ADP secretion and subsequent P2Y12 receptor signalling play a crucial role in thrombin-induced ERK2 activation in human platelets

2004 ◽  
Vol 92 (07) ◽  
pp. 114-123 ◽  
Author(s):  
Knut Fälker ◽  
Danica Lange ◽  
Peter Presek
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Detectable Effect ◽  
Receptor Signalling ◽  
Cellular Signal ◽  
Adp Receptor ◽  
High Concentrations ◽  
Erk2 Activation

SummaryStimulating human platelets with thrombin induces the activation of the extra cellular signal-regulated kinase 2 (ERK2). We demonstrate that this effect is highly dependent on ADP secretion and P2Y12 receptor signalling. AR-C69931MX (10 µM), a specific antagonist of the Gi-coupled P2Y12 ADP receptor, inhibits ERK2 activation induced by thrombin. Antagonists of the Gq-coupled P2Y1 ADP receptor, A3P5P (500 µM) and MRS2179 (100 µM), have no effect. ADP and its more potent analogue 2-methylthio-ADP alone (both up to 100 µM) do not induce ERK2 activation. Furthermore, we show that the inhibitory effect of AR-C69931MX on ERK2 activation induced by 0.1 U/ml thrombin as well as on platelet aggregation can be bypassed by epinephrine (1 and 10 µM), whereas epinephrine alone has no effect. Epinephrine acts on platelets mainly via α2A-adrenergic receptors, which, like P2Y12 receptors, couple to inhibitory G proteins. In addition, 2-methylthioADP as well as epinephrine provoke ERK2 activation at a thrombin concentration that alone has no detectable effect (0.05 U/ml). Thromboxane A2 (TXA2), which, like ADP, is released by activated platelets, acts as a positive feedback mediator. Stimulating the Gq-coupled TXA2-receptor with U46619 (10 µM), which leads to ADP secretion and P2Y12 receptor dependent platelet aggregation, also induces P2Y12-related ERK2 activation. The inhibition of U46619-induced ERK2 activation and platelet aggregation by AR-C69931MX are also rescued by epinephrine. Pretreatment with aspirin inhibits ERK2 activation induced by 0.1 U/ml thrombin, but has no effect at high concentrations of thrombin. The combination of U46619 and thrombin, at concentrations which alone have no effect, provokes ERK2 activation, suggesting that thrombin and released TXA2 act synergistically. Our data indicate that both primary signalling through Gq, which evokes ADP secretion, as well as subsequent coupling via Gi by the P2Y12 receptor are required for ERK2 activation.

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.

P2Y12 ADP receptor-dependent tyrosine phosphorylation of proteins of 27 and 31 kDa in thrombin-stimulated human platelets

2005 ◽  
Vol 93 (05) ◽  
pp. 880-888 ◽  
Author(s):  
Knut Fälker ◽  
Danica Lange ◽  
Peter Presek
Keyword(s):  
Adenylyl Cyclase ◽  
Tyrosine Phosphorylation ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
P2y12 Receptor ◽  
Dependent Manner ◽  
Adp Receptor ◽  
Txa2 Receptor Antagonist ◽  
Phosphoinositide 3 Kinase ◽  
Camp Levels

SummaryIn thrombin-stimulated human platelets several proteins undergo rapid and transient changes in tyrosine phosphorylation. We demonstrate that a set of proteins of 27, 29, 31, 34, and 39 kDa is affected by released ADP and P2Y12 receptor signaling during platelet activation. AR-C69931MX, an antagonist of the Gi2-coupled P2Y12 ADP receptor, inhibits initial tyrosine phosphorylation of p27 and p31 and prevents subsequent dephosphorylation of p29, p34, and p39. Antagonists of the Gq-coupled P2Y1 ADP receptor have no effect. Precluding integrin αIIbβ3 outside-in signaling with RGDS or S1197 does not affect the increase in tyrosine phosphorylation of the set of proteins but inhibits their subsequent dephosphorylation. Besides the ADP analogue 2-MeS-ADP, other platelet agonists such as collagen and the TXA2-mimetic U46619 also induce p27 and p31 tyrosine phosphorylation in a P2Y12 receptor-dependent manner. Tyrosine phosphorylation of p27 and p31 in response to collagen, but not thrombin, is prevented by aspirin and the TXA2 receptor antagonist SQ29548, indicating that the effect of collagen strongly relies on TXA2 signaling. Furthermore, epinephrine, acting via inhibitory Gz-coupled α2A-adrenoceptors, bypasses the inhibitory effect of AR-C69931MX on thrombin-induced p27 and p31 tyrosine phosphorylation. Finally, we demonstrate that tyrosine phosphorylation of p27 and p31 downstream of P2Y12 receptors is due to the inhibition of adenylyl cyclase but not phosphoinositide 3-kinase (PI 3-K) activation. Elevating cAMP levels with PGI2 or forskolin precludes thrombin-induced p27 and p31 tyrosine phosphorylation. Moreover, direct inhibition of adenylyl cyclase by SQ22536 reverses the effect of AR-C69931MX. Our data indicate that the observed changes in tyrosine phosphorylation are the result of both primary Gq signaling, initiating the release of ADP, as well as subsequent P2Y12 receptor-mediated Gi coupling.

