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Adenosine and forskolin inhibit platelet aggregation by collagen but not the proximal signalling events Clark, Joanne; Kavanagh, Deirdre; Watson, Stephanie; Pike, Jeremy; Andrews, Robert; Gardiner, Elizabeth E.; Poulter, Natalie; Hill, Stephen J; Watson, Steve DOI: 10.1055/s-0039-1688788 License: Other (please specify with Rights Statement)

Document Version Peer reviewed version Citation for published version (Harvard): Clark, J, Kavanagh, D, Watson, S, Pike, J, Andrews, R, Gardiner, EE, Poulter, N, Hill, SJ & Watson, S 2019, 'Adenosine and forskolin inhibit platelet aggregation by collagen but not the proximal signalling events', Thrombosis and Haemostasis, vol. 119, no. 07, pp. 1124-1137. https://doi.org/10.1055/s-0039-1688788

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Publisher Rights Statement: Adenosine and Forskolin Inhibit Platelet Aggregation by Collagen but not the Proximal Signalling Events, Joanne C. Clark et al, Thromb Haemost 2019; 119(07): 1124-1137, DOI: 10.1055/s-0039-1688788, © Georg Thieme Verlag KG 2019

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Download date: 26. Sep. 2021 Thrombosis and Haemostasis

Adenosine and forskolin inhibit platelet aggregation by collagen but not the proximal signalling events

Journal: Thrombosis and Haemostasis

Manuscript ID TH-18-10-0772.R1 Manuscript Type:ForOriginal Peer Article: Cellular Review Haemostasis and Platelets Category: Basic Science

Date Submitted by the 01-Mar-2019 Author:

Complete List of Authors: Clark, Joanne; University of Birmingham, Cardiovascular Sciences; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham Kavanagh , Deirdre; University of Birmingham, Cardiovascular Sciences; University of Birmingham, Cardiovascular Sciences Watson, Stephanie; University of Birmingham, Cardiovascular Sciences Pike , Jeremy; University of Birmingham, Cardiovascular Sciences; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham Andrews, Robert; Monash University, Australian Centre for Blood Diseases Gardiner, Elizabeth; Australian National University, Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research Poulter, Natalie ; University of Birmingham, Cardiovascular Sciences; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham Hill , Stephen ; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham; University of Nottingham, School of Life Sciences, Division of Physiology, Pharmacology and Neuroscience Watson, Steve; University of Birmingham, Cardiovascular Sciences ; Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham

Keywords: adenosine, cAMP, Collagens, GP VI, platelets

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1 2 3 Adenosine and forskolin inhibit platelet aggregation by collagen but not the proximal 4 5 signalling events 6 Joanne C. Clark1, 2, Deirdre M. Kavanagh1,2, Stephanie Watson1, Jeremy A. Pike1,2, Robert K. 7 3 4 1,2 2,5 1,2 8 Andrews , Elizabeth E. Gardiner , Natalie S. Poulter , Stephen J. Hill , Steve P. Watson 9 10 11 1 12 Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, 13 University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 14 2 15 Centre of Membrane Proteins and Receptors (COMPARE), The Universities of Birmingham 16 and Nottingham, The Midlands, UK 17 18 3Australian Centre for Blood Diseases, Monash University, Melbourne, Australia 19 4 20 Department of Cancer Biology and Therapeutics, John Curtin School of Medical Research, 21 Australian National University,For Canberra, Peer Australia Review 22 23 5Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University 24 of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, UK 25 26 27 28 Abstract word count: 242 29 30 Main Text: 4265 31 32 33 34 Correspondence: 35 36 Prof. Steve P Watson, Institute of Cardiovascular Sciences, Level 1 IBR, College of 37 Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, 38 UK. Tel: +44 (0) 121 414 6514, Email: [email protected] 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Abstract 4 5 6 Background: The G protein-coupled receptor, adenosine A2A, signals through the stimulatory 7 8 9 G protein, Gs in platelets leading to activation of adenylyl cyclase and elevation of cAMP and 10 11 inhibition of platelet activation. 12 13 14 Objective: To investigate the effect of A2A receptor activation on signalling by the collagen 15 16 receptor glycoprotein (GP) VI in platelets. 17 18 19 Methods: Washed human platelets were stimulated by collagen or the GPVI-specific agonist 20 21 collagen-related-peptide For (CRP) inPeer the presence Review of the adenosine receptor agonist, 5'-N- 22 23 24 ethylcarboxamidoadenosine (NECA) or the adenylyl cyclase activator, forskolin and analysed 25 26 for aggregation, ATP secretion, protein phosphorylation, spreading, Ca2+ mobilisation, GPVI 27 28 receptor clustering, cAMP, TxB2 and P-selectin exposure. 29 30 31 Results: NECA, a bioactive adenosine analogue, partially inhibits aggregation and secretion 32 33 34 to collagen or CRP in the absence or presence of the P2Y12 receptor antagonist, cangrelor and 35 36 the cyclooxygenase inhibitor, indomethacin. The inhibitory effect in the presence of the three 37 38 inhibitors is largely overcome at higher concentrations of collagen but not CRP. Neither NECA 39 40 nor forskolin altered clustering of GPVI, elevation of Ca2+ or spreading of platelets on a 41 42 43 collagen surface. Further, neither NECA nor forskolin, altered collagen induced tyrosine 44 45 phosphorylation of Syk, LAT nor PLCγ2. However, NECA and forskolin inhibited platelet 46 47 activation by the thromboxane A (TxA ) mimetic, U46619, but not the combination of ADP 48 2 2 49 50 and collagen. 51 52 53 Conclusion: NECA and forskolin have no effect on the proximal signalling events by collagen. 54 55 They inhibit platelet activation in a response specific manner in part through inhibition of the 56 57 feedback action of thromboxane A2. 58 59 60 Key words: adenosine, cAMP, collagen, glycoprotein VI (GPVI) receptor, platelets

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1 2 3 Summary Table 4 5 6 7 What is known about this topic? 8 9  Adenosine stimulates cAMP formation leading to inhibition of platelet activation 10 11 by collagen. 12 13 14  There are contrasting reports on whether the proximal signalling events induced by 15 16 collagen are altered in the presence of cAMP elevation. 17 18 What this paper adds? 19 20 21  Adenosine has noFor effect onPeer the proximal Review events in the GPVI signalling cascade up to 22 23 intracellular Ca2+ but inhibits the feedback action of downstream signals including 24 25 thromboxane A . 26 2 27 28 29 30 31 Introduction 32 33 34 Platelets are small anucleate cytoplasmic discs derived from megakaryocytes in the bone 35 36 marrow that circulate in the blood. They play a vital role in the control of haemostasis and 37 38 thrombus formation as well as in supporting inflammation, immunity, angiogenesis and 39 40 1 41 vascular integrity . Platelets are exposed to prostacyclin and nitric oxide at the endothelial 42 43 surface which leads to elevation of cAMP and cGMP, respectively2. This is the most potent 44 45 endogenous mechanism of platelet inhibition3. cAMP and cGMP activate the cyclic nucleotide- 46 47 48 dependent protein kinases, PKA and PKG respectively, leading to phosphorylation of a wide 49 50 variety of substrate proteins4. The consequences of phosphorylation include inhibition of Ca2+ 51 52 release from intracellular stores5, modulation of cytoskeletal actin dynamics via vasodilator- 53 54 stimulated phosphoprotein (VASP), LIM and SH3 domain protein (LASP), heat shock protein 55 56 6-8 57 (HSP) 27, filamin-A and caldesmon , and inhibition of heterotrimeric and small G proteins. 58 59 This leads to inhibition of integrin activation, secretion and adhesion9. 60

