.1182/blood.v104.11.630.630 ◽

2004 ◽

Vol 104 (11) ◽

pp. 630-630

Author(s):

Weston R. Gould ◽

Sangita Baxi ◽

Lisa A. Perrin ◽

Robert J. Leadley

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Intracellular Signaling ◽

Human Platelets ◽

Dense Granule ◽

Calcium Mobilization ◽

Dependent Manner ◽

Induced Aggregation ◽

Granule Release ◽

Stimulation Of

Abstract At the site of vascular injury, platelet activation is paramount in supporting formation of a platelet plug and generating a functional surface for the protein elements of coagulation. Recently, we demonstrated that the receptors for the α-granule constituent Gas6, support and enhance platelet aggregation and dense-granule release. The current study examined additional affects of Gas6 signaling in human platelets and sought to decipher intracellular signaling mechanisms initiated by stimulation of Axl, a Gas6 platelet receptor. Flow cytometry analyses indicated that all three Gas6 receptors, Axl, Sky, and Mer were present on the platelet surface. Blockade of Gas6, Sky, or Mer by specific antibodies not only inhibited TRAP- and ADP-induced platelet aggregation and dense granule release, but also prevented thrombin mediated clot retraction by as much as 55%. Furthermore, intracellular calcium mobilization in response to TRAP activation was greater than 80% inhibited in the presence of each of these blocking antibodies. A highly specific antibody directed toward Axl (< 2% cross reactivity with Sky and Mer) activated Axl leading to an enhancement of TRAP and ADP induced aggregation and degranulation. Stimulation of human platelets by this Axl agonist led to a modest, but sustained increase in calcium mobilization suggesting that Axl signaling incorporated activation of PLC. The increase in calcium mobilization was sensitive to wortmannin, demonstrating that PLC activation occurred concurrent with or downstream of PI3K. Indeed, additional experiments to ascertain the intracellular mediators of Axl activity identified a two-fold increase in specific phosphorylation of Akt downstream of PI3K as well as a similar increase in phosphorylation of PLCγ. TRAP stimulation of human platelets also increased the phosphorylation levels of Akt and PLCγ in a Gas6 dependent manner as a Gas6 blocking antibody reduced the levels of Akt and PLCγ phosphorylation by 50%. Overall, these studies suggest that Gas6 enhancement of human platelet activation occurs through the low-level stimulation of the intracellular signaling molecules Akt and PLCγ, serving at the juncture of several mediators of platelet activation. These events also increase levels of cytoplasmic calcium, further supporting an enhancement of activation observed in response to low levels of known platelet agonists. Thus, platelet Gas6 functions to support platelet activation at the very early stages of the hemostatic response to injury.

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In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650661 ◽

1996 ◽

Vol 76 (05) ◽

pp. 786-790 ◽

Cited By ~ 1

Author(s):

Atsuhiro Sugidachi ◽

Norbert Breiter ◽

Taketoshi Ogawa ◽

Fumitoshi Asai ◽

Hiroyuki Koike

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Anticoagulant Activity ◽

Acid Amide ◽

Human Platelets ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Free System ◽

Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

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Chlorin e6 Prevents ADP-Induced Platelet Aggregation by Decreasing PI3K-Akt Phosphorylation and Promoting cAMP Production

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/569160 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Ji Young Park ◽

Hyun Dong Ji ◽

Bo Ra Jeon ◽

Eun Ju Im ◽

Young Min Son ◽

...

Keyword(s):

Platelet Aggregation ◽

Underlying Mechanism ◽

Serotonin Release ◽

Intracellular Camp ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Akt Phosphorylation ◽

Mitogen Activated Protein ◽

Induced Aggregation ◽

Camp Levels

A number of reagents that prevent thrombosis have been developed but were found to have serious side effects. Therefore, we sought to identify complementary and alternative medicinal materials that are safe and have long-term efficacy. In the present studies, we have assessed the ability of chlorine e6 (CE6) to inhibit ADP-induced aggregation of rat platelets and elucidated the underlying mechanism. CE6 inhibited platelet aggregation induced by 10 µM ADP in a concentration-dependent manner and decreased intracellular calcium mobilization and granule secretion (i.e., ATP and serotonin release). Western blotting revealed that CE6 strongly inhibited the phosphorylations of PI3K, Akt, c-Jun N-terminal kinase (JNK), and different mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2) as well as p38-MAPK. Our study also demonstrated that CE6 significantly elevated intracellular cAMP levels and decreased thromboxane A2formation in a concentration-dependent manner. Furthermore, we determined that CE6 initiated the activation of PKA, an effector of cAMP. Taken together, our findings indicate that CE6 may inhibit ADP-induced platelet activation by elevating cAMP levels and suppressing PI3K/Akt activity. Finally, these results suggest that CE6 could be developed as therapeutic agent that helps prevent thrombosis and ischemia.

