Gap junctions are key mediators of intercellular communication,

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1 Gap Junctions and Connexin Hemichannels Underpin Hemostasis and Thrombosis Sakthivel Vaiyapuri, PhD; Chris I. Jones, PhD; Parvathy Sasikumar, MSc; Leonardo A. Moraes, PhD; Stephanie J. Munger, BS; Joy R. Wright, PhD; Marfoua S. Ali, MSc; Tanya Sage, MSc; William J. Kaiser, PhD; Katherine L. Tucker, PhD; Christopher J. Stain, PhD; Alexander P. Bye, BSc; Sarah Jones, PhD; Ernesto Oviedo-Orta, MD, PhD; Alexander M. Simon, PhD; Martyn P. Mahaut-Smith, PhD; Jonathan M. Gibbins, PhD Background Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. Methods and Results We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. Conclusions Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets. (Circulation. 2012;125: ) Key Words: blood platelets connexin 37 gap junctions hemostasis thrombosis Gap junctions are key mediators of intercellular communication, formed from the coupling of 2 hemichannels (or connexons) on adjacent cells. Each hemichannel results from the oligomerization of 6 connexin monomers in the cell membrane. On docking of 2 hemichannels on adjacent cells, a pore of 2 to 3 nm in diameter is formed that allows the bidirectional intercellular transport of small molecules up to 1000 Da. 1 Although gap junctions have recognized roles in intercellular communication in various cell types such as nerve cells, 2 oocytes, 3 bone marrow stromal cells, 4 arterial endothelial cells, 5 and cardiac myocytes, 6 hemichannels also act as conduits for the exchange of molecules with signaling properties between the cytoplasm and extracellular space. 7,8 More than 20 connexins have been identified in mammalian cells that are able to form either homomeric or heteromeric hemichannels and gap junctions with a range of conductances and varying mechanisms of channel gating regulation. 9,10 Editorial see p 2414 Clinical Perspective on p 2491 Platelets are small, anucleate blood cells derived from their precursors, megakaryocytes, and are vital for hemostasis. On blood vessel injury, platelets adhere to exposed subendothelial collagens. Platelet activation then ensues, culminating in aggregation and the formation of a platelet plug, or thrombus, to stem the loss of blood. 11 Thrombus formation involves 3 phases: initiation, extension, and perpetuation. 12 Initial interactions with collagen are indirect and mediated via glycoprotein (GP) Ib-V-IX von Willebrand factor binding, which is later stabilized by direct collagen binding to GPVI and integrin 2 1. This enables the platelets to coat the injury site and to secrete a range of prothrombotic factors (eg, ADP, serotonin, and fibrinogen). 13 Activated platelets also synthesize and release thromboxane A 2, an additional secondary agonist for platelet stimulation. During the extension phase, Received August 12, 2011; accepted March 15, From the Institute for Cardiovascular and Metabolic Research, School of Biological Sciences (S.V., C.I.J., P.S., L.A.M., M.S.A., T.S., W.J.K., K.L.T., J.M.G.) and Centre for Advanced Microscopy (C.J.S.), University of Reading, Reading, UK; Department of Physiology, University of Arizona, Tucson (S.J.M., A.B.S.); Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, UK (J.R.W., A.P.B., S.J., M.P.M.-S.); and Cardiovascular Biology Research, Faculty of Health and Medical Sciences, University of Surrey, Guildford, UK (E.O.-O.). The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Jonathan M. Gibbins, PhD, Institute for Cardiovascular and Metabolic Research, School of Biological Sciences, University of Reading, Hopkins Bldg, Reading, RG6 6UB, UK. j.m.gibbins@reading.ac.uk 2012 American Heart Association, Inc. Circulation is available at DOI: /CIRCULATIONAHA

2 2480 Circulation May 22, 2012 these secreted and synthesized molecules recruit additional platelets to the first layer by stimulating platelets in a paracrine manner. 14 At the final perpetuation stage, activation of platelets modulates the affinity of IIb 3, which then binds to the bivalent ligand fibrinogen and mediates platelet-platelet aggregation. 15 Under some pathological conditions such as atherosclerotic plaque rupture, platelets are activated inappropriately, resulting in the formation of platelet aggregates within the circulation (thrombosis), a principal trigger for heart attack and stroke. 16 The connexin hemichannels and gap junctions have been widely studied in various cell types in which sustained cell interactions are recognized. Recent reports, however, have indicated the potential for connexins to regulate the functions of some circulating cells (monocytes and T cells) Although platelets are single circulating cells under normal conditions, on activation, the formation of a thrombus brings platelets into close proximity for a prolonged period during which they function in a coordinated manner. The sustained signaling within the thrombus regulates its stability and subsequent clot retraction, which is important for tissue repair. 21 The present study uncovers the role of connexin hemichannels and gap junctions in platelet interactions during thrombus formation and function. Methods Detailed methods for transcriptomic analysis, real-time quantitative polymerase chain reaction analysis, human and mouse platelet preparation, aggregation assays, dense granule secretion, immunoblotting, immunohistochemistry, transmission and scanning electron microscopy, calcium flux, flow cytometry, clot retraction, and in vivo and in vitro thrombus formation are provided in the online-only Data Supplement. Fluorescence Recovery After Photobleaching Measurements Fluorescence recovery after photobleaching measurements were made on an Olympus inverted microscope with a confocal laser scanning module (Olympus FluoView1000) as described previously. 