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1 2 3 Collagen binds to the immunoglobulin receptor glycoprotein (GP) VI which is its major 4 5 10 6 signalling receptor in platelets, and to integrin α2β1 which supports platelet adhesion . GPVI 7 8 forms a complex in the membrane with the FcR-chain which contains an immunoreceptor 9 10 tyrosine-based activation motif (ITAM) defined by the presence of two YxxL groups (single 11 12 11 12 13 amino acid code) separated by 12 amino acids . Collagen binds selectively to dimeric GPVI 14 15 and induces phosphorylation of the conserved tyrosine residues in the ITAM through the action 16 17 of Src family kinases. This leads to binding of the tyrosine kinase Syk through its tandem SH2 18 19 20 domains and initiation of a downstream signalling pathway that culminates in activation of 21 For Peer Review 22 PLC210. Collagen activates platelets at sites of vascular lesions and so it is important to 23 24 understand how collagen is able to do this in the presence of elevated levels of cyclic 25 26 27 nucleotides. 28 29 30 Adenosine is an endogenous nucleotide that inhibits platelet activation by ADP and collagen 31 32 through A2A and A2B receptors. Both receptors activate the heterotrimeric G protein subunit, 33 13-15 34 Gs, leading to elevation of cAMP . Adenosine has been reported to inhibit platelet 35 36 aggregation, ATP secretion, P-selectin cell surface expression and adhesion to a collagen 37 38 14 14 39 surface and to inhibit thrombus formation in vivo . The inhibitory action of adenosine may 40 41 contribute to the clinical efficacy of the P2Y12 receptor antagonist, ticagrelor, which inhibits 42 43 uptake of adenosine into platelets16. 44 45 46 There are also reports that cAMP does not inhibit platelet activation by collagen. For example, 47 48 49 prostacyclin which increases cAMP levels, has been reported to inhibit thrombin but not 50 51 collagen induced tyrosine phosphorylation and signalling17-19. In addition collagen induced 52 53 shape change, which is mediated by Ca2+ mobilisation, is insensitive to cAMP 20. On the other 54 55 21 56 hand, Loyau et al reported that elevation of cAMP inhibits binding of a monoclonal antibody 57 58 (mAb), 9E18, that binds selectively to dimeric GPVI, and Takayama et al22 reported that cAMP 59 60

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1 2 3 promotes endocytosis of GPVI. Both actions should result in inhibition of platelet activation 4 5 6 by collagen. 7 8 9 In view of these contrasting reports, the aim of the present study was to investigate the effect 10 11 of adenosine and forskolin which is a more powerful stimulus of adenylyl cyclase, on platelet 12 13 activation by collagen. We report that adenosine and forskolin inhibit platelet aggregation by 14 15 low concentrations but not high concentrations of collagen downstream of the proximal 16 17 2+ 18 signalling events including elevation of intracellular Ca . 19 20 21 Methods For Peer Review 22 23 24 Materials 25 26 27 Horm collagen was obtained from Takeda (High Wycombe, UK). NECA (5'-N- 28 29 ethylcarboxamidoadenosine) was purchased from Tocris Bioscience (Bristol, UK). Cangrelor 30 31 was purchased from Medicines Company. U46619 was obtained from Cayman Chemical. 32 33 34 Chronolume and ATP were from Chrono-log Corporation (Manchester, UK). Horseradish 35 36 peroxidase (HRP)-conjugated secondary antibodies and Enhanced Chemiluminescence (ECL) 37 38 substrate were obtained from Amersham Biosciences (GE Healthcare, Bucks, UK). Oregon 39 40 green 488 BAPTA-1-AM was purchased from Invitrogen (Invitrogen, ThermoFisher 41 42 43 Scientific, Paisley, UK). Other reagents were obtained from Sigma. 44 45 46 Antibodies: PLCγ2 p1217, PLCγ2 p759, Syk p525/526, Syk p323, Syk p352, LAT p132 and 47 48 LAT p171 were from Cell Signalling Technology (Danvers, Massachusetts, United States). 49 50 LAT p200 mAb was obtained from Abcam (Cambridge, UK). Mouse anti-human anti- 51 52 53 phosphotyrosine (clone 4G10) mAb was from Millipore UK Ltd (Watford, UK). Alexa Fluor 54 55 488 phalloidin and Alexa Fluor 647 were purchased from Invitrogen (Invitrogen, 56 57 ThermoFisher Scientific, Paisley, UK). 1G5-Fab recognises monomeric and dimeric GPVI was 58 59 23 60 raised as described .

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1 2 3 Preparation of human washed platelets 4 5 6 Blood was collected from healthy and consenting volunteers by venipuncture in accordance 7 8 9 with the Declaration of Helsinki (local ethical review no: ERN_11-0175). Trisodium citrate [1 10 11 part 3.8% (w/v) stock: 9 parts blood] was used as the anticoagulant. Washed platelets were 12 13 prepared by centrifugation in the presence of prostacyclin (2.8 μM) followed by resuspension 14 15 in Tyrodes-HEPES buffer (134 mM NaCl, 0.34 mM Na HPO , 2.9 mM KCl, 12 mM NaHCO 16 2 4 3 17 24 18 20 mM HEPES, 1 mM MgCl2, 5 mM glucose, pH 7.3) as previously described . Platelet count 19 20 was measured using a Coulter counter and the suspension was diluted to the required 21 For Peer Review 22 concentration in Tyrodes-HEPES buffer. Platelets were then left for 30 min. For aggregation 23 24 8 25 and ATP secretion measurements platelets were used at 2x10 /ml; for western blotting at 26 27 5x108/ml and for spreading at 2x107/ml. 28 29 30 Aggregometry and luminescence measurement of ATP secretion 31 32 33 Aggregation was monitored using a Chrono-Log optical aggregometer (Labmedics, 34 35 Manchester, UK) at 37oC with constant stirring at 1200 rpm. Secretion of ATP was measured 36 37 using luciferin-luciferase Chrono-lume reagent (Chrono-Log). NECA (100 μM), indomethacin 38 39 40 (10 μM), cangrelor (10 μM), forskolin (10 μM), ADP (10 μM) or vehicle were added 60-120 41 42 sec before collagen, CRP or U46619. The platelets were left for 5 min before addition of ATP 43 44 (2 nM) for calibration of ATP secretion. 45 46 47 Platelet lysis and protein phosphorylation 48 49 50 Whole cell lysates were prepared from 400 μl sample of stirred platelet suspensions. 51 52 53 Eptifibatide (9 μM) was added to prevent aggregation. The reaction was terminated by the 54 55 addition of 5x SDS sample buffer (reducing conditions) to the platelets. The samples were heat 56 57 denatured at 100oC for 5 min and spun at 15000 g for 10 min. The whole cell lysate was 58 59 60 electrophoresed on 4-12% BisTris Plus acrylamide Bolt gels (Invitrogen, Paisley, UK) and

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1 2 3 transferred onto PVDF membranes using a Trans-blot Transfer Imaging system (Bio-Rad, 4 5 6 Hertfordshire, UK). Each gel included at least one lane of 10 μl of Color Prestained Protein 7 8 Standard (11–245 kDa; NEB, Ipswich, Massachusetts, United States). Membranes were 9 10 blocked in 4% (w/v) BSA dissolved in TBST and 0.1% (w/v) sodium azide for a minimum of 11 12 1 h at room temperature and incubated overnight at 4oC with anti-phosphotyrosine (clone 13 14 15 4G10; 1:1,000), PLCγ2 (Tyr 1217; 1:250, Tyr 759; 1:1,000), Syk (Tyr 525/526; 1:500, Tyr 16 17 352; 1:1,000, Tyr 323; 1:1,000) and LAT (Tyr 200; 1:500, Tyr 171; 1:1,000, Tyr 132; 1:500) 18 19 primary antibodies. Membranes were imaged using the Licor Odyssey system. Quantification 20 21 For Peer Review 22 of band intensities was performed using Image Studio Lite v5.2 where background correction 23 24 was applied. 25 26 27 Human platelet spreading 28 29 30 Glass coverslips were coated with collagen (10 μg/ml) in manufacturer-supplied diluent 31 32 overnight at 4oC. The coverslips were blocked with 5 mg/ml BSA in PBS (heat denatured, 33 34 35 filter-sterilised) for 1 h at room temperature, then washed with PBS. Washed platelets were 36 37 incubated on collagen for 30 min at 37oC and NECA and forskolin were added and left for an 38 39 additional 30 min. Pre-incubation experiments were conducted where NECA and forskolin 40 41 were incubated with platelets for 30 min and followed by 30 min spreading. Following 42 43 44 spreading the coverslips were washed in PBS and adherent platelets were fixed in 4% (w/v) 45 46 formalin for 10 min at room temperature, permeabilized with 0.1% (v/v) Triton X-100 (in PBS) 47 48 and stained with phalloidin-Alexa Fluor 488. The coverslips were mounted and imaged with 49 50 51 a Zeiss Observer 7 Epifluorescent microscope using a 63x 1.4NA oil immersion lens. Images 52 53 were acquired using Zen Pro v2.3 and processed using FIJI v1.51. Using the open-source 54 55 KNIME software25 platelet segmentation was performed and platelet count and surface area 56 57 26 58 were analysed. An ilastik pixel classifier was used to produce a binary segmentation. To 59 60 separate touching platelets the centre of individual platelets were manually selected using

