The ability to stop actions and thoughts is essential for goal-directed behaviour. Neuroimaging research has revealed that stopping actions and thoughts engage similar cortical mechanisms, including the ventro- and dorso-lateral prefrontal cortex. However, whether and how these abilities require similar subcortical mechanisms remains unexplored. Specifically of interest are the basal ganglia, subcortical structures long-known for their motor functions, but less so for their role in cognition. To investigate the potential common mechanisms in the basal ganglia underlying action and thought stopping, we conducted meta-analyses using fMRI data from the Go/No-Go, Stop-signal, and Think/No-Think tasks. All three tasks require active stopping of prepotent actions or thoughts. To localise basal ganglia activations, we performed high-resolution manual segmentations of striatal subregions. We found that all three tasks recovered clusters in the basal ganglia, although the specific localisation of these clusters differed. Although the Go/No-Go and Stop-signal tasks are often interchangeably used for measuring action stopping, their cluster locations in the basal ganglia did not significantly overlap. These different localised clusters suggest that the Go/No-Go and Stop-signal tasks may recruit distinct basal ganglia stopping processes, and therefore should not be treated equivalently. More importantly, the basal ganglia cluster recovered from the Think/No-Think task largely co-localised with that from the Stop-signal task, but not the Go/No-Go task, possibly indicating that the Think/No-Think and Stop-signal tasks share a common striatal circuitry involved in the cancellation of unwanted thoughts and actions. The greater similarity of the Think/No-Think task to the Stop-Signal rather than Go/No-Go task also was echoed at the cortical level, which revealed highly overlapping and largely right lateralized set of regions including the anterior DLPFC, VLPFC, Pre-SMA and ACC. Overall, we provide novel evidence suggesting not only that the basal ganglia are critical for thought stopping, but also that they are involved in specific stopping subprocesses that can be engaged by tasks in different domains. These findings raise the possibility that the basal ganglia may be part of a supramodal network responsible for stopping unwanted processes more broadly. •Stopping actions and thoughts both consistently activate the basal ganglia.•Action prevention and action cancellation engage distinct basal ganglia processes.•Thought stopping co-localises with action cancellation, but not prevention.•Basal ganglia may support a supramodal process cancellation mechanism. Much research has sought to understand how people stop prepotent responses when needed, a process known as inhibitory control. Although research on inhibitory control has often focused on stopping motor actions, there has also been significant interest in how people stop higher-level cognitive processes, such as memory retrieval. Recent evidence from neuroimaging studies suggests that inhibiting motor actions and memory retrieval may engage similar cortical mechanisms, and that a supramodal inhibition mechanism may be supported in part by the right dorsolateral and ventrolateral prefrontal cortices , the subthalamic nuclei (STN), and output via the internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr), the hyperdirect pathway instead involves direct cortical input to the STN and output through the GPi/SNr. Critically, human neuroimaging studies of motor inhibition have provided converging evidence for the importance of basal ganglia structures, such as the STN . HoweverAlthough basal ganglia mechanisms are clearly essential for motor inhibition, it is unclear to what extent they are dedicated to motor inhibition per se. It is possible, for example, that some functions performed by the basal ganglia during stopping may apply more broadly to higher-order cognitive processes . Both paIn addition to localising consistent basal ganglia activations, the current meta-analysis may also help to deduce cortical regions contributing to basal ganglia involvement during inhibitory control. Different subregions in the basal ganglia receive largely topographical projections from cortical areas with different functional roles . For exaTo address the preceding objectives, we conducted quantitative meta-analyses of brain activations from functional magnetic resonance imaging (fMRI) studies of motor and memory inhibition, including the Go/No-Go, Stop-signal, and Think/No-Think tasks . In a tyAll three of the foregoing tasks share the feature of having to stop an active process in either the memory or the motor domain. These tasks thus provide the opportunity to investigate the possibility that a supramodal inhibitory control mechanism contributes to stopping processes in general. If so, the meta-analytic clustering observed in each task may co-localise in the basal ganglia. However, each task may also engage different sub-processes through which stopping is achieved. For example, for motor inhibition, Unlike motor inhibition, there have been no formal studies of whether the basal ganglia consistently contribute to memory inhibition. We therefore used this meta-analysis to examine basal ganglia activations during the Think/No-Think task, and to compare any findings to activations observed during the Go/No-Go and Stop-signal tasks. If memory inhibition engaged the basal ganglia, it is unclear which motor inhibition task it might most resemble see . It is pTo examine the inhibitory control mechanisms in the basal ganglia in memory and motor inhibition, we compared meta-analytic activations both qualitatively by localising the clusters to specific basal ganglia structures, and quantitatively by computing conjunction and contrast maps between tasks. This coordinate-based meta-analysis approach is convenient for illustrating common activations across studies and task modalities. Nevertheless, this analysis method is, by design, more sensitive to spatially