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
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Deirdre M. Kavanagh
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Stephanie Watson
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
,
Jeremy A. Pike
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Robert K. Andrews
3  Australian Centre for Blood Diseases, Monash University, Melbourne, Australia
,
4  Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, Australian National University, Canberra, Australia
,
Natalie S. Poulter
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Stephen J. Hill
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
5  Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham Medical School, Queen's Medical Centre, Nottingham, United Kingdom
,
Steve P. Watson
1  Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2  Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
› Author Affiliations
Funding This study was supported by a grant from the Centre of Membrane and Protein and Receptors, University of Birmingham and University of Nottingham, Midlands, UK. S.P.W. is a British Heart Foundation Professor (CH 03/003).
Further Information

Publication History

30 October 2018

20 March 2019

Publication Date:
26 May 2019 (eFirst)

Abstract

Background The G protein-coupled receptor, adenosine A2A, signals through the stimulatory G protein, Gs, 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 A2A 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.

Authors' Contributions

J.C.C. has performed experiments, analysed data, wrote and edited the manuscript. S.W. performed experiments and analysed the data. D.M.K. provided training in super resolution microscopy experiments and expertise for the study design and cluster analysis. J.A.P. has analysed the data and developed the programmes for image and cluster analysis. N.S.P. provided expertise for super resolution microscopy experiment study design and cluster analysis. R.K.A. and E.E.G. provided key materials. S.J.H. provided supervision, concept, funding and contributed to manuscript editing. S.P.W. provided supervision, funding, study design and concept, reviewed data, wrote and edited the manuscript. All authors have read the manuscript.


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