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1 2 3 KNIME. These centre coordinates were then used as seeds for a watershed transform to 4 5 2 6 produce the final segmentation result. Objects smaller than 1 μm were discarded and platelet 7 8 statistics including platelet area were calculated. 9 10 11 Single cell Ca2+ measurements 12 13 14 For Ca2+ measurements, washed platelets were incubated for 45 min at 37°C with Oregon green 15 16 488 BAPTA-1-AM (1 μM) in Tyrodes-HEPES buffer and centrifuged at 1000 g for 10 min 17 18 with prostacyclin (2.8 μM) and resuspended in Tyrodes-HEPES buffer. Glass bottomed MatTek 19 20 21 dishes (No. 1.5 (0.17 mm)For coverslips) Peer (MatTek, Review Ashland, MA, USA) were prepared as 22 23 previously described27. Washed platelets were prepared and left to rest for 30 min. The platelets 24 25 were diluted to 2x107/ml and prior to exposure to an immobilized collagen surface were 26 27 28 incubated with forskolin or indomethacin and cangrelor or all three inhibitors for 2 min. Real- 29 30 time platelet Ca2+ flux was monitored using a Zeiss Observer 7 Epifluorescent microscope 31 32 using a 63x 1.4NA oil immersion lens, Colibri 7 LED light source, Zeiss Filter set 38 for 33 34 35 GFP/FITC, and Hammamatsu ORCA Flash 4 LT sCMOS camera where images were taken 36 37 every 2 sec for 100 cycles. Images were acquired using Zen Pro v2.3 and processed using FIJI 38 39 v1.51. The number of peaks and peak fluorescence intensity were analyzed in 75 platelets for 40 41 each condition. Ca2+ spikes were identified where an increase in fluorescence intensity greater 42 43 2+ 44 than 100 (a.u.) above baseline was observed. Where prolonged Ca signals were detected, 45 46 peaks were subdivided into multiple peaks when a clear change in direction of the trace was 47 48 observed. 49 50 51 Total internal reflection microscopy (TIRFM)/direct STocastic Optical Reconstruction 52 53 54 Microscopy (dSTORM) 55 56 57 Glass bottomed MatTek dishes (No. 1.5 (0.17 mm) coverslips) were coated with collagen (10 58 59 μg/ml) as previously described27. 1G5 Fab (pan-GPVI)-labelled (2 μg/ml) washed platelets 60

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1 2 3 (2x107/ml) were allowed to adhere and spread for 30 min to the collagen-coated MatTek dishes. 4 5 6 NECA (100 μM) and forskolin (10 μM) were added and platelets were spread for an additional 7 8 30 min. Adherent platelets were fixed in 4% (w/v) formalin for 10 min at room temperature, 9 10 permeabilized with 0.1% (v/v) Triton X-100 (in PBS), blocked for 30 min in 2% (v/v) goat 11 12 serum and 1% BSA (w/v) and then stained with phalloidin-Alexa Fluor 488 and Alexa Fluor 13 14 15 647-conjugated IgG secondary antibodies (diluted 1:300 in block buffer). Adhesion of 1G5 16 17 Fab-labelled washed platelets (2x107/ml) to immobilised collagenous substrate was imaged in 18 19 TIRF using a fully motorised Nikon TIRF combine mounted on a NIKON N-STORM 20 21 For Peer Review 22 microscope on a Ti-E stand equipped with a Nikon 100x 1.49 NA TIRF oil objective, Perfect 23 24 Focus System, Agilent MLC400 laser bed with 405 nm (50 mW*), 488 nm (80 mW*), 561 nm 25 26 (80 mW*) and 640 nm (125 mW*) solid-state lasers and Andor iXon Ultra EM-CCD camera 27 28 27 29 as previously described . Samples were maintained in an OKO environmental chamber at 30 31 28oC for maximum system stability during imaging. All dSTORM experiments were 32 33 performed in TIRF mode on a NIKON N-STORM microscope as described previously27. 34 35 During dSTORM acquisition, the sample was continuously illuminated at 640 nm for 20,000 36 37 38 frames (40 x 40 μm, 9.2 ms exposure time) and the 405 nm laser was used for back pumping. 39 40 Samples were imaged in switching buffer (0.5 mg/ml glucose oxidase, 40 µg/ml catalase, 10% 41 42 (w/v) glucose and 100 mM β-mercaptoethylamine in PBS, pH 7.4) to induce Alexa 647 43 44 28 45 blinking . The 20,000 frames were captured with Nikon NIS ELEMENTS v4.5 and 46 47 reconstructed in ThunderSTORM imageJ plugin29 where approximate molecule positions are 48 49 determined. The settings used were Gaussian PSF modelling and maximum likelihood fitting. 50 51 52 Further post processing analysis include: lateral drift correction, local density filtering where 53 54 the distance ratio was 50 and minimum number of neighbors was 5 and merging of reappearing 55 56 localizations. All detections in sequential within 20 nm were merged. Gaussian rendering was 57 58 59 60

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1 2 3 implemented for visualization. Points in the reconstructed images represent individually 4 5 6 identified fluorescent blinking events, which are referred to as detections. 7 8 9 Cluster analysis 10 11 12 After localising detections, density-based spatial clustering of applications with 13 14 noise (DBSCAN) 30 was used to group detections into clusters and segment clusters of arbitrary 15 16 shape such as on collagen fibres. For DBSCAN the radius of the local neighbourhood was set 17 18 to 50 nm and the minimum number of directly reachable points was set to 10. Edge points were 19 20 21 included in clusters. ClusterFor area wasPeer calculated Review by placing a circle with a radius of 50 nm over 22 23 every detection in a cluster and calculating the union of these circles. This was estimated using 24 25 a grid with a pixel size of 5 nm and image based dilation. Cluster density was defined as the 26 27 28 number of detections within a cluster divided by the cluster area. This analysis was performed 29 30 on whole fields of view using the open-source software KNIME25. DBSCAN was implemented 31 32 within KNIME using the R package ‘dbscan’. 33 34 35 Statistical analysis 36 37 38 Each experiment was performed at least three times and results are shown as mean± SEM. Data 39 40 were analysed using PRISM v7.04 (GraphPad, San Diego, CA.) and statistical analysis was by 41 42 43 one-way ANOVA with a Bonferroni post-hoc test. Significance was set at p ≤ 0.05. 44 45 46 Results 47 48 49 The effect of NECA and inhibitors of feedback agonists on aggregation and secretion 50 51 induced by collagen. 52 53 31 54 We investigated the effect of the A2A receptor agonist NECA (1, 10 and 100 μM) on platelet 55 56 aggregation induced by low (2 μg/ml) and intermediate (10 μg/ml) concentrations of collagen 57 58 59 (Supplementary Fig. 1). Both concentrations of collagen stimulated aggregation after a delay 60

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1 2 3 of approximately 15 sec which reached approximately 95% of the maximal aggregation within 4 5 6 2 min (Supplementary Fig. 1). In the presence of NECA (1-100 μM), aggregation to a low 7 8 concentration of collagen (2 μg/ml) was partially reduced and began to slowly return to the 9 10 resting level. In contrast, NECA (1 μM) had no significant effect on the response to an 11 12 intermediate concentration (10 μg/ml) of collagen whereas the maximal level of aggregation, 13 14 15 but not the time course of response, was partially inhibited in the presence of NECA (10 and 16 17 100 μM). For these reasons, 100 μM NECA was chosen for further experiments. 18 19 20 To investigate whether the mechanism of inhibition of the response to collagen by NECA was 21 For Peer Review 22 due to inhibition of the action of the secondary agonists, ADP and thromboxane A (TxA ), we 23 2 2 24 25 first monitored aggregation and dense granule secretion (ATP release) in the presence of 26 27 maximally effective concentrations of the P2Y12 receptor antagonist, cangrelor (10 μM) and 28 29 the cyclooxygenase inhibitor indomethacin (10 μM). Both agents caused a partial inhibition of 30 31 32 aggregation to collagen (10 μg/ml) which was similar to that induced by NECA (100 μM) but 33 34 neither agent, separately or in combination with NECA had an effect on ATP secretion 35 36 (Supplementary Fig. 2). A slightly greater level of inhibition of secretion was seen when 37 38 cangrelor and indomethacin were used in combination, or when given with NECA, which did 39 40 41 reach statistical significance (Fig. 1A). All three inhibitors blocked aggregation and secretion 42 43 to a lower concentration of collagen (2 μg/ml) (Fig. 1B). The lack of an additional effect of 44 45 NECA in the presence of cangrelor and indomethacin on aggregation and secretion to the 46 47 48 higher concentration of collagen suggests that its inhibitory action is mediated predominantly 49 50 by inhibition of the secondary feedback agonists. 51 52 53 One explanation for the relatively weak effect of NECA on aggregation and secretion to 54 55 collagen (10 μg/ml) is that it induces only a small increase in cAMP. To address this, we used 56 57 58 forskolin which induces powerful activation of adenylyl cyclase and therefore a much larger 59 60 increase in cAMP (Supplementary Fig. 3A). Forskolin induced a similar effect to the