clustered activations than it is to activations that are more dispersed. Thus, if a given task does not yield significant activations in a region, by this analysis, it could either be because few activations occurred in that structure across studies, or instead because the activations were spatially dispersed. To distinguish these possibilities, we therefore subsequently visualised the activation foci in the basal ganglia, and computed descriptive summary statistics from the visualised data. These descriptive results give an impression of the dispersion of basal ganglia activations in each of the Go/No-Go, Stop-signal, and Think/No-Think tasks, and with that, provide clues as to whether an absence of significant activation clusters from the meta-analysis of a given task is driven by the absence of activity in the basal ganglia, or instead by a lack of clustered activity.1To characterise the specific localisation of basal ganglia activations, we manually segmented the caudate head, body, tail, and putamen subregions of the striatum, as existing atlases either do not have these subregions available, or have imprecise segmentations. For the other fine nuclei in the basal ganglia, we used an existing ultra-high resolution basal ganglia atlas . As sugg22.11.Only fMRI studies reporting results from whole brain analyses in a standardised coordinate space  were included;2.Only data from healthy adults were included;3.Only Stop-signal and Go/No-Go tasks where participants responded by hand were included;4.Only contrasts concerning differences between inhibition and an active condition were included, i.e. No-Think>Think, Stop>Go, and No-Go>Go. We requested the relevant data from each author if they were not already reported in the original article.Studies using the Go/No-Go and Stop-signal tasks were selected for the motor inhibition meta-analyses, whereas studies using the Think/No-Think task were selected for the memory inhibition meta-analysis. For the motor inhibition meta-analyses, we first identified studies from existing ALE meta-analyses . We thenAccording to these criteria, 16 Think/No-Think, 39 Stop-signal, and 30 Go/No-Go studies were identified  and inclp<.05, cluster-forming threshold uncorrected p<.001, threshold permutations=1000). Secondly, to examine the spatial convergence and divergence between different task domains, contrast analyses . The segmentations were performed using ITK-SNAP v3.2 (www.itksnap.org) from the high-resolution ICBM 2009b template structural image .To examine how clusters recovered from the meta-analyses of the memory and motor inhibition tasks related to the subregional anatomy of the basal ganglia, we projected the clusters onto 3D renderings of the subregions. These regions of interest (ROIs) in the basal ganglia were defined with both manual segmentation and an existing atlas . See The nucleus accumbens was excluded from the caudate head following guidelines provided by the Centre for Morphometric Analysis (CMA) for creating the Harvard-Oxford Subcortical Atlas  task, whereas significant activation clustering was localised to the right hemisphere for action cancellation  and memory inhibition (Think/No-Think) tasks. The following results sections will elaborate on findings in the basal ganglia. For a summary of all the basal ganglia results from the task-specific, conjunction, and contrast analyses, please see section 3.3.3.2.1On the whole, our analyses indicated that both action cancellation and prevention yielded clusters of activation in the basal ganglia. However, action cancellation yielded more spatially extensive clusters, which scarcely overlapped with the clusters from action prevention. The largely distinct localisation of basal ganglia clusters suggests that action cancellation and action prevention may engage distinct stopping processes that should not be assumed to be equivalent. This section illustrates these findings by detailing and comparing the clusters from the Stop-signal (cancellation) and Go/No-Go (prevention) tasks.3.2.1.1Across the 39 Stop-signal studies included in the analysis, cancelling a motor action yielded a consistent cluster in the right basal ganglia . First, 3.2.1.2Across the 30 Go/No-Go studies included in the analysis, preventing a motor action yielded a cluster in the left basal ganglia, including anterior putamen, spanning into anterior GPe, only touching on the medial wall of the caudate head . The put3.2.1.3From the meta-analyses of individual task types, it is striking that action cancellation and prevention shared so few clusters in the basal ganglia, given that the Stop-signal and the Go/No-Go tasks are often used interchangeably to measure response inhibition. To formally test whether action cancellation and action prevention engaged similar basal ganglia structures, we computed a conjunction analysis between the Go/No-Go and Stop-signal tasks. No overlapping clusters were identified in the basal ganglia at the current threshold, although subthreshold clustering might exist in the Go/No-Go task . It is unlikely that this lack of similarity between these tasks within the basal ganglia arises from insufficient statistical power, given the large number of studies included in the analysis. To the extent that interactions between cortical and basal ganglia structures are considered critical to the character of the stopping mechanism, these findings suggest that overlap at the cortical level between the Stop-signal and Go/No-Go tasks  may not Some have suggested that putative differences between the two tasks may be due to the variations in the administration of the Go/No-Go task . Typical3.2.1.4Visual comparison of the clusters yielded by the Go/No-Go and Stop-signal tasks suggests that action cancellation engages both STN and SN, but that action prevention does not. To determine whether these differences are reliable, we computed a contrast analysis between the Stop-signal and Go/No-Go tasks. The results confirmed significantly greater clustered activation during action cancellation in bilateral