scholarly journals The aggregation of isolated human platelets in the presence of lipoproteins and prostacyclin

1983 ◽  
Vol 216 (1) ◽  
pp. 43-49 ◽  
Author(s):  
D G Hassall ◽  
J S Owen ◽  
K R Bruckdorfer
Keyword(s):  
Platelet Aggregation ◽  
Normal Subjects ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Low Density Lipoproteins ◽  
High Density Lipoproteins ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
High Concentrations ◽  
Very High

Addition of prostacyclin (PGI2) temporarily inhibits platelet aggregation and permits the isolation of platelets free from plasma proteins, which have the same sensitivity as those in plasma [Moncada, Radomski & Vargas (1982) Br. J. Pharmacol. 75, 165P]. By using a modification of this technique we have established that platelets isolated from normal subjects aggregate more readily in response to ADP and adrenaline when physiological concentrations of low-density lipoproteins (LDL) are present. At high LDL concentrations spontaneous aggregation occurs. High-density lipoproteins (HDL) and very-low-density lipoproteins (VLDL) had no effect on agonist-induced platelet aggregation at normal concentrations, but HDL sensitized at higher concentrations. These effects by lipoproteins are not accompanied by changes in platelet lipid content. Cyclohexanedione treatment of LDL to modify apolipoproteins appeared to abolish the sensitization effect, indicating that binding to receptors was essential for the effects of LDL. LDL, but not HDL, overcame the inhibitory effect of PGI2 on platelet aggregation, except at very high concentrations of PGI2. PGI2 raised the cyclic AMP content of isolated platelets, but LDL only partially prevented this rise. These results suggest that LDL may have a greater role in platelet aggregation than previously recognized and may also regulate effects of PGI2. These findings may be of relevance to an understanding of cardiovascular diseases.

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

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.

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

1987 ◽  
Vol 58 (02) ◽  
pp. 744-748 ◽  
Author(s):  
A R Saniabadi ◽  
G D O Lowe ◽  
J C Barbenel ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Correlation Coefficient ◽  
Whole Blood ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Time Interval ◽  
Adp Receptor ◽  
Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

Effects of Halothane on Platelet Function

1980 ◽  
Vol 44 (03) ◽  
pp. 143-145 ◽  
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.

Inhibition of Platelet Function by Antiarrhythmic Drugs, Verapamil and Disopyramide

1982 ◽  
Vol 47 (02) ◽  
pp. 150-153 ◽  
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.

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

1982 ◽  
Vol 48 (01) ◽  
pp. 078-083 ◽  
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.

Functional Involvement of Thrombospondin in Platelet Aggregation Induced by Low Versus High Concentrations of Thrombin

1986 ◽  
Vol 55 (01) ◽  
pp. 136-142 ◽  
Author(s):  
K J Kao ◽  
David M Shaut ◽  
Paul A Klein
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Inhibitory Effect ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Platelet Binding ◽  
Functional Involvement ◽  
Thrombin Activation ◽  
Platelet Aggregates ◽  
High Concentrations

SummaryThrombospondin (TSP) is a major platelet secretory glycoprotein. Earlier studies of various investigators demonstrated that TSP is the endogenous platelet lectin and is responsible for the hemagglutinating activity expressed on formaldehyde-fixed thrombin-treated platelets. The direct effect of highly purified TSP on thrombin-induced platelet aggregation was studied. It was observed that aggregation of gel-filtered platelets induced by low concentrations of thrombin (≤0.05 U/ml) was progressively inhibited by increasing concentrations of exogenous TSP (≥60 μg/ml). However, inhibition of platelet aggregation by TSP was not observed when higher than 0.1 U/ml thrombin was used to activate platelets. To exclude the possibility that TSP inhibits platelet aggregation by affecting thrombin activation of platelets, three different approaches were utilized. First, by using a chromogenic substrate assay it was shown that TSP does not inhibit the proteolytic activity of thrombin. Second, thromboxane B2 synthesis by thrombin-stimulated platelets was not affected by exogenous TSP. Finally, electron microscopy of thrombin-induced platelet aggregates showed that platelets were activated by thrombin regardless of the presence or absence of exogenous TSP. The results indicate that high concentrations of exogenous TSP (≥60 μg/ml) directly interfere with interplatelet recognition among thrombin-activated platelets. This inhibitory effect of TSP can be neutralized by anti-TSP Fab. In addition, anti-TSP Fab directly inhibits platelet aggregation induced by a low (0.02 U/ml) but not by a high (0.1 U/ml) concentration of thrombin. In conclusion, our findings demonstrate that TSP is functionally important for platelet aggregation induced by low (≤0.05 U/ml) but not high (≥0.1 U/ml) concentrations of thrombin. High concentrations of exogenous TSP may univalently saturate all its platelet binding sites consequently interfering with TSP-crosslinking of thrombin-activated platelets.

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

Blood ◽  
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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