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1 2 3 combination of cangrelor and indomethacin on collagen induced aggregation and ATP 4 5 6 secretion (Fig. 1C). The combination of all three agents caused a slightly greater level of 7 8 inhibition of aggregation and ATP secretion to that seen with NECA, cangrelor and 9 10 indomethacin. 11 12 13 To investigate whether the inhibitory effect of NECA was due to blockade of signalling by 14 15 GPVI, we investigated the effect on aggregation and secretion induced by the GPVI-specific 16 17 18 ligand, CRP. NECA or the combination of indomethacin and cangrelor has no significant effect 19 20 on platelet aggregation to a high concentration of CRP (10 μg/ml), although the combination 21 For Peer Review 22 of indomethacin and cangrelor partially reduced secretion (Fig. 2A). Surprisingly however, 23 24 25 both responses were markedly inhibited in the presence of all three inhibitors. In contrast, 26 27 NECA alone and the combination of indomethacin and cangrelor blocked the response to a 28 29 lower concentration of CRP (1 μg/ml) (Fig. 2B). This demonstrates that NECA is able to inhibit 30 31 32 aggregation and secretion to CRP through a pathway that is unmasked by the absence of 33 34 signalling by secondary agonists. 35 36 37 We extended the studies on secretion to CRP (10 μg/ml) to measure the α-granule marker P- 38 39 selectin (Supplementary Fig 3B). CRP causes a significant increase in P-selectin exposure 40 41 which is reduced in the presence of NECA and forskolin. 42 43 44 Together these results show that NECA and forskolin block the response to threshold activation 45 46 47 of GPVI in the presence of inhibition of the feedback agonists TxA2 and ADP. The inhibitory 48 49 effect of NECA is largely overcome at higher concentrations of collagen but not CRP. 50 51 52 The effect of NECA and forskolin on collagen induced protein tyrosine phosphorylation 53 54 55 To investigate the mechanism underlying the inhibitory effect of the adenosine A2A receptor 56 57 58 agonist NECA, we measured phosphorylation of key signalling proteins in the GPVI pathway 59 60 by western blotting using phosphospecific antibodies. We chose to focus on three proteins that

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1 2 3 are phosphorylated at multiple sites and which play key roles in GPVI signalling, namely Syk, 4 5 6 LAT and PLC2. We elected to use forskolin in the phosphorylation studies because it induces 7 8 a greater increase in cAMP (Supplementary Fig. 3A). Phosphorylation was measured in the 9 10 presence of maximally-effective concentrations of indomethacin and apyrase, inhibitors of the 11 12 13 TxA2 and ADP feedback pathways, respectively. Incubation times used are in-line with 14 15 previously published work18, 32. Strikingly, forskolin caused a minimal reduction in tyrosine 16 17 phosphorylation of Syk at Y323 (docking), Y352 (docking) and Y525/526 (activation), LAT 18 19 20 at Y132 (docking) and Y200 (docking), and PLC2 at Y759 (activation and docking) and 21 For Peer Review 22 Y1217 (activation) (Fig. 3ii). Similarly, NECA had no significant effect on phosphorylation of 23 24 Syk, LAT and PLC2 at Y525/526, Y200 and Y1217, respectively (Supplementary Fig. 4). 25 26 27 These results demonstrate that neither elevation of cAMP via stimulation of the adenosine A2A 28 29 receptor or using the adenylyl cyclase activator, forskolin had a major effect on tyrosine kinase 30 31 signalling downstream of platelet activation by collagen. 32 33 34 Adenosine and forskolin have no effect on platelet spreading or Ca2+ elevation induced 35 36 37 by collagen 38 39 40 The adhesion of platelets to a collagen surface leads to a rapid reorganization of the actin 41 42 cytoskeleton resulting in formation of filopodia and eventually lamellipodia and stress fibres. 43 44 Addition of NECA or forskolin to platelets that had been incubated a collagen-coated surface 45 46 for 30 min had no significant effect on the degree of adhesion or platelet morphology as shown 47 48 49 by measurement of surface area (Fig. 4A). A similar result was seen when NECA and forskolin 50 51 were given prior to the onset of spreading (Fig. 4B) although the degree of platelet adhesion 52 53 was markedly reduced. This suggests that adenosine and forskolin have an inhibitory effect 54 55 56 prior to spreading but this is lost once platelet spreading and collagen signalling has begun. 57 58 59 60

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1 2 3 To investigate if cAMP is inhibiting Ca2+ mobilization following collagen stimulation, we 4 5 2+ 6 measured the frequency and fluorescence intensity of Ca spikes in platelets spread on a 7 8 collagen surface following treatment with forskolin, indomethacin and cangrelor. We elected 9 10 to use forskolin in the single cell Ca2+ studies because it induces a greater increase in cAMP 11 12 (Supplementary Fig. 3A). Forskolin either alone or in combination with indomethacin and 13 14 15 cangrelor had no effect on the frequency of Ca2+ spikes or the fluorescence intensity of these 16 17 spikes (Fig. 5). This suggests that Ca2+ elevation induced by collagen is not affected by 18 19 elevation of cAMP. 20 21 For Peer Review 22 NECA and forskolin do not change GPVI receptor clustering 23 24 25 To determine whether NECA or forskolin had an effect on endogenous GPVI clusters which 26 27 28 form in platelets spreading on immobilized collagen, single molecule super resolution 29 30 microscopy (dSTORM) was used. GPVI was labelled using a Fab fragment of the monoclonal 31 32 antibody to pan-GPVI, 1G523. The platelets were allowed to adhere to collagen for 30 min 33 34 35 before being treated with NECA or forskolin for a further 30 min to determine whether GPVI 36 37 distribution was affected. Diffraction limited TIRF microscopy showed a characteristic GPVI 38 39 distribution with enrichment of GPVI along collagen fibres, as has been shown previously27. 40 41 dSTORM images show the localization of single molecule detections and the DBSCAN cluster 42 43 44 plot shows clustering of the detections in the image where different clusters are represented by 45 46 different colors. The largest clusters of GPVI can be seen along the collagen fibres. Results 47 48 from the quantitative cluster analysis (Fig. 6Bi-iv) show that neither NECA nor forskolin 49 50 51 altered GPVI clustering, with the number of clusters, number of detections per cluster and 52 53 cluster area and density all similar to control values. 54 55 56 NECA and forskolin inhibit TxA2 induced platelet aggregation 57 58 59 60