STN and the left SN than during action prevention , indicat3.2.1.53 in the right hemisphere and 216 mm3 in the left hemisphere) than preventing a motor action .As mentioned previously, we observed significant clustering in the right striatum and GPe, bilateral STN, and left SN in the action cancellation task. By contrast, significant clustering was limited to the left striatum and GPe in the action prevention task. To quantify the extensiveness of basal ganglia clusters yielded by these tasks, we compared the total volumes of the clusters from the task-specific analyses. At our current threshold , cancelling a motor action yielded more extensive basal ganglia activation clusters overall  than did action cancellation (1120 mm3).Across the 16 Think/No-Think studies included in the analysis, memory inhibition yielded a significant activation cluster in the right basal ganglia. This cluster was primarily located in the caudate head, spanning into caudate body, anterior putamen, and anterior GPe . This cl3.2.2.23) with the task-specific Think/No-Think and Stop-signal basal ganglia clusters, constituting 33% of the basal ganglia cluster volumes activated by memory inhibition, and 49% of those activated by action cancellation in the right hemisphere, or 41% overall. When considered together with the shared cortical activations in the right anterior and posterior DLPFC , we were unable to detect significant basal ganglia co-localised clusters between the Think/No-Think and Go/No-Go tasks. The lack of shared activations in the basal ganglia accords well with the minimal overlap with action prevention and memory inhibition at the cortical level . These f3.2.2.4To quantify the differences between memory inhibition, action cancellation, and action prevention, we computed contrast analyses between the Think/No-Think and Stop-signal tasks, and between the Think/No-Think and Go/No-Go tasks. Comparing the Think/No-Think and Stop-signal tasks, although both tasks yielded activation clusters in similar regions in the right basal ganglia, memory inhibition engaged the right anteromedial putamen and anterior GPe more than did action cancellation . This fiWhen we compared the Think/No-Think and Go/No-Go tasks , memory 3.2.2.5We also examined which regions yielded greater activation clustering during action cancellation than by memory inhibition. Our individual analyses had revealed bilateral STN and left SN activation clusters in action cancellation but not in memory inhibition. To formally test these differences, we computed a contrast analysis between the Stop-signal and Think/No-Think tasks. Our results revealed that action cancellation yielded reliably greater activation clustering in bilateral STN and ventral thalamus than did memory inhibition . Again, 3.2.3, including activations from the task-specific, conjunction, and contrast analyses. This summary shows that whereas the Go/No-Go task primarily engages the left putamen and GPe, the Stop-signal and Think/No-Think tasks primarily engage the right caudate, putamen, and GPe. In other words, our findings suggest that the cancellation of actions and thoughts engages similar basal ganglia structures, and that these are distinct from the basal ganglia structures engaged in the prevention of actions. This does not necessarily mean that none of the action prevention studies activated the same basal ganglia structures as action cancellation and memory inhibition. However, this does suggest that, if action prevention studies did activate the same structures , the activation peaks were not sufficiently spatially clustered to be detected with the ALE algorithm. We explore these considerations further in The basal ganglia ALE results are summarised in Fig. 3.113.2.4By definition, the ALE analyses are sensitive to common clusters of activation across studies. Activation peaks that are more spatially dispersed, either because a particular cognitive function is not associated with localised activation, or because the included studies are few in number, therefore might not be detected as common across studies. To explore this possibility further, we visualised the spatial distribution of peak activations across studies on 3D renders of the pre-defined basal ganglia ROIs , and cou4The current investigation examined the potential existence of common mechanisms in the basal ganglia that underlie the inhibition of actions and thoughts. Although the basal ganglia have an established role in motor inhibition, whether and how this structure is involved in memory inhibition remains unexplored. To address these issues, we conducted a set of meta-analyses using fMRI data from the Go/No-Go, Stop-signal, and Think/No-Think tasks. Whereas the first two tasks require inhibiting motor actions, the last task requires inhibition of episodic memory retrieval. After examining the ALE maps for each task, we computed conjunction and contrast analyses to formally examine the similarities and differences between the locations of significant basal ganglia clusters recovered in each task. Moreover, because the ALE analysis may be more sensitive to spatially clustered than spatially distributed activations, we also examined basal ganglia peak coordinates from the studies included to illustrate the prevalence and dispersion of basal ganglia activations yielded by each task. We localised the observed basal ganglia clusters as precisely as possible by manually segmenting the striatal sub-regions from a high-resolution template brain, including the caudate head, body, and tail, and the putamen. This is the first segmentation to our knowledge that has individual compartments of the striatum at this level of spatial resolution. Our key observations and their implications are discussed below.On the cortical level, our motor inhibition meta-analyses resembled prior meta-analytic findings , includiOn the subcortical level, although we observed basal ganglia clusters in all three tasks, the specific localisation of these clusters differed. Strikingly, the Go/No-Go and Stop-Signal