ADP receptor P2y12 prevents excessive primitive hematopoiesis in zebrafish by inhibiting Gata1

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.

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The Effect of Agonists and Antagonists of Platelet Aggregation on von Willebrand Factor-Mediated Platelet Agglutination

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648192 ◽

1991 ◽

Vol 65 (05) ◽

pp. 573-577 ◽

Cited By ~ 1

Author(s):

Jean McPherson ◽

Marjorie B Zucker ◽

Evelyn A Mauss ◽

Sandra Brownlea

Keyword(s):

Receptor Antagonist ◽

Intracellular Calcium ◽

Calcium Ions ◽

Inhibitory Action ◽

Platelet Rich Plasma ◽

Cytoskeletal Proteins ◽

A 23187 ◽

Sulphydryl Groups ◽

Affinity Receptor ◽

Adp Receptor

SummaryRistocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 ± 11% by 1.25 ΜM ADP, 41 ± 14% by 1 ΜM A 23187, and 26 ± 7% by 0.1 Μg/ml platelet activating factor (PAF). The effect of 5-110 ΜM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 ΜM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A 23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 ΜM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A 23187, but not phorbol myristate acetate (0.1 ΜM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 ΜM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition. N-ethylmaleimide (0.25-0.5 mM), an agent that can penetrate cell membranes and block sulphydryl groups, prevented or reversed ADP, A 23187- and PAF-induced inhibition of RIPA, but 0.5 mM dithionitrobisbenzoic acid, a non-penetrating sulphydryl blocker, had no effect. Diamide (0.1-0.5 mM), an agent that can crosslink cytoskeletal proteins by oxidation of sulphydryl groups, reduced RIPA. Thus an increase in intracellular calcium ions with resultant cytoskeletal changes and reorganisation of intracellular sulphydryl groups may mediate the inhibitory action of agonists on RIPA.

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Deficiency of (33P)2MeS-ADP Binding Sites on Platelets with Secretion Defect, Normal Granule Stores and Normal Thromboxane A2 Production

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656090 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0986-0990 ◽

Cited By ~ 66

Author(s):

Marco Cattaneo ◽

Rossana Lombardi ◽

Maddalena L Zighetti ◽

Christian Gachet ◽

Philippe Ohlmann ◽

...

Keyword(s):

Binding Sites ◽

Specific Binding ◽

Thromboxane A2 ◽

Normal Subjects ◽

Congenital Defects ◽

Normal Production ◽

Adp Receptor ◽

Partial Deficiency ◽

Induced Aggregation ◽

Platelet Secretion

SummaryBy the term “Primary Secretion Defect” (PSD), we mean a common heterogeneous group of congenital defects of platelet secretion, characterized by a normal primary wave of platelet aggregation induced by ADP and other agonists, a normal concentration of platelet granule contents, and normal production of thromboxane A2. The biochemical abnormalities responsible for PSD are not well known. Since a secretion defect similar to PSD is found in platelets that are severely deficient of binding sites for the ADP analogue 2MeS-ADP and do not aggregate in response to ADP, we tested the hypothesis that PSD platelets have moderately decreased 2MeS-ADP binding sites, which may be sufficient for normal ADP-induced aggregation but not for potentiating platelet secretion. The specific binding of [33P]2MeS-ADP to platelets from 3 PSD patients (347,443 and 490 sites/platelet; KD 2.8-3.9 nM) was lower than to platelets from 24 normal subjects (647 [530-1102]; KD = 3.8 [2.3-7.3]) (median [range]). Normal values were found in a fourth PSD patient (710; KD 3.7). The degree of inhibition of PGE1- induced cAMP increase by 0.1 |μM ADP was lower in patients than in controls. The secretion induced by the endoperoxide analogue U46619 from normal, acetylsalicylic acid-treated platelets under conditions that prevented the formation of large aggregates was potentiated by 1 fimol/1 ADP and inhibited by apyrase. These findings indicate that a partial deficiency of the platelet ADP receptor(s) might be responsible for the defect of platelet secretion in some PSD patients and that ADP potentiates platelet secretion independently of the formation of large aggregates and thromboxane A2 production.

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Personalized ADP-receptor inhibition strategy and outcomes following primary PCI for STEMI (PASTOR study)

International Journal of Cardiology ◽

10.1016/j.ijcard.2015.09.074 ◽

2016 ◽

Vol 202 ◽

pp. 463-466 ◽

Cited By ~ 2

Author(s):

Jussi Mikkelsson ◽

Tuomas Paana ◽

Aino Lepantalo ◽

Pasi P. Karjalainen

Keyword(s):

Primary Pci ◽

Receptor Inhibition ◽

Adp Receptor

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P2Y13: Identification and Characterization of a Novel Gαi-Coupled ADP Receptor from Human and Mouse

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.301.2.705 ◽

2002 ◽

Vol 301 (2) ◽

pp. 705-713 ◽

Cited By ~ 125

Author(s):

Fang L. Zhang ◽

Lin Luo ◽

Eric Gustafson ◽

Kyle Palmer ◽

Xudong Qiao ◽

...

Keyword(s):

Adp Receptor ◽

Identification And Characterization ◽

Human And Mouse

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Clinical pharmacology of the adenosine diphosphate (ADP) receptor antagonist, clopidogrel

Vascular Medicine ◽

10.1177/1358836x9800300312 ◽

1998 ◽

Vol 3 (3) ◽

pp. 247-251 ◽

Cited By ~ 28

Author(s):

Karsten Schrör

Keyword(s):

Clinical Pharmacology ◽

Receptor Antagonist ◽

Adenosine Diphosphate ◽

Adp Receptor

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Interaction of retinoic acid and scl controls primitive blood development

Blood ◽

10.1182/blood-2009-10-249557 ◽

2010 ◽

Vol 116 (2) ◽

pp. 201-209 ◽

Cited By ~ 28

Author(s):

Jill L. O. de Jong ◽

Alan J. Davidson ◽

Yuan Wang ◽

James Palis ◽

Praise Opara ◽

...