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1 2 3 The effect of NECA and forskolin on TxB2 formation and platelet aggregation induced by the 4 5 6 TxA2 mimetic, U46619, was investigated to determine the basis of loss of platelet activation. 7 8 The thromboxane A2 (TP) receptor agonist U46619 caused ~60% maximal platelet 9 10 aggregation. However, both NECA and forskolin caused a significant reduction in U46619 11 12 induced platelet aggregation (Fig. 7A) with NECA reducing aggregation to ~10% and forskolin 13 14 15 causing complete blockade. This suggests that cAMP is inhibiting thromboxane receptor 16 17 signalling. In addition both NECA and forskolin also partially decreased TxB2 formation by 18 19 collagen (Supplementary Fig. 3C) demonstrating that elevation of cAMP causes inhibition both 20 21 For Peer Review 22 upstream and downstream of TxA2. 23 24 25 ADP does not cause aggregation in washed platelets due to desensitisation of the P2Y1 receptor 26 27 which prevents synergy with the P2Y12 receptor. To investigate the effect of NECA on the 28 29 response to P2Y12 receptor activation, we monitored platelet aggregation induced by a low 30 31 32 concentration of collagen (2 μg/ml) in the absence and presence of ADP (Fig. 7C&D). On its 33 34 own, ADP had no effect on aggregation. NECA inhibited the response to the low dose of 35 36 collagen as described in Fig.1, but had no effect on the response to collagen in the presence of 37 38 ADP (Fig. 7C&D). This demonstrates that NECA has a greater effect on the TxA arm of the 39 2 40 41 feedback agonists. 42 43 44 Discussion 45 46 47 In the present study, we show that signalling via the adenosine A2A receptor and stimulation of 48 49 adenylyl cyclase blocked platelet aggregation and ATP secretion induced by low 50 51 concentrations of collagen but only had a partial effect against higher concentrations and 52 53 54 present evidence that this is mediated in part by inhibition of TxA2 receptor signalling. 55 56 Adenosine also blocked platelet aggregation and ATP secretion by a low concentration of the 57 58 GPVI-selective agonist CRP, but only inhibited the response to a higher concentration in the 59 60

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1 2 3 presence of the combination of a P2Y12 antagonist and cyclooxygenase inhibitor. Together 4 5 6 these results show that platelet activation induced by high concentrations of collagen and the 7 8 GPVI-specific CRP is relatively refractory to the inhibitory adenosine A2A receptor unless 9 10 inhibitors of the feedback agonists ADP and TxA2 are also present. 11 12 13 The observation that adenosine or the more powerful inhibitor forskolin, which induces a 14 15 greater increase in cAMP, had no effect on clustering of GPVI, tyrosine phosphorylation of 16 17 18 Syk, LAT and PLC2, elevation of Ca2+ and spreading of platelets on collagen demonstrates 19 20 that elevation of cAMP has a minimal effect on the proximal signalling events by GPVI. This 21 For Peer Review 22 23 therefore explains why collagen in able to initiate platelet activation at sites of lesion in the 24 25 vessel wall despite the increase in cAMP. The inhibitory effect of NECA against lower 26 27 concentrations of collagen is therefore mediated downstream of Ca2+ signalling, including 28 29 inhibition of the formation and action of TxA and secretion of ADP as shown. 30 2 31 32 Loyau et al21 have proposed that elevation of cAMP maintains GPVI in a monomeric form as 33 34 35 shown using the mAb 9E18 which recognises dimeric but not monomeric GPVI. This suggests 36 37 that elevation of cAMP should induce powerful inhibition of platelet activation by collagen as 38 39 this binds selectively to the dimeric form of GPVI12. The observation that the proximal events 40 41 42 in GPVI signalling are not altered by NECA or by forskolin therefore suggests the effect of 43 44 cAMP on 9E18 binding may not be directly related to GPVI/FcR-dependent signalling. 45 46 Takayama et al22 reported that elevation of cAMP leads to internalisation of GPVI in human 47 48 49 platelets, but the lack of effect on signalling indicates that this is a relatively slow event. Taken 50 51 together, our data suggests that the inhibitory effect of cAMP occurs downstream of GPVI 52 53 signalling, including inhibition of the feedback agonist TxA2 and possibly via additional effects 54 55 3, 33, 34 56 on integrin IIb3 activation and secretion . 57 58 59 60

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1 2 3 Adenosine has been previously reported to inhibit platelet activation by a number of G protein- 4 5 13, 14, 31, 35 6 coupled receptor agonists including thrombin, ADP and TxA2 . In all cases, inhibition 7 8 was mediated by loss of PLC activation in combination with elevation of cGMP and inhibition 9 10 of IP -induced Ca2+ release via the PKG-IRAG-IP receptor complex. In our study, single cell 11 3 3 12 2+ 13 Ca measurements during collagen induced signalling in the presence of forskolin however 14 15 revealed no effect on Ca2+ release signalling, questioning the significance of this mechanism. 16 17 18 In conclusion, the observation that the proximal events in collagen-mediated signalling 19 20 including Ca2+ flux are not altered by elevation of cAMP with adenosine or forskolin is 21 For Peer Review 22 23 consistent with a critical role for collagen/GPVI in mediating platelet activation at sites of 24 25 injury to the vasculature, despite the presence of the cAMP-elevating agent, prostacyclin. 26 27 Inhibition of platelet activation by adenosine and forskolin is overcome at higher 28 29 concentrations of collagen which mimics the situation in the vessel wall. In addition, these 30 31 32 results may have important implications for the therapeutic action of the P2Y12 inhibitor 33 34 ticagrelor which also inhibits adenosine uptake, and this would further inhibit platelet 35 36 signalling notably by low concentrations of collagen. Similar interactions may benefit the 37 38 39 therapeutic action of the clinically-used PDE inhibitors such as cilostazol and dipyridamole. 40 41 42 Authors’ Contributions: J. C. Clark has performed experiments, analysed data, wrote and 43 44 edited manuscript. S. Watson performed experiments and analysed data. D. M. Kavanagh 45 46 provided training in super resolution microscopy experiments and expertise for the study 47 48 design and cluster analysis. J. A. Pike has analysed data and developed the programmes for 49 50 51 image and cluster analysis. N. S. Poulter provided expertise for super resolution microscopy 52 53 experiment study design and cluster analysis. R. K. Andrews and E. E. Gardiner provided key 54 55 materials. S. J. Hill provided supervision, concept, funding and contributed to manuscript 56 57 58 editing. S. P. Watson provided supervision, funding, study design and concept, reviewed data, 59 60 wrote and edited manuscript. All authors have read the manuscript.

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1 2 3 Acknowledgements: This study was supported by a grant from the Centre of Membrane and 4 5 6 Protein and Receptors, University of Birmingham and University of Nottingham, Midlands, 7 8 UK). SPW is a British Heart Foundation Professor (CH 03/003). We thank Steve Thomas and 9 10 Chiara Pallini, for help with the Ca2+ measurements and dSTORM experiments, respectively. 11 12 13 Disclosure of Conflict of Interests: The authors declare no conflict of interest 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 References 4 5 6 1Borst S, Sim X, Poncz M, French DL, Gadue P. Induced Pluripotent Stem Cell-Derived 7 8 Megakaryocytes and Platelets for Disease Modeling and Future Clinical Applications. Arterioscler 9 10 Thromb Vasc Biol. 2017; 37: 2007-13. 11 12 2Siess W. Cross-talk of cGMP- and cAMP-signaling pathways in human platelets. Blood. 2003; 101: 13 14 4230. 15 16 3 17 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol. 18 19 2001; 62: 1153-61. 20 21 4Eigenthaler M, Nolte C, HalbruggeFor M,Peer Walter U. Review Concentration and regulation of cyclic nucleotides, 22 23 cyclic-nucleotide-dependent protein kinases and one of their major substrates in human platelets. 24 25 Estimating the rate of cAMP-regulated and cGMP-regulated protein phosphorylation in intact cells. Eur 26 27 J Biochem. 1992; 205: 471-81. 28 29 5Geiger J, Nolte C, Walter U. Regulation of calcium mobilization and entry in human platelets by 30 31 endothelium-derived factors. Am J Physiol Cell Physiol. 1994; 267: C236-44. 32 33 6Butt E, Immler D, Meyer HE, Kotlyarov A, Laass K, Gaestel M. Heat shock protein 27 is a substrate 34 35 36 of cGMP-dependent protein kinase in intact human platelets: phosphorylation-induced actin 37 38 polymerization caused by HSP27 mutants. J Biol Chem. 2001; 276: 7108-13. 39 40 7Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, Illenberger S. Phosphorylation of the vasodilator- 41 42 stimulated phosphoprotein regulates its interaction with actin. J Biol Chem. 2000; 275: 30817-25. 43 44 8Hettasch JM, Sellers JR. Caldesmon phosphorylation in intact human platelets by cAMP-dependent 45 46 protein kinase and protein kinase C. J Biol Chem. 1991; 266: 11876-81. 47 48 9Bennett JS, Zigmond S, Vilaire G, Cunningham ME, Bednar B. The platelet cytoskeleton regulates the 49 50 affinity of the integrin alpha(IIb)beta(3) for fibrinogen. J Biol Chem. 1999; 274: 25301-7 51 52 10Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor? Blood. 2003; 53 54 55 102: 449-61. 56 57 58 59 60