tasks – two of the most widely studied forms of motor stopping that are often assumed to engage similar functions – showed clusters in different basal ganglia regions. Whereas the Go/No-Go task consistently activated the left anterior putamen (spanning into anterior GPe), the Stop-signal task yielded more extensive right-lateralised spatial clusters of activation mainly in the caudate head/body, anterodorsal putamen, anterior GPe. A formal conjunction analysis revealed that overlap between the activation clusters observed in these tasks was not statistically significant. These findings hold both when we included all 30 Go/No-Go studies, and when we excluded those with equiprobable Go and No-Go trials. To ensure that inhibitory processes are taking place, future Go/No-Go studies should always have infrequent No-Go trials in the design . The difAfter comparing the Go/No-Go and Stop-signal tasks, we examined whether the basal ganglia were involved in stopping memory retrieval. Interestingly, we found that, like stopping actions, stopping thoughts also engages the basal ganglia. Memory inhibition in the Think/No-Think showed a consistent cluster of activation in the right caudate head/body, anterodorsal putamen, and anterior GPe. This cluster of activations was exclusively right lateralised, and was more spatially extensive than the analogous clusters from the motor stopping tasks. This clearly indicates that basal ganglia structures play an important role in stopping retrieval, perhaps akin to its role in stopping actions. This commonality raises the possibility that basal ganglia structures are involved in stopping in a more general way than is usually assumed in research on motor inhibition. A similar supramodal hypothesis was discussed by Although both memory and motor inhibition activated the basal ganglia, the pattern of activations in that structure provides converging evidence that memory inhibition in the Think/No-Think task may be more similar to action cancellation in the Stop-signal task than it is to action prevention in the Go/No-Go task. Consistent with their strong overlap at the cortical level , our conDespite the foregoing between-task differences in the STN activation clustering, the overall similarity between the clusters observed for memory inhibition and action cancellation in the striatum and GPe suggests that inhibiting thoughts may require active cancellation. This observation argues against the possibility that people prevent retrieval of an unwanted item by simply directing the retrieval process to distracting thoughts, or, instead, by passively failing to engage retrieval. Rather, the recruitment of cancellation-related striatal processes suggests that retrieval is being actively stopped. This interpretation converges with findings indicating that the engagement of inhibitory mechanisms during retrieval stopping is particularly robust when memories intrude into awareness and need to be purged . Using tOur findings raise questions about the connectivity underlying these dynamic interactions. Of particular interest is the connectivity of these basal ganglia regions with other putative supramodal areas associated with inhibitory control , and also with domain-specific regions involved in memory and action, such as the hippocampus and the primary motor cortex (M1) respectively. For example, in our meta-analyses, by localising clusters within the basal ganglia, we observed that all of the Go/No-Go, Stop-signal, and Think/No-Think tasks recovered clusters in the centromedial striatum, including the caudate head/body, spanning across the internal capsule into medial putamen. This cluster roughly coincides with the region identified by Despite its similarity to action cancellation, the memory inhibition cluster extended to parts of the right putamen and GPe more than did motor stopping in general. It is unclear what functions these potentially memory-inhibition-specific activations of putamen and GPe may be performing, or whether these functions are unique to this process or simply a more robust and spatially extensive engagement of putamen processes observed during action cancellation. The possibility that parts of the putamen may serve functions specific to memory control should be considered. It is worth noting, for example, that although the putamen is often seen as a motor structure , recent On top of the established network of motor control involving the basal ganglia, several authors have discussed potential interactions between the basal ganglia and the hippocampus. While some found that the basal ganglia and the hippocampus may be largely independent from each other , others Despite this evidence that the basal ganglia and the hippocampal systems are independent or interact through the prefrontal cortex, other evidence has suggested that the basal ganglia and hippocampus may interact in other ways. For example, Sabatino and colleagues found evidence that basal ganglia activity influences hippocampal oscillations. Specifically, whereas caudate stimulation appeared to influence the hippocampal theta rhythm by inhibiting the hippocampal spikes , pallidaUltimately, determining the extent to which a supramodal cancellation process truly exists will depend on whether intrinsic basal ganglia pathways are engaged in similar ways for memory and motor inhibition tasks, including the direct, indirect, and hyperdirect pathways. Unfortunately, meta-analytic activations do allow us to determine which specific pathways are required by these tasks. For example, increased striatal activity may imply engagement of either the direct or indirect pathway, or an interaction between the two. Similarly, increased STN activity may indicate engagement of either the hyperdirect or the indirect pathway. Despite these limits on our data, it is worth considering how a supramodal cancellation process might emerge from these pathways. In one recent effort  analysedAlthough we sought to localise basal ganglia clusters in memory and motor inhibition tasks, our approach is not