22 Briefly, human citrated whole blood was incubated with calcein AM (2 g/ml; Sigma Aldrich) for 20 minutes at 30 C, with exposure to gap junction blockers (100 g/ml 37,43 Gap27 23 ; 100 mol/l carbenoxolone or 18 -glycyrrhetinic acid [18 -GA)]) or appropriate control during the final 10 minutes. The blood was then perfused over a collagen 100 g/ml coated coverslip in a laminar flow chamber at a shear rate of 1000 seconds 1, and thrombi were allowed to form for 3 minutes. The unbound dye and free blood cells were washed away by perfusing platelet-poor plasma for 2 minutes. After the absence of free dye was confirmed, thrombi were selected via a 60 oil-immersion objective lens (UPLSAPO 60XO, NA 1.35, UK), and an 8- m-diameter circular region at the center of the thrombus was exposed to high-intensity 488-nm laser light for 300 milliseconds to achieve 85% photobleach of fluorescence. Fluorescence images were continuously acquired 8 seconds before and 72 seconds after photobleach. Five thrombi were analyzed for each of 4 donors for each of the gap junction blockers. A 40-mW multiline argon laser was used for both imaging and photobleach, with outputs of 0.8% and 20%, respectively, of the 488-nm line. Average fluorescence intensities for bleached, nonbleached, and background regions were recorded for each time point. Fluorescence signals were corrected for background and steady loss of signal owing to imaging illumination and expressed as F/F0 ratios to normalize fluorescence levels (F) against starting fluorescence (F0). Fluorescence intensities were measured with FluoView software and further analyzed with Slidebook5 software (Intelligent Imaging Innovations). Statistical Analysis The data obtained from fluorescence recovery after photobleaching and in vitro thrombus formation experiments in the presence and absence of various gap junction blockers were analyzed by general linear model with repeated measures through the use of the SPSS (version 17) statistical package (IBM). The data obtained from clot retraction, aggregation, fibrinogen binding, calcium mobilization, and granule secretions in human samples with gap junction blockers were analyzed simultaneously by use of nonparametric Kruskal- Wallis global statistical method, and each inhibited sample was compared with the control by use of the Dunn multiple-comparison test using GraphPad Prism (version 5.04) from GraphPad Software Inc. Data obtained from control and Cx37 / mice were analyzed with the nonparametric Mann-Whitney test using GraphPad Prism. Results Presence of Connexins in Platelets To study potential connexin expression in blood cells and megakaryocytes, we analyzed transcriptomics data obtained using an Illumina bead chip-based array by the Bloodomics Consortium. 24 Varying levels of expression of mrna for 16 different connexins were found in human megakaryocytes. The transcript for connexin37 (Cx37) was most highly expressed compared with the other connexins and other human blood cells (Figure 1A). Notable levels of Cx62 and Cx40 mrnas were also detected in megakaryocytes. Quantitative polymerase chain reaction analysis with mrna obtained from a highly purified population of human platelets also confirmed the expression of Cx37 (Figure 1B). Connexin protein expression was investigated in human platelets. Immunoblot analysis of human platelet lysates along with positive control cell lysate (human endothelial cells) confirmed the presence of Cx37 (Figure 1C). Stimulation and activation of platelets with collagen-related peptide (CRP-XL) resulted in no change in the levels of Cx37 detected. In addition to Cx37, expression of Cx32 (Figure 1D) and Cx43 (Figure 1E) was also detected in human platelets. The identification of Cx43 in platelets is consistent with the identification of this protein in megakaryocytes. 25,26 In accordance with previous reports, 27 Cx32 and Cx43 were not detected in HeLa cell lysates. It was not possible to assess the expression of Cx62 because of the lack of specific antibodies against this protein. The presence of connexins on the surface of platelets was confirmed by immunohistochemistry with antibodies that recognize the extracellular regions of Cx32, Cx37, Cx40, and Cx Washed platelets were allowed to spread on fibrinogen-coated coverslips, immunolabeled with specific antibodies, and analyzed with fluorescence microscopy. Connexins were detected on the surface of platelets (Figure 1F), an observation that was confirmed by flow cytometry (Figure 1G). Gap Junctions in Platelets Transmission electron microscopy was used to determine whether gap junction like structures form between platelets. Washed human platelets were aggregated with thrombin, formed into pellets, and processed for epoxy resin embed-

3 Vaiyapuri et al Role of Gap Junctions in Platelet Activation 2481 ding. The pellet was sectioned (90-nm thickness) with an ultramicrotome and stained before analysis by transmission electron microscopy. Electron micrographs show apposite membrane structures between platelets (Figure 2A) typical of gap junction like structures well established in other cell types. 1,7,29 To further explore the existence of connexins and gap junction-mediated intercellular communication between platelets in a thrombus, we used fluorescence recovery after photobleaching, which allowed the study of the migration of the fluorescent dye calcein from 1 cell to another through gap junction channels in the presence or absence of gap junction blockers after photobleaching of recipient cells. 