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1 2 3 11Watson SP, Auger JM, McCarty OJ, Pearce AC. GPVI and integrin alphaIIb beta3 signaling in 4 5 platelets. J Thromb Haemost 2005; 3: 1752-62. 6 7 12Miura Y, Takahashi T, Jung SM, Moroi M. Analysis of the interaction of platelet collagen receptor 8 9 10 glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows 11 12 affinity to fibrous collagen. J Biol Chem. 2002; 277: 46197-204. 13 14 13Cooper JA, Hill SJ, Alexander SP, Rubin PC, Horn EH. Adenosine receptor-induced cyclic AMP 15 16 generation and inhibition of 5-hydroxytryptamine release in human platelets. Br J Clin Pharmacol. 17 18 1995; 40: 43-50. 19 20 14Fuentes E, Pereira J, Mezzano D, Alarcón M, Caballero J, Palomo I. Inhibition of platelet activation 21 For Peer Review 22 and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular 23 24 modeling. PLOS ONE. 2014; 9: e112741. 25 26 15Amisten S, Braun OO, Bengtsson A, Erlinge D. Gene expression profiling for the identification of G- 27 28 protein coupled receptors in human platelets. Thromb Res. 2008; 122: 47-57. 29 30 31 16Cattaneo M, Schulz R, Nylander S. Adenosine-mediated effects of ticagrelor: evidence and potential 32 33 clinical relevance. J Am Coll Cardiol 2014; 63: 2503-9. 34 35 17Smith JB, Dangelmaier C, Daniel JL. Elevation of cAMP in human platelets inhibits thrombin- but 36 37 38 not collagen-induced tyrosine phosphorylation. Biochem Biophys Res Commun. 1993; 191: 695-700. 39 18 40 Smith JB, Dangelmaier C, Selak MA, Ashby B, Daniel J. Cyclic AMP does not inhibit collagen- 41 42 induced platelet signal transduction. Biochem J. 1992; 283: 889-92. 43 44 19Ichinohe T, Takayama H, Ezumi Y, Yanagi S, Yamamura H, Okuma M. Cyclic AMP-insensitive 45 46 activation of c-Src and Syk protein-tyrosine kinases through platelet membrane glycoprotein VI. J Biol 47 48 Chem. 1995; 270: 28029-36. 49 50 20Riondino S, Lotti LV, Cutini L, Pulcinelli FM. Collagen-induced Platelet Shape Change Is Not 51 52 Affected by Positive Feedback Pathway Inhibitors and cAMP-elevating Agents. J Biol Chem. 2005; 53 54 280: 6504-10. 55 56 57 58 59 60

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1 2 3 21Loyau S, Dumont B, Ollivier V, Boulaftali Y, Feldman L, Ajzenberg N, et al. Platelet glycoprotein 4 5 VI dimerization, an active process inducing receptor competence, is an indicator of platelet reactivity. 6 7 Arterioscler Thromb Vasc Biol. 2012; 32: 778-85. 8

9 22 10 Takayama H, Hosaka Y, Nakayama K, Shirakawa K, Naitoh K, Matsusue T, et al. A novel antiplatelet 11 12 antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain 13 14 complex. J Clin Invest. 2008; 118: 1785-95. 15 16 23Al-Tamimi M, Mu F-T, Arthur JF, Shen Y, Moroi M, Berndt MC, et al. Anti-glycoprotein VI 17 18 monoclonal antibodies directly aggregate platelets independently of FcγRIIa and induce GPVI 19 20 ectodomain shedding. Platelets. 2009; 20: 75-82. 21 For Peer Review 22 24Grygielska B, Hughes CE, Watson SP. Molecular basis of platelet activation by an alphaIIbbeta3- 23 24 CHAMPS peptide. J Thromb Haemost. 2009; 7: 339-46. 25 26 25Berthold M, Cebron N, Dill F, Gabriel T, Kötter T, Meinl T, et al. KNIME - The Konstanz information 27 28 miner : Version 2.0 and Beyond 2009. 26-31 p. 29 30 26 31 Sommer C, Straehle C, Köthe U, Hamprecht FA, editors. Ilastik: Interactive learning and 32 33 segmentation toolkit. 2011 IEEE International Symposium on Biomedical Imaging: From Nano to 34 35 Macro; pp 230-233. 36 37 27Poulter NS, Pollitt AY, Owen DM, Gardiner EE, Andrews RK, Shimizu H, et al. Clustering of 38 39 glycoprotein VI (GPVI) dimers upon adhesion to collagen as a mechanism to regulate GPVI signaling 40 41 in platelets. J Thromb Haemost. 2017; 15: 549-64. 42 43 28van de Linde S, Loschberger A, Klein T, Heidbreder M, Wolter S, Heilemann M, et al. Direct 44 45 stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc. 2011; 6: 991- 46 47 1009. 48 49 29 50 Ovesný M, Křížek P, Borkovec J, Švindrych Z, Hagen GM. ThunderSTORM: a comprehensive 51 52 ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics. 53 54 2014; 30: 2389-90. 55 56 30Ester M, Kriegel H-P, Sander r, Xu X. A density-based algorithm for discovering clusters a density- 57 58 based algorithm for discovering clusters in large spatial databases with noise. Proceedings of the 59 60

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1 2 3 Second International Conference on Knowledge Discovery and Data Mining; Portland, Oregon. 4 5 3001507: AAAI Press; 1996. p. 226-31. 6 7 31Cusack NJ, Hourani SM. 5'-N-ethylcarboxamidoadenosine: a potent inhibitor of human platelet 8 9 10 aggregation. Br J Pharmcol. 1981; 72: 443-7. 11 32 12 Adnot S, Desmier M, Ferry N, Hanoune J, Sevenet T. Forskolin (a powerful inhibitor of human 13 14 platelet aggregation). Biochem Pharmacol. 1982; 31: 4071-4 15 16 33Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost. 2012; 17 18 10: 167-76. 19 20 34Kroll MH, Schafer AI. Biochemical mechanisms of platelet activation. Blood. 1989; 74: 1181-95. 21 For Peer Review 22 35Quattrin S, Genovese A, Cirillo R, Formisano S, Marone G. Functional and biochemical evidence of 23 24 a specific adenosine A2/Ra receptor on human platelets. La Ricerca in clinica e in laboratorio. 1988; 25 26 18: 105-18. 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Figure Captions 4 5 6 7 Fig. 1 The effect of NECA, forskolin, indomethacin (Indo) and cangrelor (Cang) on collagen 8 9 induced platelet aggregation and ATP secretion. Platelet aggregation induced by (A, C) intermediate 10 11 (10 μg/ml) and (B) low (2 μg/ml) concentrations of collagen was monitored by light transmission 12 13 aggregometry at 37oC with constant stirring at 1200 rpm. Secretion of ATP was measured using 14 15 luciferin-luciferase Chromo-lume reagent. The effect of NECA (100 μM), indomethacin (10 μM) and 16 17 cangrelor (10 μM) on (Ai) intermediate (10 μg/ml) and (Bi) low (2 μg/ml) concentration collagen 18 19 induced aggregation. The effect of NECA (100 μM), indomethacin (10 μM) and cangrelor (10 μM) on 20 21 For Peer Review 22 (Aii) intermediate (10 μg/ml) and (Bii) low (2 μg/ml) concentration collagen induced ATP secretion. 23 24 Representative (Aiii) aggregation and (Aiv) secretion traces showing the effect of the inhibitors on 25 26 intermediate concentration (10 μg/ml) collagen induced platelet aggregation and ATP secretion. 27 28 Representative (Biii) aggregation and (Biv) secretion traces showing the effect of the inhibitors on low 29 30 concentration (2 μg/ml) collagen induced platelet aggregation and ATP secretion. (C) The effect of 31 32 forskolin (10 μM), indomethacin (10 μM) and cangrelor (10 μM) on intermediate (10 μg/ml) 33 34 concentration collagen induced aggregation and ATP secretion. Representative (Ciii) aggregation and 35 36 (Civ) secretion traces showing the effect of the inhibitors on intermediate concentration (10 μg/ml) 37 38 collagen induced platelet aggregation and ATP secretion. Significance was measured using one-way 39 40 41 ANOVA with a Bonferroni post-hoc test where, P<0.05. Data presented as mean±SEM (n=6). 42 43 Fig. 2 The effect of NECA, indomethacin (Indo) and cangrelor (Cang) on collagen-related-peptide 44 45 (CRP) induced platelet aggregation and ATP secretion. Platelet aggregation induced by (A) high 46 47 48 (10 μg/ml) and (B) low (1 μg/ml) concentrations of CRP was monitored by light transmission 49 o 50 aggregometry at 37 C with constant stirring at 1200 rpm. Secretion of ATP was measured using 51 52 luciferin-luciferase Chromo-lume reagent. The effect of NECA (100 μM), indomethacin (10 μM) and 53 54 cangrelor (10 μM) on (Ai) high (10 μg/ml) and (Bi) low (1 μg/ml) concentration CRP induced 55 56 aggregation. The effect of NECA (100 μM), indomethacin (10 μM) and cangrelor (10 μM) on (Aii) 57 58 high (10 μg/ml) and (Bii) low (1 μg/ml) concentration CRP induced ATP secretion. Representative 59 60 (Aiii) aggregation and (Aiv) secretion traces showing the effect of the inhibitors on high concentration