without caveats. For example, Furthermore, the ALE activation maps, rightfully, model the spatial uncertainty of the reported peak coordinates from each study, which introduces a certain level of spatial smoothness. These factors also recommend caution when drawing conclusions about the precise localisation of our observed activations, given limitations on spatial resolution inherent to the meta-analytic method. Reporting bias is also a consideration, because some researchers may choose to omit activation peaks that do not fit prior expectations for a task, especially if the spatial extent of the activation is small, as would be true for some of the structures of key interest within the basal ganglia. These caveats have led some to argue that results from coordinate-based meta-analysis should be treated as an integration of existing knowledge instead of the absolute truth , as moreOne final caveat, applicable to this and all other ALE meta-analyses, concerns how to interpret lack of significant clusters in a structure of interest. One hand, failing to find a significant cluster for a particular task may indicate that the structure is genuinely not engaged in the task. On the other hand, because the ALE algorithm seeks to identify clusters of activation, lack of a significant cluster may also be consistent with the presence of more dispersed activation peaks that fail to constitute a significant cluster. Indeed, the visualisation and counts of activation peaks in the left and right basal ganglia show clearly that there exist activations in basal ganglia structures in both hemispheres, especially for our two motor stopping tasks see . Thus, wDespite these limitations, our meta-analyses have provided the first meta-analytic evidence that memory and motor inhibition (action cancellation in particular) engage overlapping regions within the basal ganglia. These patterns suggest that similar frontostriatal pathways may be involved when people stop thoughts or actions. Moreover, by localising the observed clusters within our high-resolution manual segmentation of striatal subregions, we hope that our results can serve as a useful reference against which the results of future studies may be compared.5The current meta-analyses demonstrate that the basal ganglia are consistently activated in the inhibition of both actions and thoughts. This basic finding is broadly congruent with recent literature indicating that the basal ganglia are not merely involved in motor control, but also in higher-level cognitive processes, such as memory. Importantly, however, the surprising similarity of memory inhibition to action cancellation more than action prevention suggests that the nature of the stopping processes that are recruited may dictate the localisation of basal ganglia activity more so than does task domain, at least for the tasks we studied. Our data indicate that, during cancellation, similar cortical and basal ganglia regions are engaged, irrespective of the domain of the process that is controlled, consistent with the possibility of a supramodal cancellation process. Meanwhile, the differences in activation clusters between the Go/No-Go and Stop-signal tasks suggest that they may engage different stopping processes and that it may imprudent to treat these tasks equivalently. However, it bears emphasis that the current ALE meta-analysis is more sensitive to clustered activations than to dispersed ones. The inference that motor cancellation and motor prevention are distinctly localised in these data depends on the assumption that highly clustered activations (as detected by ALE) provide a more informative signature of functional specialization in the basal ganglia than more dispersed activations would, an assumption that deserves to be critically examined when more data is available. Importantly, future studies should characterise the specific basal ganglia engagement in memory and motor inhibition and investigate how the frontal, basal ganglia, and domain specific target regions  interact to perform specific stopping processes in different task domains. Extending the study of the role of the basal ganglia in inhibitory control to measure the stopping of both actions and thoughts will provide a valuable source of constraint on hypotheses about the computational functions that the basal ganglia perform.


We measured photosynthetic rates, oxidative stress levels, antioxidant contents, antioxidant enzyme activities, and activities of oxidative metabolic enzymes in S. vulgare growing at a natural acidified site (pH 6.7) compared to samples from a site with current pH (pH 8.2), used as a control one. These variables were also tested in plants transplanted from the control to the acidified site and vice-versa. After short-term exposure, photosynthetic rates and energy metabolism were increased in S. vulgare together with oxidative damage. However, in natural populations under long-term conditions photosynthetic rates were similar, the activity of oxidative metabolic enzymes was maintained, and no sign of oxidative damages was observed. The differences in the response of the macroalga indicate that the natural population at the acidified site is adapted to live at the lowered pH. The results suggest that this macroalga can adopt biochemical and physiological strategies to grow in future acidified oceans.Studies regarding macroalgal responses to ocean acidification (OA) are mostly limited to short-term experiments in controlled conditions, which hamper the possibility to scale up the observations to long-term effects in the natural environment. To gain a broader perspective, we utilized volcanic CO Marine macroalgae are a large and diverse group of photoautotrophs that contribute significantly to global primary production and to blue carbon sequestration . In addi2 concentrations in the atmosphere and in the oceans. Theoretically, elevated CO2 stimulates photosynthesis, and therefore OA should benefit marine autotrophs (2 at low percentage (1%), carbonate (CO32-), and the most abundant