22 Fluorescence recovery was 14% in control-treated thrombi but reduced to 2% in the presence of 37,43 Gap27, a connexin mimetic peptide (SRPTEKTIFII) designed to target the extracellular loops of Cx37 and Cx43, and the non isotypeselective connexin inhibitors carbenoxolone (100 mol/l) or Figure 1. Presence of connexins in megakaryocytes and platelets. A, Connexin mrna levels were quantified in human megakaryocytes and a range of blood cells by analysis of gene array data. Samples were clustered with the mean normalized intensity values for the probes; 108, low-level expression (blue); 315, moderate-level expression (yellow); and 800, high-level expression (red). B, Expression of connexin37 (Cx37) mrna levels was quantified in cultured megakaryocyte cell line, MEG-01, and human platelets with quantitative polymerase chain reaction and normalized to ACTB ( -actin) gene expression. Data represent mean SD; n 10. C, Presence of Cx37 in human platelets was confirmed by immunoblot analysis with 2 different antibodies obtained from Epitomics and Invitrogen (human endothelial cells [a] and resting [b] or collagen-related peptide [CRP-XL] stimulated human platelets for 90 seconds [c] and 5 minutes [d]). D, Presence of Cx32 and Cx43 (E) in human platelets was confirmed by immunoblot analysis using antibodies obtained from Invitrogen (Cx32 and Cx43) and Epitomics (Cx43) (HeLa cell lysate was used as negative control in blots D and E [lane a]; human liver [D] and heart [E] tissue lysates [b], and resting [c] or CRP-XL stimulated human platelets for 90 seconds [d] and 5 minutes [e]). GAPDH was used as a loading control. Presence of connexins on the surface of platelets was detected with extracellular loop specific anti-connexin antibody by fluorescence microscopy (F) and flow cytometry (G). MW indicates molecular weight. 18 -GA (100 mol/l; Figure 2B). These observations are consistent with previous fluorescence recovery after photobleaching studies of gap junctions between adipose tissue derived mesenchymal stem cells 30 and provide strong evidence for the involvement of gap junction mediated intercellular communication between platelets within a thrombus. We hypothesized that such communication may help regulate the coordinated response of platelets within the thrombus. After the binding of fibrinogen, the integrin IIb 3 transduces signals into the cell, triggering platelet spreading and, in the latter phase of thrombus formation, clot retraction. 31 Such outside-in integrin signaling through IIb 3 may be isolated through the measurement of clot retraction in vitro. 32 The effect of gap junction blockers on clot retraction was therefore measured. Platelet clots were initiated by the addition of thrombin to platelet-rich plasma in the absence or presence of gap junction blockers (100 g/ml 37,43 Gap27;

4 2482 Circulation May 22, mol/l carbenoxolone or 18 -GA), and the retraction rate of the clot was monitored over 5 hours. Initial clot formation (clot weight 15 minutes after addition of thrombin) was not altered in the presence of the gap junction blockers (Figure I in the online-only Data Supplement). Notably, clot retraction was 3-fold slower in the presence of each gap junction blocker at 90 minutes compared with control-treated samples (Figure 2C). However, after 5 hours, the clots formed in the presence of carbenoxolone and 18 -GA were retracted similar to control samples, whereas the rate of clot retraction was still slower with 37,43 Gap27. These data suggest that outside-in signaling through IIb 3, which controls the coordinated process of clot retraction, is influenced by gap junction function. Gap Junction Blockers Inhibit Platelet Activation The effect of 37,43 Gap27, carbenoxolone, and 18 -GA on platelet aggregation in response to various activators of platelet function was also explored. Different concentrations Figure 2. Gap junctions in platelets. A, Presence of gap junction like structures between activated platelet membranes was analyzed with transmission electron microscopy. Arrows indicate gap junction like structures similar to other cell types. Inset, Enlarged regions. B, Fluorescence recovery after photobleaching analysis was performed in thrombi formed with calcein-am dye loaded platelets in the presence or absence of gap junction blockers and fluorescence recovery after photobleaching measured for 72 seconds. Data represent mean SD (n 20 from 4 donors). Data were analyzed by general linear model with repeated measures with the SPSS statistical package, and a statistically significant (P ) effect of inhibitor treatment was observed. The comparison of time factor (P ) and time and inhibitor interactions (P ) between samples was also significant. Pairwise comparison between each inhibitor and control was found to be significant (P 0.05). C, Effect of gap junction blockers on clot retraction was analyzed in vitro. Data represent mean SD (n 6). Inset, Representative image of clot retraction at 90 minutes. The P values obtained with the nonparametric Kruskal-Wallis global test are shown, and P values obtained by the Dunn multiple comparison test were 0.05 for all the samples at 90 minutes and 37,43 Gap27 (for carbenoxolone and 18 -glycyrrhetinic acid[ 18 -GA], the P value was 0.05) at 5 hours. of each of the gap junction blockers were tested with a GPVI-selective ligand, CRP-XL (0.5 and 1 g/ml), as an agonist, and the effect was recorded with optical aggregometry. Aggregation induced by a low concentration of CRP-XL was reduced at all concentrations of the gap junction blockers. Inhibition of 40%, 50%, and 75% was observed with 37,43 Gap27 (100 g/ml), carbenoxolone (100 mol/l), and 18 -GA (100 mol/l), respectively, when 0.5 g/ml CRP-XL was used (Figure 3A). Lower levels of inhibition were noted with 1 g/ml CRP-XL (Figure 3B). Aggregation was monitored over an extended period of 10 minutes, during which time inhibition was maintained (Figure IIA in the online-only Data Supplement). Similarly, 37,43 Gap27-, carbenoxolone-, and 18 -GA mediated inhibition of platelet aggregation to collagen and ADP was also observed (Figure IIC and IID in the online-only Data Supplement). A scrambled peptide (REKIITSFIPT) was used in platelet aggregation analysis as a control for 37,43 Gap27 and showed no inhibitory effects (Figure III in the online-only Data Supplement).