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Inhibition of Thrombin-, Epinephrine- and Collagen-Induced Platelet Aggregation by α1-Acid Glycoprotein

10.1055/s-0039-1687235 ◽

1979 ◽

Author(s):

P. Andersen ◽

C. Eika

Keyword(s):

Platelet Aggregation ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

Inhibitory Effect ◽

Human Platelets ◽

Clotting Time ◽

Acid Glycoprotein ◽

High Concentrations ◽

Spontaneous Platelet Aggregation ◽

Induced Aggregation

α1-Acid glycoprotein (α1,-acid GP) isolated from human plasma was found to inhibit thrombin-induced aggregation of washed human platelets (0.05 NIH U/ml final conc.), and inhibition was complete with physiological concentrations of α1-acid GP (1.0-1.5 g/1 final conc.). The inhibitory effect seemed to occur immediately on thrombin addition, thus similar to the effect of heparin previously observed. As opposed to heparin, however, α1-acid GP did not affect spontaneous platelet aggregation. Furthermore, α1-acid GP (in optimal cone.) reduced the combined inhibitory effect of heparin and antithrombin III on thrombin-induced platelet aggregation, thus consistent with the previous findings using heparin thrombin clotting time.Snyder and Coodley (1976) found α1-acid GP to inhibit platelet aggregation induced by epinephrine and adenosine diphosphate in platelet-rich plasma. As we also found α1-acid GP to inhibit collagen-induced platelet aggregation, α1-acid GP may possibly act as an inhibitor of the release reaction though fairly high concentrations (10 mg/ml final cone.) was needed for complete inhibition.

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Adenosine Receptor Identification for Controlling Platelet Aggregation

Blood ◽

10.1182/blood-2018-99-116213 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3733-3733

Author(s):

Bunyan Teng ◽

Daniel N Darlington ◽

Andrew P Cap

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Adenosine Receptor ◽

Receptor Subtype ◽

Intracellular Camp ◽

Dependent Manner ◽

Inhibition Of Platelet Aggregation ◽

A2a Receptor ◽

A2b Receptor ◽

Stimulation Of

Abstract Introduction: Adenosine, an autacoid and metabolite of ATP, has been known to have anti-platelet properties. Of the 4 adenosine receptors, both A2A and/or A2B have been implicated in adenosine-mediated anti-platelet properties, while the roles of A1 and A3 have not been clearly defined in humans. In addition, previous studies show that A2A/A2B on platelets are G-Protein Coupled Receptors and are coupled to a stimulatory G-protein that activate adenylyl cyclase and subsequently increase intracellular cAMP. An elevation of cAMP in platelets inhibits aggregation. In this study, we set out to determine which adenosine receptor subtype leads to inhibition of platelet aggregation, and change in intracellular cAMP. Materials and Methods: Platelet-rich plasma (PRP) was isolated from whole blood of human volunteers, and centrifuged at 200g for 10min. Light transmission aggregometry was performed by 100uM ADP with or without NECA (non-specific AR agonist), CCPA (A1 AR agonist), CGS 21680 (A2A AR agonist), BAY 60-6583 (A2B AR agonist), DPCPX (A1 AR antagonist), Sch 58261 (A2A AR antagonist), GS 6201 (A2B AR antagonist), and MRS 1220 (A3 AR antagonist). Cyclic AMP was extracted from 100ul of PRP after adding 1ml of EtOH, 10mM ammonium formate, with 10ug/ml cGMP-Br as an internal control, and measured by liquid chromatography/ Tandem Mass Spectroscopy (Quantiva, ThrermoFisher). Results: ADP-induced platelet aggregation was inhibited in a dose dependent manner by the non-specific adenosine agonist, NECA (Figure 1). This inhibition of platelet aggregation was likely mediated by A2A receptor as the specific A2A receptor agonist had a similar effect (Figure 2). Furthermore, A2A antagonist blocked the effects of NECA (Figure 5). Stimulation of A1 receptor had no effect on the ADP-induced platelet aggregation, except at the highest concentration (250 µM), and is likely due to its non-specific effect on A2A AR (Figures 3 and 4). Blockade of A1 enhanced the effects of NECA (Figure 5). This suggest that A2A and A1 may have opposing roles for control of platelet aggregation. Stimulation of A2B receptor, had no effect on ADP-induced platelet aggregation, except at the highest concentration (250 µM), which was likely due to the non-specific vehicle effects (2.5% DMSO, Figure 6). Blockade of A2B receptor had no effect on NECA, while A3 blockade showed slight inhibition on NECA's anti-platelet effect (data not shown). NECA inhibition of platelet aggregation was likely due to elevation of intracellular cAMP as incubation for 5min with NECA stimulated intracellular cAMP (Figure 7). This effect was blocked by A2A, not by A1 antagonist. Conclusion: Our results support previous findings that adenosine receptor A2A mediates adenosine-induced anti-platelet properties in human platelets. Adenosine and its analogs inhibit platelet aggregation to the natural stimulus, ADP. The mechanism appears to be due to elevation in intracellular cAMP. We did not find evidence that A2B played a significant role in platelet aggregation. A1 and A3. however, demonstrated modulatory effects that has not been previously described. Disclosures No relevant conflicts of interest to declare.

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