bicarbonate 2 via diffusion; however, this mechanism is around 10,000 times slower in water than in air  . All macn in air . Therefoynthesis . Hence, estments  to allowestments . When thgy costs . Howevertrations . Consisttrations .2 concentration could also affect other physiological processes, including reproduction, ion homeostasis, energy metabolism, and nutrient uptake ; Environmental changes often affect the production of reactive oxygen (ROS) and nitrogen (RNS) species in macroalgae . The acclower pH . The pose stress . Elevatetiolecta . Howevertiolecta . These sMost of our understanding of the effect of OA on macroalgae is obtained in confined short-term studies. In contrast to natural environments, such studies mainly involve a single species and conditions of constant and stable carbonate chemistry parameters. Based on these studies, it is difficult to predict how macroalgae will respond to OA in natural ecosystems. Moreover, the relevance of short-term studies to understand longer-time scale adaptive responses is questionable. Consequently, there is an increasing interest to assess adaptive response and potential of marine organisms to face climate change stressors over longer-time scales . The mosSargassum vulgare, whose settlement dates back at least three decades  and Lacco Ameno  . Castello Aragonese is the site where underwater CO2 vents lower the local pH. These venting activities date back to nearly 2000 years (2 (90.1–95.3%) in absence of harmful sulfur gas or effects on seawater temperature , wave exposition (sheltered bays), PAR transmission properties of the water, temperature and salinity  in Lacco Ameno were tied in a net and moved to the acidified site (A) in Castello Aragonese (C-A), and vice-versa (A-C). In order to evaluate the stress effect due to the transplant itself, other thalli were also transplanted in their respective natural site (C-C and A-A) and used as controls.in situ using a Diving-PAM (Pulse Amplitude Modulated) fluorometer  on natural populations from both sites and on the reciprocal transplants (2 weeks after transplantation). To avoid differences due to variable environmental conditions, all analyses were performed on the same day between 11 am and 1 pm.Photosynthetic parameters were measured 2. In both locations, a total of nine thalli of similar size (8–10 cm frond length) were handpicked in three different patches by snorkeling along a coastal stretch of 15 m to cover the natural variability of the two local populations. For the analyses on in situ reciprocal transplants, samples were collected in the same way at 2 weeks after transplantation.The samples for biochemical analyses on natural populations were collected in March and July on the same day at approximately the same time (between 11 am and 1 pm) in order to avoid effects of environmental fluctuations other than pH and COThe collected samples were maintained onboard in water of their respective sites and brought to the laboratory where the tissues were washed with filtered sea water, using a soft paint brush to remove visible epiphytes. The samples were either processed immediately or snap frozen in liquid nitrogen and stored at -80°C for further analysis.S. vulgare (settled in the control and the acidified site) to use 2 is so low that it limits photosynthesis for obligate CO2 using macroalgae (2 vents at the time of harvest). Actinic light was supplied at a level of ca. 200 μmol photons m-2s-1 at 25°C. After 24 h, the algae were removed from the containers and pH was recorded. The containers were left open for 24 h before the pH was measured again.A preliminary experiment was conducted in the lab in order to determine the ability of the two populations of croalgae . To test-2 s-1, produced by the Diving-PAM lamp, and lasting 10 s after each 10 min dark adaptation period. Photosynthetic parameters, such as relative maximum electron transport rate (rETRmax), initial slope of the curve (α), and the saturating irradiance (Ek) were calculated by fitting empirical data to an exponential function of Fv/Fm) of the two populations. Chlorophyll a and c content was determined in tissues extracted in 80% acetone according to The collection and processing of fluorescence data, obtained using a Diving-PAM fluorometer , were performed fundamentally following the guidelines suggested by 2O2) content was measured in the frozen algal tissues by the FOX1 assay, based on the peroxide mediated oxidation of Fe2+, followed by reaction of Fe3+ with xylenol orange. Absorbance of the Fe3+-xylenol orange complex was measured at 560 nm . Total ASC (ASCt) and GSH (GSHt) concentrations (reduced + oxidized) were determined after reduction with 0.04 M DTT for 10 min at room temperature according to Intracellular ROS in the algal tissues were quantified by measuring the oxidation of 2′,7′-dichlorohydrofluorescein diacetate  according to t 560 nm . Lipid pFor antioxidant enzymatic assays, protein extracts were prepared according to n = 3) by spectrophotometric methods. NADH-DH activity was measured according to L-3,4-dihydroxyphenylalanine (L-DOPA) to suppress NADH-DH activity were used as negative controls. The absorbance was measured at 410 nm and NADH-DH activity was calculated using an extinction coefficient of 1 mM-1 cm-1. COX activity was determined according to The activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (COX) were measured in the algal tissues (n = 5) were measured with the Griess reagent according to S-nitrosothiol content was determined according to Nitric oxide levels in the algal tissues .Student’s S. vulgare to use Samples collected from the acidified site and incubated for 24 h in sea water at pH 8.08 and 7.0 in closed containers raised the pH to 9.14 ± 0.01 SE and 9.05 ± 0.05 SE, respectively. Samples from the control site, treated following the same protocol, showed an increase of the pH values to 9.16 ± 0.03 SE and 9.1 ± 0.04 SE, respectively. The ability to raise pH above 9.0 demonstrates the capacity of Fv/Fm in samples transplanted from the acidified to the