5 Vaiyapuri et al Role of Gap Junctions in Platelet Activation 2483 Platelet aggregation is dependent on modulation of the conformation of IIb 3 through inside-out signaling to increase its affinity for fibrinogen binding. 21 Thus, as a marker for inside-out signaling, fibrinogen binding was measured on the surface of the platelets by flow cytometry. Platelet-rich plasma (diluted 100-fold) was stimulated with CRP-XL (1 g/ml) in the absence or presence of gap junction blockers for 90 seconds, and fibrinogen binding was measured. CRP- XL stimulated fibrinogen binding was reduced significantly in the presence of gap junction blockers: 30% with 37,43 Gap27 (100 g/ml), 60% with carbenoxolone (100 mol/l), and 70% with 18 -GA (100 mol/l; Figure 3C). Lower concentrations of 37,43 Gap27 (10 and 50 g/ml), carbenoxolone (10 and 50 mol/l), and 18 -GA (10 and 50 mol/l) also showed a clear reduction in fibrinogen binding (Figure IV in the online-only Data Supplement). This illustrates the involvement of connexins in inside-out integrin signaling in platelets that results in platelet aggregation. Because this assay was performed with flow cytometry gating on the Figure 3. Gap junction blockers inhibit platelet activation. Aggregation performed in the presence or absence of gap junction blockers was recorded for 90 seconds after stimulation with collagen-related peptide (CRP-XL) (0.5 g/ml [A] and 1 g/ml [B]) or thrombin (0.09 U/mL [F] and 0.18 U/mL [G]). V represents vehicle alone, and numbers represent the concentrations of gap junction blockers. Cumulative data represent mean SD (n 4). The level of aggregation obtained with vehicle was taken as 100%. C, The effect of gap junction blockers on fibrinogen binding was measured by flow cytometry. Data represent mean SD (n 4). The level of fibrinogen binding obtained with vehicle was taken as 100%. Platelets stimulated with CRP-XL (0.5 g/ml; D) or thrombin (0.09 U/mL; H) in the presence or absence of gap junction blockers were analyzed by immunoblotting with antiphosphotyrosine antibody. E, Phosphospecific antibodies against proteins involved in the glycoprotein VI (GPVI) pathway (1, resting; 2 6, platelets stimulated with CRP-XL for 90 seconds and treated with vehicle-tyrode [2], 37,43 Gap27 [100 g/ml; 3], carbenoxolone [100 mol/l; 4], vehicle dimethyl sulfoxide [5], and 18 -glycyrrhetinic acid [18 -GA; 100 mol/l; 6]). Nonparametric Kruskal-Wallis global (P value is shown) and Dunn multiple-comparison (P 0.05) tests were performed. population of individual platelets, inhibition of platelet function suggests the potential role for connexin hemichannels in platelets. Functions of hemichannels in various other cell types have been reported, the inhibition of which may be achieved with gap junction blockers such as carbenoxolone, 18 -GA, and 37,43 Gap27. 7,8 Because aggregation was largely inhibited by the gap junction blockers at low concentrations of CRP-XL, the phosphorylation levels of various proteins involved in the GPVI pathway, which is stimulated on binding at collagen or CRP-XL, were analyzed. Platelet lysates were prepared after stimulation with CRP-XL (0.5 g/ml) in the absence or presence of gap junction blockers (100 g/ml 37,43 Gap27; 100 mol/l carbenoxolone or 18 -GA). Phosphotyrosine and phosphospecific antibodies against Lyn, LAT, Vav, PLC 2, and AKT proteins were used to assess phosphorylation status by immunoblot analysis. The total (Figure 3D) and individual protein (Figure 3E) phosphorylation levels were unaffected after treatment with gap junction blockers, sug-

6 2484 Circulation May 22, 2012 gesting that connexins are not involved in the control of receptor proximal events but instead can modulate the signaling further downstream, which may be shared with activation mechanisms stimulated by other agonists. This is consistent with the observed effects of gap junction blockers on the response of platelets to a range of agonists. The effect of gap junction blockers on platelet activation stimulated by thrombin, which stimulates human platelet activation via the protease activated G-protein coupled receptors protease-activated receptor-1 and -4, 33 was measured. Thrombin-stimulated (0.09 and 0.18 U/mL) aggregation was also inhibited by each of the gap junction blockers (100 g/ml 37,43 Gap27; 100 mol/l carbenoxolone or 18 -GA). Inhibition of 60%, 70%, and 80% with 37,43 Gap27, carbenoxolone, and 18 -GA, respectively, was recorded when 0.09 U/mL thrombin was used (Figure 3F) and maintained over 10 minutes (Figure IIB in the online-only Data Supplement). Inhibition was also maintained at lower concentrations of gap junction blockers (Figure V in the online-only Data Supplement). Inhibition was markedly less pronounced with each gap junction blocker at a higher concentration of thrombin (Figure 3G). As observed with CRP-XL, the levels of thrombin-stimulated total protein tyrosine phosphorylation remained unaffected after treatment with gap junction blockers (Figure 3H). These data suggest that the connexins play a key role in platelet functional responses in both GPVI- and GPCR-stimulated activation pathways, although they do not appear to modulate early kinase-mediated signaling events. Platelet Connexins and Calcium Signaling Immediately downstream of the early signaling events, elevation of intracellular calcium levels is essential for platelet activation and thus is important in thrombus formation. Calcium mobilization plays a paramount role in various platelet functions, including reorganization of the actin cytoskeleton necessary for shape change, 34 degranulation, and integrin IIb 3 affinity modulation. 35 In platelets, a major central mechanism for the elevation of cytosolic Ca 2 is release from intracellular stores after phospholipase C mediated generation of inositol-1,4,5-trisphosphate (IP 3 ), which releases calcium from a dense tubular system. 36 In addition, platelets express several pathways for Ca 2 influx across the plasma membrane. 