Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

Joanne C. Clark; Deirdre M. Kavanagh; Stephanie Watson; Jeremy A. Pike; Robert K. Andrews; Elizabeth E. Gardiner; Natalie S. Poulter; Stephen J. Hill; Steve P. Watson

doi:10.1055/s-0039-1688788

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2019; 119(07): 1124-1137
DOI: 10.1055/s-0039-1688788

Cellular Haemostasis and Platelets

Georg Thieme Verlag KG Stuttgart · New York

Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events

Joanne C. Clark

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

,

Deirdre M. Kavanagh

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

,

Stephanie Watson

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

,

Jeremy A. Pike

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

,

Robert K. Andrews

³Australian Centre for Blood Diseases, Monash University, Melbourne, Australia

,

Elizabeth E. Gardiner

⁴Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra, Australia

,

Natalie S. Poulter

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

,

Stephen J. Hill

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

⁵Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, United Kingdom

,

Steve P. Watson

¹Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

²Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom

› Author Affiliations

Abstract

Background The G protein-coupled receptor, adenosine A_2A, signals through the stimulatory G protein, G_s, in platelets leading to activation of adenylyl cyclase and elevation of cyclic adenosine monophosphate (cAMP) and inhibition of platelet activation.

Objective This article investigates the effect of A_2A receptor activation on signalling by the collagen receptor glycoprotein (GP) VI in platelets.

Methods Washed human platelets were stimulated by collagen or the GPVI-specific agonist collagen-related peptide (CRP) in the presence of the adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed for aggregation, adenosine triphosphate secretion, protein phosphorylation, spreading, Ca²⁺ mobilisation, GPVI receptor clustering, cAMP, thromboxane B₂ (TxB₂) and P-selectin exposure.

Results NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion to collagen or CRP in the absence or presence of the P2Y₁₂ receptor antagonist, cangrelor and the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA nor forskolin altered clustering of GPVI, elevation of Ca²⁺ or spreading of platelets on a collagen surface. Further, neither NECA nor forskolin, altered collagen-induced tyrosine phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet activation by the TxA₂ mimetic, U46619, but not the combination of adenosine diphosphate and collagen.

Conclusion NECA and forskolin have no effect on the proximal signalling events by collagen. They inhibit platelet activation in a response-specific manner in part through inhibition of the feedback action of TxA₂.

Keywords

adenosine - cAMP - collagen - glycoprotein VI (GPVI) receptor - platelets

Full Text

References

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Supplementary Material

Supplementary Material