control site (Table 1). The concentration of chlorophyll c was higher in algae from the acidified site and in samples transplanted from the acidified to the control site (Table 1).We did not observe any significant differences in the photosynthetic performance between algae naturally growing at the acidified and the control site, respectively. However, in the transplants there was a significant increase in rETRmax and Ek and a decrease in α in plants transplanted from the control to the acidified site in comparison to those transplanted in the same site. We also noticed a decrease in S. vulgare, we examined the cellular redox state by measuring ROS, H2O2, lipid peroxidation, total antioxidant capacity, nitric oxide, and protein nitrosothiol levels. Total ROS was lower in the algae grown at the acidified site compared to those from the control site , but no differences in H2O2, MDA, and total antioxidant capacity were observed between the two sites . In samples transplanted from control to acidified site, there was a significant increase in total ROS, H2O2, MDA, and total antioxidant capacity . Inversely, H2O2 and total antioxidant capacity decreased in algae transplanted from acidified to control site . No significant variation was observed in nitric oxide levels , whereas lower levels of S-nitrosylated proteins were detected in algae living at the acidified site compared to those of the control site . No changes in nitrosylation were found in transplants .In order to understand if OA induces oxidative and nitrosative stress in Table 2). ASCt and ASC redox states were generally lower in the acidified site; however, the difference was significant only in samples collected in March. ASCt was increased in samples transplanted from the acidified to the control site. GSHt and GSH redox states were affected only in natural samples collected in March only. The level of α-tocopherol was higher in the algae at the acidified site in both seasons. α-tocopherol levels consistently increased in transplants from the control to the acidified site and decreased in samples transplanted from the acidified site to the control conditions. β- and γ-tocopherols were increased only in samples transplanted from the acidified to the control site. Polyphenol and flavonoid levels were lower in the algae living for a long time at the CO2 vents site. Moreover, polyphenol levels decreased when algae were transferred from the acidified to the control site.As the total antioxidant capacity reflects overall changes in small molecular antioxidants, we separately quantified the levels of the major antioxidant molecules, including ASC, GSH, tocopherols, polyphenols, and flavonoids  and in samples transplanted from the control to the acidified site, while the activity decreased in transplants from the acidified site to the control one . The activities of H2O2 scavenging enzymes showed variable responses . In algae living at the acidified site, the activities of APX, GPX, and TRX were higher in both seasons compared to control samples, while in the case of PRX higher values were observed only in March samples. Inversely, levels of CAT, MDHAR, POX, GST, PRX, and GRX activities in samples collected at the acidified site were lower than the control samples. This trend was observed in both seasons for MDHAR, only in March for CAT, and only in July for the other enzymatic activities . Upon short-term transplants of algae from the control to the acidified site, an increase in the activities of CAT, APX, DHAR, GPX, PRX, and TRX was observed. Inversely, APX, DHAR, POX, PRX, and TRX activities decreased when samples were moved from the acidified to the control site.To understand the mechanisms responsible for the maintenance of the cellular redox state in the algae living under contrasting conditions, we investigated the enzymatic components of the antioxidant machinery. SOD activity was higher in samples at the acidified site . Transplantation from the control to the acidified site also caused increase of these enzymatic activities , whereas the inverse transplantation caused a decrease in COX activity .To examine whether OA affects the energy metabolism in this species, we measured the activities of oxidative metabolic enzymes. Higher activities of NADH-DH and COX were detected in algae living at the acidified site  could be related to the presence of carbon concentration mechanisms which rely on S. vulgare was indeed 2 into the cells under acidified conditions, the capacity for in situ on the brown alga Lobophora variegata (Posidonia oceanica (L.) Delile  revealed an increase in rETRmax, in line with the positive photophysiological response observed in Cystoseira compressa after short-term transplant at CO2 vents off the island of Vulcano , suggesting again that the algal population at the vents is adapted to grow under the acidified conditions.The lack of significant changes in the photosynthetic parameters of the lization . The resariegata  and the ) Delile . In contniculata  and in tw Guinea . Our sho Vulcano . The finnean Sea . Further2 causes seawater acidification which has the potential to affect metabolism and cellular homeostasis, thereby impairing cellular function or increasing energy demand, as it has been found in cyanobacteria and phytoplankton . However, in natural populations under long-term conditions ROS values were lower in the acidified site than those in the control one, whilst H2O2 and lipid peroxidation were comparable. These results could be explained with a higher efficiency of energy transfer reactions, suggesting the capability of the acidified population to overcome the negative effects of lowered pH and to sustain itself in the future acidified ocean, if acclimatized for longer periods. This is also supported by the finding that the levels of nitric oxide, which has been reported to be involved in different physiological responses in marine photosynthetic organisms . SOD represents the first line of antioxidant defense in marine algae (2O2 in S. vulgare in short-term acidification may be