37 These include store-operated Ca 2 entry through Orai store-operated Ca 2 channels, ATP-gated P2X receptors, and TRPC6 44 channels activated by diacylglycerol or phosphatidylinositol 4,5-bisphosphate depletion. To assess whether connexins play a role in regulating calcium mobilization, intracellular calcium levels were measured in fluo4 NW dye loaded platelets (platelet-rich plasma) in the absence or presence of gap junction blockers (100 g/ml 37,43 Gap27, 100 mol/l carbenoxolone) for 90 seconds after stimulation with CRP-XL (0.5 or 1 g/ml) by spectrofluorimetry. Similar to their effects on aggregation, peak calcium levels were reduced by 25% and 40% by 37,43 Gap27 and carbenoxolone, respectively, after stimulation with CRP-XL (0.5 g/ml; Figure 4A and 4C; 18 -GA was not used in this assay because of its incompatibility with the dye, fluo4 NW). The inhibition was maintained at lower concentrations of gap junction blockers on stimulation with 0.5 g/ml CRP-XL (Figure VIA and VIB in the online-only Data Supplement). The inhibitory effects were maintained at the higher concentrations of CRP-XL (Figure 4B and 4D). Similar experiments were performed in the presence of EGTA to block calcium influx and the formation of platelet aggregates after platelet stimulation. The overall cytosolic calcium levels were reduced compared with the levels obtained in the absence of EGTA, although the gap junction blockers maintained their ability to inhibit cytosolic calcium elevation (15% by 37,43 Gap27 and 25% by carbenoxolone) stimulated by CRP-XL (Figure 4E 4H). The inhibitory effects were still observed at lower concentrations of gap junction blockers on stimulation with 0.5 g/ml CRP-XL (Figure VIC and VID in the online-only Data Supplement). These results suggest that connexins predominantly influence release of calcium from intracellular stores in platelets, although they may also control influx of calcium across the plasma membrane. Although the exact mechanism is unknown, it is interesting to note that extracellular Ca 2 levels are known to modulate hemichannel opening in other cell types. 7 Therefore, connexins could operate to promote intercellular signaling by diffusion of messengers such as IP 3 and Ca 2 between platelets after contact or in enhancing membrane-membrane interactions to promote outside-in signaling, as shown for Eph kinases and ephrins, 45 or indeed through sustained hemichannel function within a thrombus. Role of Connexins in Platelet Granule Secretion One way in which platelets influence the formation of thrombus is through the release of granule contents. To analyze the involvement of connexins on platelet granule secretion, both - and dense granule secretion was assayed in the absence or presence of gap junction blockers (100 g/ml 37,43 Gap27; 100 mol/l carbenoxolone or 18 -GA). -Granule secretion was assessed by measuring the levels of P-selectin exposed on the surface of platelets after stimulation with CRP-XL with the use of flow cytometry analysis in washed platelets. CRP-XL 0.5 g/ml stimulated -granule secretion was reduced by 30%, 70%, and 75% with 37,43 Gap27, carbenoxolone, and 18 -GA, respectively (Figure 5A). Similar levels of inhibition were seen with higher concentrations of CRP-XL (eg, 1 g/ml; Figure 5B). Furthermore, lower concentrations of gap junction blockers inhibited the level of P-selectin exposed on the surface (Figure VIIA VIID in the online-only Data Supplement). To determine the role of hemichannels separate from the gap junctions formed during platelet-platelet contact, the effect of the connexin blockers was also assessed on -granule secretion under conditions that disfavor aggregation (presence of EGTA 1 mmol/l, indomethacin 10 mol/l [to prevent the thromboxane A 2 synthesis], and apyrase 2 U/mL [to prevent ATP and ADP activation of platelets]). This, combined with flow cytometry analysis, enabled their effects on individual platelets to be studied. The maintenance of single platelets was confirmed by microscopy. -Granule secretion was reduced by 30%, 70%, and 80% with 37,43 Gap27, carbenoxolone, and 18 -GA, respectively, at a low concentration of CRP-XL (0.5 g/ml; Figure 5C), and

7 Vaiyapuri et al Role of Gap Junctions in Platelet Activation 2485 Figure 4. Platelet connexins and calcium mobilization. Calcium mobilization was measured in fluo4 NW dye loaded platelets by spectrofluorimetry in the presence or absence of gap junction blockers. Platelets were stimulated with collagen-related peptide (CRP; 0.5 g/ml [A], 1 g/ml [B]), and fluorescence was measured for 90 seconds. Similar experiments were performed in the presence of EGTA (1 mmol/l; E and F). The traces shown here are representative of 4 separate experiments. Data (C, D, G, and H) represent mean SD (n 4). The calcium levels obtained with vehicle was taken as 100%. Nonparametric Kruskal-Wallis global (P value is shown) and Dunn multiple comparison (P 0.05) tests were performed. the inhibition was maintained at a higher concentration of CRP-XL (1 g/ml; Figure 5D). Similarly, the level of inhibition was clearly observed when low concentrations of gap junction blockers used (Figure VIIE VIIH in the online-only Data Supplement). The effect of gap junction blockers on dense granule secretion was assessed by measuring ATP secretion after platelet activation with CRP-XL using a luciferin-luciferase luminescence assay. Platelets were activated with CRP-XL (0.5 and 1 g/ml) in the absence or presence of gap junction blockers, and both aggregation and ATP secretion were measured simultaneously. ATP secretion was reduced by 20% in the presence of 37,43 Gap27 (100 g/ml) and 60% and 80% in the presence of carbenoxolone (100 mol/l) and 18 -GA (100 mol/l), respectively, at 0.5 g/ml of CRP-XL (Figure 5E). The inhibition of dense granule secretion by gap junction blockers was still observed at higher concentration of CRP-XL, although the aggregation effects were largely overcome (Figures 3B and 5F). Similar experiments were performed to assess the effect of gap junction blockers under conditions that disfavor aggregation (as above). ATP secretion was reduced by 25% in the presence of 37,43 Gap27 (100 g/ml) and 60% and 75% in the presence of carbenoxolone (100 mol/l) and 18 -GA (100 mol/l), respectively (Figure 5G). The inhibitory effects were maintained at higher concentration of CRP-XL (Figure 5H). The ability of gap junction blockers to inhibit granule secretion on activation and under conditions that disfavor aggregation indicates that connexins regulate platelet activation even in the absence of gap junction formation. Effect of Gap Junction Blockers on Thrombus Formation Given the presence of connexins on platelets, their ability to form gap junctions, and the importance of hemichannels in regulating platelet function, we speculated that connexins might make an important contribution to thrombus formation. Thus, thrombus formation was analyzed in vitro by fluorescence microscopy in whole blood under arterial flow conditions in the presence or absence of gap junction blockers. Captured images were analyzed by calculating the size and number of thrombi formed, the sum intensity of fluorescence, and the total thrombus volume. The results indicate that the size, number, and volume of thrombi were reduced by all 3 gap junction blockers (Figure 6A). The sum intensity of thrombi was reduced by 40% (at 10 minutes) with 37,43 Gap27 (100 g/ml) and 18 -GA (100 mol/l), whereas carbenoxolone (100 mol/l) inhibited by 65% (Figure 6B 6E). To assess the impact of gap junctions in vivo, the effect of 37,43 Gap27 on arterial thrombosis was measured with a laser