Keyword(s):

Stem Cell ◽

Retinoic Acid ◽

Cell Fate ◽

Embryonic Stem ◽

Yolk Sac ◽

Erythroid Progenitors ◽

Self Renewal ◽

Hematopoietic Development ◽

Blood Island ◽

Primitive Hematopoiesis

Abstract Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1+ erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4−/− mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

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Revival of PFA-100 – how far is it useful for the monitoring of ADP receptor antagonists?

Thrombosis and Haemostasis ◽

10.1160/th12-08-0548 ◽

2013 ◽

Vol 109 (03) ◽

pp. 564-565

Author(s):

Kamila Syska ◽

Cezary Watala ◽

Jacek Golanski

Keyword(s):

Receptor Antagonists ◽

Adp Receptor ◽

Adp Receptor Antagonists

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Comparison of VASP-phosphorylation assay to light-transmission aggregometry in assessing inhibition of the platelet ADP P2Y12 receptor

Thrombosis and Haemostasis ◽

10.1160/th06-09-0491 ◽

2006 ◽

Vol 96 (12) ◽

pp. 767-773 ◽

Cited By ~ 29

Author(s):

Agnieszka Pampuch ◽

Giovanni de Gaetano ◽

Chiara Cerletti

Keyword(s):

Light Transmission ◽

Platelet Reactivity ◽

Flow Cytometric Analysis ◽

Individual Variability ◽

P2y12 Receptor ◽

Reactivity Index ◽

Drug Induced ◽

Light Transmission Aggregometry ◽

Adp Receptor

SummaryThere is need to improve platelet function testing to monitor the response to antiplatelet drugs. We compared flow-cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) to light-transmission aggregometry for the detection of drug-induced in-vitro inhibition of the platelet P2Y12 ADP receptor on 22 healthy subjects (10 males, 12 females, 28.5 ± 6.6 years). The platelet reactivity index (PRI) of VASP was calculated both from mean fluorescence intensity (MFI) and percent of fluorescence-positive platelets in the presence of PGE1 with or without ADP (10 µM). Platelet aggregation was induced by ADP (1.25, 2.5 and 5 µM). Cangrelor, a competitive inhibitor of the P2Y12 receptor, preincubated 5 minutes, induced a concentration-dependent inhibition of platelet ADP-receptor function in both tests. Indeed PRI (%) based on either MFI or percent platelets gated were highly correlated with each other (r = 0.97, p<0.0001) and with aggregation in- duced by ADP. The IC50 of cangrelor against each of the three ADP concentrations used in aggregometry increased from 5.8 ± 3.4 nM to 23.1 ± 4.0 nM and to 98 ± 25 nM, respectively. The IC50 of cangrelor based on VASP-P was within the same range (25.5 ± 7.7 nM). No correlation was observed between IC50 values of cangrelor and ADP concentrations giving 50% effect (EC50) in the absence of the drug. However, at 10 nM cangrelor seven subjects could be identified by the VASP-P assay as “low responders” to the drug (PRI> 50%), and six of them also had an aggregation response to 5 µM ADP > 50%. These six subjects showed the lowest ADP EC50 values in the absence of the drug, possibly reflecting high sensitivity of their platelet P2Y12 receptors to ADP. In conclusion, both the VASP-P assay and light-transmission aggregometry detect in a comparable way in-vitro pharmacological inhibition of the platelet P2Y12 ADP receptor and its individual variability.

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Modulation of TGF-β signaling by endoglin in murine hemangioblast development and primitive hematopoiesis

Blood ◽

10.1182/blood-2010-12-325019 ◽

2011 ◽

Vol 118 (1) ◽

pp. 88-97 ◽

Cited By ~ 31

Author(s):

Liying Zhang ◽

Alessandro Magli ◽

Jacquelyn Catanese ◽

Zhaohui Xu ◽

Michael Kyba ◽

...

Keyword(s):

Signaling Pathway ◽

Transforming Growth Factor ◽

Es Cells ◽

Transforming Growth Factor Β ◽

Early Developmental Stage ◽

Type I ◽

Hematopoietic Development ◽

Downstream Target ◽

Primitive Hematopoiesis ◽

Alk 5

Abstract Endoglin (Eng), an accessory receptor for the transforming growth factor β (TGF-β) superfamily, is required for proper hemangioblast and primitive hematopoietic development. However the mechanism by which endoglin functions at this early developmental stage is currently unknown. Transcriptional analyses of differentiating eng−/− and eng+/+ ES cells revealed that lack of endoglin leads to profound reductions in the levels of key hematopoietic regulators, including Scl, Lmo2, and Gata2. We also detected lower levels of phosphorylated Smad1 (pSmad1), a downstream target signaling molecule associated with the TGF-β pathway. Using doxycycline-inducible ES cell lines, we interrogated the TGF-β signaling pathway by expressing activated forms of ALK-1 and ALK-5, type I receptors for TGF-β. Our results indicate that ALK-1 signaling promotes hemangioblast development and hematopoiesis, as evidenced by colony assays, gene expression and FACS analyses, whereas signaling by ALK-5 leads to the opposite effect, inhibition of hemangioblast and hematopoietic development. In Eng−/− ES cells, ALK-1 rescued both the defective hemangioblast development, and primitive erythropoiesis, indicating that ALK-1 signaling can compensate for the absence of endoglin. We propose that endoglin regulates primitive hematopoiesis by modulating the activity of the Smad1/5 signaling pathway in early stages of development.

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