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1 2 3 (10 μg/ml) CRP induced platelet aggregation and ATP secretion. Representative (Biii) aggregation and 4 5 (Biv) secretion traces showing the effect of the inhibitors on low (1 μg/ml) concentration CRP induced 6 7 platelet aggregation and ATP secretion. Significance was measured using one-way ANOVA with a 8 9 10 Bonferroni post-hoc test where: P<0.05. Data presented as mean±SEM (n=3-6). 11 12 Fig. 3 The effect of forskolin on collagen induced PLCγ2, Syk and LAT phosphorylation. The 13 14 15 effect of forskolin (10 μM) in the presence of maximally-effective concentrations of indomethacin (10 16 17 μM) and apyrase (10 μM) on collagen induced (10 μg/ml) phosphorylation investigated by western 18 19 blotting with (i) the phosphotyrosine antibody 4G10 and (ii) phosphospecific antibodies to PLCγ2, Syk 20 21 and LAT. (iii) QuantificationFor of band Peer intensities where Review the blue line represents control and the red line 22 23 represents phosphorylation in the presence of forskolin. Data presented as mean±SEM (n=5). 24 25 26 Fig. 4 NECA and forskolin have no significant effect on platelet spreading on collagen. (A) 27 28 Washed platelets (2x107/ml) were spread on a collagen-coated surface for 30 min at 37oC and then 29 30 NECA (100 μM) and forskolin (10 μM) were added and platelets spread for an additional 30 min. Five 31 32 fields of view per treatment per experiment were captured using epifluorescence microscopy and 33 34 analysed with KMINE software (Field of view = 211 μm x 211 μm). (Ai) Representative zoomed-in 35 36 images of platelet spreading with the indicated treatments. (Field of view= 94 μm x 94 μm) (Scale bar: 37 38 5 μm). Quantification of (Aii) surface area and (Aiii) platelet adhesion. (B) Washed platelets (2x107/ml) 39 40 41 were pre-incubated with NECA (100 μM) and forskolin (10 μM) for 30 min followed by spreading on 42 o 43 a collagen-coated surface for 30 min at 37 C. Five fields of view per treatment per experiment were 44 45 captured using epifluorescence microscopy and analysed with KMINE software (Field of view = 211 46 47 μm x 211 μm). (Bi) Representative zoomed-in images of platelet spreading with the indicated 48 49 treatments. (Field of view= 94 μm x 94 μm) (Scale bar: 5 μm). Quantification of (Bii) surface area and 50 51 (Biii) platelet adhesion. Significance was measured using one-way ANOVA with a Bonferroni post- 52 53 hoc test where: P<0.05. Data presented as mean±SEM (n=3). 54 55 56 Fig. 5 Forskolin, indomethacin and cangrelor have no significant effect on collagen induced Ca2+ 57 58 mobilisation. Washed platelets were loaded with Oregon green 488 BAPTA-1-AM and diluted to 59 60 2x107/ml. Prior to spreading the platelets were incubated with forskolin (10 μM) or indomethacin (10

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1 2 3 μM) and cangrelor (10 μM) or all three inhibitors for 2 min and then spread on a collagen-coated surface 4 5 for 20 min at 37°C. Real-time platelet Ca2+ flux was monitored using epifluorescence microscopy for 6 7 200 sec with images taken every 2 sec for 100 cycles. Five fields of view per treatment per experiment 8 9 10 were captured and (A) the number of peaks and (B) peak fluorescence intensity were analysed in 75 11 2+ 12 platelets for each condition. (C) Representative Ca traces in a single platelet for (i) control, (ii) 13 14 forskolin, (iii) indomethacin and cangrelor and (iv) forskolin, indomethacin and cangrelor. Data 15 16 presented as mean±SEM (n=3). 17 18 19 Fig. 6 NECA and forskolin do not change GPVI receptor clustering on collagen. Washed platelets 20 21 (2x107/ml) were incubated Forwith 1G5-Fab Peer and allowed Review to spread on collagen for 30 min at 37oC. NECA 22 23 (100 μM) and forskolin (10 μM) were then added to the platelet suspensions and platelets were spread 24 25 for an additional 30 min. All STORM experiments were performed on a NIKON N-STORM 26 27 microscope. Seven fields of view per treatment per experiment were captured and the 20,000 frames 28 29 captured with Nikon NIS ELEMENTS v4.5 were reconstructed in ThunderSTORM imageJ plugin. 30 31 Cluster analysis was performed in KMINE software with an algorithm based on DBSCAN with an 32 33 additional setting based on image dilation. (A) Representative TIRF and reconstructed STORM images 34 35 of platelet spreading and detections with the indicated treatments. Cluster plots produced in MATLAB 36 37 38 show the clusters, each represented by a different arbitrary colour. (Field of view = 40 x 40 μm) (scale 39 40 bar: 2 μm). (B) Relative quantification of (i) number of clusters, (ii) number of detections per cluster 41 42 and (iii) cluster area and (iv) density from seven fields of view per treatment per experiment. Data 43 44 presented as mean±SEM (n=3). 45 46 47 Fig. 7 The effect of NECA and forskolin on U46619 induced platelet aggregation and the effect of 48 49 ADP in combination with NECA on collagen induced platelet aggregation. Platelet aggregation was 50 51 monitored by light transmission aggregometry at 37oC with constant stirring at 1200 rpm. (A) The effect 52 53 of the inhibitors: NECA (100 μM) and forskolin (10 μM) on U46619 (3 μM) induced aggregation. (B) 54 55 Representative aggregation traces showing the effect of the inhibitors on platelet aggregation. (C) The 56 57 effect of NECA (100 μM) alone, ADP (10 μM) alone and in the presence of NECA on collagen induced 58 59 aggregation. (D) Representative aggregation traces showing the effect of ADP, NECA, and both in 60

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1 2 3 combination on platelet aggregation. Trace showing ADP alone does not cause aggregation of washed 4 5 platelets. Significance was measured using one-way ANOVA with a Bonferroni post-hoc test where 6 7 P<0.05. Data presented as mean±SEM (n=3-6). 8 9 10 11 12 13 14 15 16 17 18 19 20 21 For Peer Review 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 1 The effect of NECA, forskolin, indomethacin (Indo) and cangrelor (Cang) on collagen induced platelet 46 aggregation and ATP secretion. Platelet aggregation induced by (A, C) intermediate (10 μg/ml) and (B) low 47 (2 μg/ml) concentrations of collagen was monitored by light transmission aggregometry at 37oC with 48 constant stirring at 1200 rpm. Secretion of ATP was measured using luciferin-luciferase Chromo-lume 49 reagent. The effect of NECA (100 μM), indomethacin (10 μM) and cangrelor (10 μM) on (Ai) intermediate (10 μg/ml) and (Bi) low (2 μg/ml) concentration collagen induced aggregation. The effect of NECA (100 μM), 50 indomethacin (10 μM) and cangrelor (10 μM) on (Aii) intermediate (10 μg/ml) and (Bii) low (2 μg/ml) 51 concentration collagen induced ATP secretion. Representative (Aiii) aggregation and (Aiv) secretion traces 52 showing the effect of the inhibitors on intermediate concentration (10 μg/ml) collagen induced platelet 53 aggregation and ATP secretion. Representative (Biii) aggregation and (Biv) secretion traces showing the 54 effect of the inhibitors on low concentration (2 μg/ml) collagen induced platelet aggregation and ATP 55 secretion. (C) The effect of forskolin (10 μM), indomethacin (10 μM) and cangrelor (10 μM) on intermediate (10 μg/ml) concentration collagen induced aggregation and ATP secretion. Representative (Ciii) aggregation 56 57 58 59 60 Thrombosis and Haemostasis Page 28 of 33