8 2486 Circulation May 22, 2012 Figure 5. Role of connexins in platelet granule secretion. Granule secretion was measured by stimulating platelets with collagen-related peptide (CRP; 0.5 g/ml and 1 g/ml) for 90 seconds. Platelets were stimulated, and the level of P-selectin exposed on the surface was measured by flow cytometry (A and B). P-selectin exposure was also measured in the presence of EGTA (1 mmol/l; C and D). The level of P-selectin exposure with vehicle was taken as 100%. ATP release in platelets was measured by use of luminescence-luciferase substrate in the absence (E and F) or presence (G and H) of EGTA (1 mmol/l). The level of ATP released in vehicle-treated samples was taken as 100%. Data represent mean SD (n 3). Nonparametric Kruskal-Wallis global (P value is shown) and Dunn multiple comparison (P 0.05) tests were performed. injury model in mice ,43 Gap27 also inhibited mouse platelet aggregation, as observed with human platelets (data not shown and Figure 3). After injury, subendothelial collagens are exposed to the blood, and thrombus formation is induced at the site of injury. Platelets were labeled with an anti-mouse GPIb Alexa fluor-488 antibody. Thrombus formation was monitored over a period of 180 seconds at the site of injury, and the rate and size of thrombus development were analyzed by calculating the fluorescence intensity. Data analysis was performed for multiple thrombi obtained from 4 control- or 37,43 Gap27 (100 g/ml of blood) treated mice. The initial adherence of platelets at the site of injury was relatively unaffected; thus, the initial kinetics of thrombus formation was similar in both control- and 37,43 Gap27-treated animals. Overall thrombus growth, however, was reduced by 70% in 37,43 Gap27-treated mice (Figure 6F and 6G). Together, these data confirm the involvement of connexins in thrombus formation under in vivo conditions. Characterization of Cx37-Deficient Mouse Platelets Because the connexin mimetic peptide 37,43 Gap27 binds Cx37 and Cx43, to further assess the specific roles of Cx37 in platelet function, we used a genetic approach and examined the function of platelets from Cx37-knockout (Cx37 / ) mice. 47 Developmental defects in Cx37-deficient platelets were excluded through analysis of cellular and subcellular morphology and receptor expression levels. Electron microscopy analysis revealed that the morphology of platelets from Cx37 / and Cx37 / was indistinguishable, with - and dense granules of normal appearance and frequency (data not shown). Flow cytometry was used to measure the expression of platelet surface receptors such as IIb 3 (Figure 7A), GPVI (Figure 7B), and GPIb (Figure 7C), which were found to be unchanged in Cx37 / platelets. Similarly, the expression level of the integrin 2 subunit was confirmed to be normal by immunoblot analysis (Figure 7D). It is possible that deletion of the Cx37 gene (Gja4) may result in compensatory effects on the expression of other platelet connexins. The levels of other platelet connexins such as Cx32 and Cx43 (identified in this study) were also examined by immunoblot analysis. As expected, Cx37 (Figure 7E) was confirmed to be absent in Cx37 / mouse platelets. Cx32 (Figure 7F) and Cx43 (Figure 7G) were detected at similar levels in platelets from Cx37 / and Cx37 / mice. Effect of Cx37 Deficiency on Murine Platelet Function Aggregation assays were performed with washed mouse platelets at the density of /ml with an optical aggregometer. The aggregation of Cx37 / platelets was reduced by 60% at the concentration of 0.5 g/ml CRP-XL (Figure 8A and 8B) compared with Cx37 / platelets. At the higher concentration of CRP-XL (1 g/ml), the reduction was 20% (Figure 8C and 8D). These results are consistent with data obtained with human platelets in the presence of the connexin mimetic peptide inhibitor 37,43 Gap27 (Figure 3A and 3B). Fibrinogen binding on the surface of platelets (in citrated whole blood) was measured on activation by CRP-XL. This was also reduced (by 50% at 0.5 g/ml CRP-XL and by 30% at 1 g/ml CRP-XL) in Cx37 / mice platelets compared with Cx37 / platelets (Figure 8E and 8F). To

9 Vaiyapuri et al Role of Gap Junctions in Platelet Activation 2487 Figure 6. Effect of gap junction blockers in thrombus formation. DIOC6-labeled human blood was preincubated with vehicle or gap junction blockers and perfused over a collagen-coated Vena8 BioChip. Images were obtained for every 30 seconds up to 10 minutes; representative images are shown in A. Fluorescence intensity levels of thrombi obtained in the presence of gap junction blockers, 37,43Gap27 (B), carbenoxolone (C), and 18 -glycyrrhetinic acid (18 -GA; D) were compared with vehicle. P values were calculated by a general linear model with repeated measures (P [37,43Gap27], [carbenoxolone], and [18 -GA]). The fluorescence intensity obtained at 10 minutes with vehicle was taken as 100% to calculate the percent inhibition of gap junction blockers on thrombus formation (E). Data represent mean SD (n 8 from 4 donors). Nonparametric Kruskal-Wallis global (P value is shown) and Dunn multiple comparison (P 0.05) tests were performed. In vivo thrombosis assay was performed by use of intravital microscopy as described previously. Representative images of thrombi obtained at different time intervals are shown (F). Arrow indicates the direction of blood flow. Median fluorescence intensity was measured from 24 thrombi (from 4 mice) for each of the control and treated experiments (G). assess the role of Cx37 on granule secretion, P-selectin was measured with flow cytometry. Granule secretion was also reduced (by 50% at 0.5 g/ml CRP-XL and 40% at 1 g/ml CRP-XL) in Cx37 / platelets compared with Cx37 / platelets (Figure 8G and 8H). Given the effects of gap junction blockers on clot retraction, similar assays were also performed with Cx37 / and Cx37 / platelets. The weight of the remaining clot measured (after 5 hours) in Cx37 / was 3 times higher than in Cx37 / platelets, indicating the reduced retraction in Cx37 / platelets (Figure 8I [image shown was taken at 90 minutes] and 8J). This is consistent with the inhibitory actions of carbenoxolone, 18 -GA, and 37,43Gap27 on clot retraction in human platelet-rich plasma (Figure 2C) and point to a fundamental role for gap junction mediated intercellular communication in the stimulation of platelet thrombus function. Discussion In the intact circulation, platelets normally circulate as individual discoid bodies but show a marked ability to adhere to each other to form a thrombus via the support of the integrin IIb 3 and the adhesion proteins fibrinogen and von Willebrand factor.11 Recent studies45 have indicated that sustained signaling is required to maintain thrombus stability, although little is known of how this is mediated. The contraction of the clot then ensues, a coordinated response driven by platelet integrins,21 that results in the shrinking of the thrombus and the drawing together of wound edges. Given that platelet function may therefore be acutely regulated while in isolation in the plasma and in concert within a thrombus, the possibility was explored that connexin hemichannels and gap junctions may be present in platelets and regulate the different phases of platelet function.

10 2488 Circulation May 22, 2012 Figure 7. Characterization of Cx37 / mouse platelets. The expression levels of IIb 3 (A), glycoprotein (GP) VI (B), and GPIb (C) were analyzed by flow cytometry with citrated mouse blood. Data represent mean SD (n 6 Cx37 / and Cx37 / mice). P values calculated by the nonparametric Mann-Whitney test are shown (P 0.05). The expression level of 2 was measured by immunoblot analysis (D) using detection of 14 to 3 3 as a loading control. The expression of Cx37 (E), Cx32 (F), and Cx43 (G) was analyzed by immunoblots in Cx37 / and Cx37 / mouse platelets. Blots are representative of 4 separate experiments. We report the expression of several members of the connexin family in human megakaryocytes and platelets in which Cx37 expression was found to be notably abundant. Consistent with the formation and function of gap junctions within the thrombus, dye transfer between cells was observed and blocked by pharmacological connexin-blocking agents, including the Cx37- and Cx43-selective mimetic peptide 37,43 Gap27. The retraction of platelet-rich clots was also Figure 8. Effect of Cx37 deficiency on mouse platelet function. Aggregation was recorded for 180 seconds after stimulation with collagen-related peptide (CRP-XL; 0.5 g/ml [A] and 1 g/ml [C]). Cumulative data (B and D) represent mean SD. The level of aggregation obtained with Cx37 / was taken as 100%. The level of fibrinogen binding in Cx37 / and Cx37 / was measured by flow cytometry after stimulation with CRP-XL (0.5 g/ml [E] and 1 g/ml [F]). Data represent mean SD of median fluorescence intensity values. P-selectin exposure on activation by CRP-XL (0.5 g/ml [G] and 1 g/ml [H]) was measured with citrated mouse blood by flow cytometry. Effect of Cx37 deficiency on clot retraction was analyzed in vitro with Cx37 / mouse platelets (I; image shown was taken at 90 minutes). Data represent mean SD (n 4 Cx37 / and Cx37 / mice) of clot weights measured after 5 hours (J). P values calculated by the nonparametric Mann-Whitney test are shown (P 0.03; n 4 Cx37 / and Cx37 / mice).

11 Vaiyapuri et al Role of Gap Junctions in Platelet Activation 2489 reduced in the presence of these agents, strongly supporting the notion that gap junction dependent intercellular signaling within a thrombus is required for synchronized platelet function. Connexin blocking agents were found to inhibit a range of platelet functions, including aggregation, -granule secretion, and fibrinogen binding, suggesting that before the association of platelets within a thrombus, hemichannels are also important for platelet function. Although calcium signaling was diminished in the presence of gap junction blockers, it is unclear whether the mode of hemichannel function may be through ion channel functionality (eg, by cooperation with Orai1, TRPC6, 44 and P2X ) or through interaction with other components of the platelet cell machinery. As platelets become activated, the formation of initial microthrombi leading to macrothrombi may result in complex combinations of hemichannel and gap junction function, although the effect of gap junction blockers on platelets treated under conditions that disfavor platelet-platelet aggregation strongly suggests that initial platelet reactivity is modulated through hemichannels and later events within the thrombus are governed by gap junctions. The potent inhibition of both thrombus formation in vitro with human blood under arterial flow conditions and thrombosis in mice by gap junction blockers suggests that gap junctions may offer new avenues for therapeutic intervention. Our detailed characterization of platelet function using 3 unrelated gap junction blockers in parallel point strongly to an activatory role for connexins in platelets. Because of the inability to truly inhibit the action of specific connexins, it is possible that this represents the compound actions of several connexins, each of which may exhibit different roles and regulation as heteromeric or homomeric connexons in the context of platelet or platelet thrombi. To begin to dissect the roles of connexins, and particularly to focus on the family member most notably expressed (at transcript level) in megakaryocytes, we explored the function of Cx37 / mouse platelets. Cx37 / platelets were confirmed to lack Cx37 protein; however, no abnormalities in platelet morphology or in their receptor expression levels were observed compared with Cx37 / platelets. Further functional characterization of Cx37 / platelets revealed clear inhibition in platelet function compared with control platelets. These results are consistent with the data obtained from human platelets in the presence of various gap junction blockers. Our dose-response experiments using multiple approaches in human platelets along with detailed characterization (including confirmation of genotype, lack of expressed protein) of Cx37 / mouse platelets confirm Cx37 to positively influence platelet function. It is important to note that although these experiments reveal a positive role for Cx37 in platelet function, the potential contributions of other connexins identified in this study in the regulation of platelets (positive or negative) cannot be excluded. It is interesting to reflect that recent studies implicate Cx37 on macrophages in inflammatory responses that lead to atherogenesis in susceptible mice. 19 Indeed, a single-nucleotide polymorphism (C1019T) within the human Cx37 gene (GJA4) has been reported to be a potential prognostic marker for atherosclerosis and myocardial infarction. 