1 2 3 and (Civ) secretion traces showing the effect of the inhibitors on intermediate concentration (10 μg/ml) 4 collagen induced platelet aggregation and ATP secretion. Significance was measured using one-way ANOVA 5 with a Bonferroni post-hoc test where, P<0.05. Data presented as mean±SEM (n=6). 6 7 171x342mm (120 x 120 DPI) 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 33 Thrombosis and Haemostasis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 2 The effect of NECA, indomethacin (Indo) and cangrelor (Cang) on collagen-related-peptide (CRP) 46 induced platelet aggregation and ATP secretion. Platelet aggregation induced by (A) high (10 μg/ml) and (B) 47 low (1 μg/ml) concentrations of CRP was monitored by light transmission aggregometry at 37oC with 48 constant stirring at 1200 rpm. Secretion of ATP was measured using luciferin-luciferase Chromo-lume 49 reagent. The effect of NECA (100 μM), indomethacin (10 μM) and cangrelor (10 μM) on (Ai) high (10 μg/ml) and (Bi) low (1 μg/ml) concentration CRP induced aggregation. The effect of NECA (100 μM), indomethacin 50 (10 μM) and cangrelor (10 μM) on (Aii) high (10 μg/ml) and (Bii) low (1 μg/ml) concentration CRP induced 51 ATP secretion. Representative (Aiii) aggregation and (Aiv) secretion traces showing the effect of the 52 inhibitors on high concentration (10 μg/ml) CRP induced platelet aggregation and ATP secretion. 53 Representative (Biii) aggregation and (Biv) secretion traces showing the effect of the inhibitors on low (1 54 μg/ml) concentration CRP induced platelet aggregation and ATP secretion. Significance was measured using 55 one-way ANOVA with a Bonferroni post-hoc test where: P<0.05. Data presented as mean±SEM (n=3-6). 56 57 58 59 60 Thrombosis and Haemostasis Page 30 of 33

1 2 3 216x297mm (120 x 120 DPI) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 31 of 33 Thrombosis and Haemostasis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 3 The effect of forskolin on collagen induced PLCγ2, Syk and LAT phosphorylation. The effect of 46 forskolin (10 μM) in the presence of maximally-effective concentrations of indomethacin (10 μM) and 47 apyrase (10 μM) on collagen induced (10 μg/ml) phosphorylation investigated by western blotting with (i) the phosphotyrosine antibody 4G10 and (ii) phosphospecific antibodies to PLCγ2, Syk and LAT. (iii) 48 Quantification of band intensities where the blue line represents control and the red line represents 49 phosphorylation in the presence of forskolin. Data presented as mean±SEM (n=5). 50 51 207x272mm (120 x 120 DPI) 52 53 54 55 56 57 58 59 60 Thrombosis and Haemostasis Page 32 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 4 NECA and forskolin have no significant effect on platelet spreading on collagen. (A) Washed platelets 46 (2x107/ml) were spread on a collagen-coated surface for 30 min at 37oC and then NECA (100 μM) and 47 forskolin (10 μM) were added and platelets spread for an additional 30 min. Five fields of view per treatment 48 per experiment were captured using epifluorescence microscopy and analysed with KMINE software (Field of 49 view = 211 μm x 211 μm). (Ai) Representative zoomed-in images of platelet spreading with the indicated treatments. (Field of view= 94 μm x 94 μm) (Scale bar: 5 μm). Quantification of (Aii) surface area and 50 (Aiii) platelet adhesion. (B) Washed platelets (2x107/ml) were pre-incubated with NECA (100 μM) and 51 forskolin (10 μM) for 30 min followed by spreading on a collagen-coated surface for 30 min at 37oC. Five 52 fields of view per treatment per experiment were captured using epifluorescence microscopy and analysed 53 with KMINE software (Field of view = 211 μm x 211 μm). (Bi) Representative zoomed-in images of platelet 54 spreading with the indicated treatments. (Field of view= 94 μm x 94 μm) (Scale bar: 5 μm). Quantification 55 of (Bii) surface area and (Biii) platelet adhesion. Significance was measured using one-way ANOVA with a 56 Bonferroni post-hoc test where: P<0.05. Data presented as mean±SEM (n=3). 57 58 59 60 Page 33 of 33 Thrombosis and Haemostasis

1 2 3 4 165x212mm (120 x 120 DPI) 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Thrombosis and Haemostasis Page 34 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Fig. 5 Forskolin, indomethacin and cangrelor have no significant effect on collagen induced Ca2+ 41 mobilisation. Washed platelets were loaded with Oregon green 488 BAPTA-1-AM and diluted to 2x107/ml. 42 Prior to spreading the platelets were incubated with forskolin (10 μM) or indomethacin (10 μM) and 43 cangrelor (10 μM) or all three inhibitors for 2 min and then spread on a collagen-coated surface for 20 min 2+ 44 at 37°C. Real-time platelet Ca flux was monitored using epifluorescence microscopy for 200 sec with 45 images taken every 2 sec for 100 cycles. Five fields of view per treatment per experiment were captured and (A) the number of peaks and (B) peak fluorescence intensity were analysed in 75 platelets for each 46 condition. (C) Representative Ca2+ traces in a single platelet for (i) control, (ii) forskolin, (iii) indomethacin 47 and cangrelor and (iv) forskolin, indomethacin and cangrelor. Data presented as mean±SEM (n=3). 48 49 206x213mm (120 x 120 DPI) 50 51 52 53 54 55 56 57 58 59 60 Page 35 of 33 Thrombosis and Haemostasis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Fig. 6 NECA and forskolin do not change GPVI receptor clustering on collagen. Washed platelets (2x107/ml) 46 were incubated with 1G5-Fab and allowed to spread on collagen for 30 min at 37oC. NECA (100 μM) and 47 forskolin (10 μM) were then added to the platelet suspensions and platelets were spread for an additional 30 48 min. All STORM experiments were performed on a NIKON N-STORM microscope. Seven fields of view per 49 treatment per experiment were captured and the 20,000 frames captured with Nikon NIS ELEMENTS v4.5 50 were reconstructed in ThunderSTORM imageJ plugin. Cluster analysis was performed in KMINE software with an algorithm based on DBSCAN with an additional setting based on image dilation. (A) Representative TIRF 51 and reconstructed STORM images of platelet spreading and detections with the indicated treatments. Cluster 52 plots produced in MATLAB show the clusters, each represented by a different arbitrary colour. (Field of view 53 = 40 x 40 μm) (scale bar: 2 μm). (B) Relative quantification of (i) number of clusters, (ii) number of 54 detections per cluster and (iii) cluster area and (iv) density from seven fields of view per treatment per 55 experiment. Data presented as mean±SEM (n=3). 56 57 58 59 60 Thrombosis and Haemostasis Page 36 of 33

1 2 3 149x284mm (120 x 120 DPI) 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 37 of 33 Thrombosis and Haemostasis

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Fig. 7 The effect of NECA and forskolin on U46619 induced platelet aggregation and the effect of ADP in 36 combination with NECA on collagen induced platelet aggregation. Platelet aggregation was monitored by 37 light transmission aggregometry at 37oC with constant stirring at 1200 rpm. (A) The effect of the inhibitors: 38 NECA (100 μM) and forskolin (10 μM) on U46619 (3 μM) induced aggregation. (B) Representative 39 aggregation traces showing the effect of the inhibitors on platelet aggregation. (C) The effect of NECA (100 40 μM) alone, ADP (10 μM) alone and in the presence of NECA on collagen induced aggregation. (D) 41 Representative aggregation traces showing the effect of ADP, NECA, and both in combination on platelet 42 aggregation. Trace showing ADP alone does not cause aggregation of washed platelets. Significance was 43 measured using one-way ANOVA with a Bonferroni post-hoc test where P<0.05. Data presented as mean±SEM (n=3-6). 44 45 180x162mm (120 x 120 DPI) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60