48,49 Given the established role of platelet-monocyte adhesion and the initiation or progression of atherosclerosis, it is tempting to speculate that the role of platelet connexin function may extend to platelet-immune cell intercellular signaling in a range of pathological conditions. Before this study, Angelillo-Scherrer et al 50 also reported the expression of Cx37 (and no other connexins) in platelets in which it was proposed to be involved in the regulation of platelet function. The prominent expression of Cx37 in human and mouse platelets is indeed consistent with our findings. In contrast to our work, however, they concluded that this connexin serves to inhibit platelet function. The reasons for this discrepancy are not entirely clear. We can conclude that platelets possess a range of connexin family members and that therefore the situation may be more complex than originally suspected, although in some cases similar experimental approaches were used. Moreover, in the above study, increased human platelet reactivity was reported with a single inhibitor, 18 -GA, which we have found to be inappropriate for the analysis of platelet function because of its ability to activate platelet function in the absence of agonists (Figure VIII in the online-only Data Supplement). Indeed, in some aggregation data (presented in the previous study), concentrations of agonists were used that in themselves did not cause aggregation in many donors, calling into question whether increased responses in the presence of 18 -GA represented increased platelet sensitivity or were due to nonspecific effects of the agent. To overcome this, in the present study, a range of pharmacological agents were used that in the absence of a platelet agonist do not cause platelet activation, allowing the study of connexin function in the presence of physiologically relevant levels of platelet activating factors. The substantial variability in response in the platelets from different donors in the previously published study was also a concern; therefore, we have presented cumulative data with appropriate statistical analysis. Angelillo-Scherrer et al 50 also reported increased aggregation responses using the platelets from Cx37 / mice. We have scrutinized the methodologies used in both studies because the experiments reported in the present study were obtained with the same mouse line. It is possible that differences in anticoagulants used may underlie apparent discrepancies in reactivity, although ambiguity in the methodology previously presented makes this difficult to confirm. It is interesting to note that given the high agonist concentrations used in the study of Angelillo-Scherrer et al 50 (eg, collagen at 5 g/ml), very slow aggregation responses are observed, particularly for analysis in platelet-rich plasma, with no aggregation apparent until 100 seconds after stimulation. Furthermore, this is reversible, a characteristic of weak stimulation in which secretion has not been stimulated. The analysis of hemostasis and thrombosis in Cx37 / mice may present useful insights into the function of this protein and potentially other connexins in platelets in vivo. Indeed, Angelillo-Scherrer et al 50 report a reduction in bleeding time and increased thromboembolism, although the effects of systemic deficiency of Cx37 on endothelial cell/blood vessel susceptibility to injury have not been established. It

12 2490 Circulation May 22, 2012 may also be pertinent to note that Cx37 plays an important roles in lung function 51,52 that may present further confounding issues in the thromboembolism model. Our analysis of thrombosis in vivo indicates that on the general inhibition of connexin function thrombi formed are less stable. It is therefore possible that thrombi formed in Cx37 / mice are less stable compared with Cx37 / mice and more susceptible to embolization, resulting in reduced survival time. It is likely that cell (platelet)-specific deletion of Cx37 and the other connexins detected in platelets will be necessary to completely unravel these inconsistencies between the studies and to examine potential differential roles of different platelet connexins. Conclusions Our study provides detailed evidence for a fundamental role of connexins in platelet function. The connexins, particularly Cx37, contribute to the early phases of platelet activation through formation of hemichannels. In addition, they drive thrombus formation and subsequent clot retraction after gap junction formation. Further research is required to establish the molecular nature of the signaling mechanisms through which platelet connexins exert their effects. It is possible that they contribute to calcium signaling, although additional possibilities suggested in other cell systems 53 such as the dilution of cgmp levels between platelets within a thrombus and consequent reduction in inhibitory signaling may also contribute to the effects. Finally, the identification and function of connexin hemichannels and gap junctions in platelets may represent potential targets for novel antiplatelet therapies. Acknowledgments We thank Professor Willem H. Ouwehand and Dr Nicholas A. Watkins for providing the gene array data. We are grateful to Professor William Howard Evans and Dr Michael Fry for advice and critical evaluation during the preparation of this manuscript. We thank John Kanady for his support during clot retraction assays. We are grateful for the AZCC/ARL, Division of Biotechnology Cytometry Core facility at the University of Arizona for support with flow cytometry analysis. Sources of Funding This work was supported by the British Heart Foundation (grants RG/05/007, PG/08/100/26125, PG/05/014, and PG/09/094); Medical Research Council (grant G ); Wellcome Trust, UK (grant /Z/07/Z); BBSRC studentship, Leicester NIHR Biomedical Research Unit in Cardiovascular Disease, Glenfield Hospital, Leicester, UK; and the National Institutes of Health (grant HL64232). None. Disclosures References 1. Goodenough DA, Paul DL. Gap junctions. Cold Spring Harb Perspect Biol. 2009;1:a Bruzzone RDR. Structure and functions of gap junctions in the developing brain. Cell Tissue Res. 2006;326: Kidder GM, Mhawi AA. Gap junctions and ovarian folliculogenesis. Reproduction. 2002;123: Dorshkind K, Green L, Godwin A, Fletcher WH. 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