removed by increased CAT activity. The ascorbate-glutathione (ASC/GSH) cycle, a major mechanism of H2O2 control in autotrophs  when transferred to the control site, further supporting the idea that populations living for some decades at the vents site are adapted to grow under acidified conditions. The occurrence of oxidative stress in short-term conditions suggests that macroalgae need longer time to overcome the effects of acidification. However, utilizing molecular and enzymatic antioxidants, S. vulgare is capable to mitigate stress effects and adapt to acidified conditions.The different response of S. vulgare at the acidified site . The increase in the photosynthetic performance, and a higher energy production would be useful in maintaining ion-homeostasis and for enhancing growth. ROS, nitric oxide, and other redox molecules would be contributing toward maintaining cellular signaling and genetic regulation under acidified conditions. The short-term acclimation responses seem to allow S. vulgare to adapt to the acidified conditions, resulting in a population with a more active energy metabolism, without signs of oxidative stress and changes in photosynthetic efficiency. Overall, the results obtained in this study suggest that S. vulgare could be expected to be among the species benefitting from future acidified ocean.Based on these results, a series of events have likely happened in in situ transplant experiments, AK and ML performed in situ photosynthetic measurements, AK and HAE performed biochemical and physiological experiments and collected data, AK, HAE, IC, MCB, and AP analyzed output data and results, GB and HA provided Materials and Methods for biochemical tests, AK wrote the first draft of the manuscript, and all the authors contributed substantially to the interpretation and final version of the paper.AK, AP, MCB, IC, and MD designed the study, AK and MCB performed sample collection and The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. We present an integrated approach for highly sensitive identification and validation of substrate-specific kinases as cancer biomarkers. Our approach combines phosphoproteomics for high throughput cancer-related biomarker discovery from patient tissues and an impedimetric kinase activity biosensor for sensitive validation. Using non-small-cell lung cancer (NSCLC) as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed 198 phosphoproteins that are over-phosphorylated in NSCLC. Among the differentially regulated phosphorylation sites, the most significant alteration was in residue S165 in the Hepatoma Derived Growth Factor (HDGF) protein. Hence, HDGF was selected as a model system for the electrochemical studies. Further motif-based analysis of this altered phosphorylation site revealed that extracellular-signal-regulated kinase 1/2 (ERK1/2) are most likely to be the corresponding kinases. For validation of the kinase–substrate pair, densely packed peptide monolayers corresponding to the HDGF phosphorylation site were coupled to a gold electrode. Phosphorylation of the monolayer by ERK2 and dephosphorylation by alkaline phosphatase (AP) were detected by electrochemical impedance spectroscopy (EIS) and surface roughness analysis. Compared to other methods for quantification of kinase concentration, this label-free electrochemical assay offers the advantages of ultra-sensitivity as well as higher specificity for the detection of cancer-related kinase–substrate pair. With implementation of multiple kinase–substrate biomarker pairs, we expect this integrated approach to become a high throughput platform for discovery and validation of phosphorylation-mediated biomarkers. Developing kinase-based diagnostic tools for cancer involves the identification of the target kinase whose expression is altered in cancer, followed by the development of sensitive methods for detecting the activity of this specific kinase.12 The aberrant kinase could ubiquitously affect the signal transduction networks across multiple pathways. A kinase detection method capable of detecting its phenotype-dependent substrates may provide better specificity to monitor a disease related to a specific pathway. Due to their medical and biological significance, many methods for the detection of kinase activity have been previously demonstrated. These include radiometric assays,13 spectroscopic assays15 and electrochemical methods. Electrochemical methods have the advantages of avoiding work with radioactive materials, higher detection sensitivities and consumption of lower amounts of analyte without the need for amplification systems.17 Diverse applications of electrochemical methods for detecting kinase activity were reported. These include the use of ferrocene labeled ATP10 or detection with antiferrocene antibodies in an immunoarray format.18 In label-free methods, the detection is based on repulsion between the negatively charged phosphate groups and negatively charged redox species in the solution that is translated in EIS measurements to increase in the resistance to charge transfer (RCT).19 Alternatively, there is attraction of a cationic redox species to the phosphate group on the peptide that is translated in cyclic voltammetry (CV) measurements to increase in current.20 We have previously reported a label-free electrochemical kinase detection method, which is based on a peptide monolayer that changes its conformation upon phosphorylation from a densely packed monolayer into a disordered one, causing a sharp drop in RCT. This approach has a built-in amplification mechanism, which is based on a reversible order–disorder transition that the peptide monolayer undergoes due to its phosphorylation. This order–disorder transition changes the electronic properties of the layer from a good ionic insulator to an ion-permeable peptide layer and does not rely only on ionic repulsion/attraction from the phosphorylated peptide layer.20The human kinome contains over 500 identified kinases22 Using NSCLC as a proof-of-concept study, label-free quantitative phosphoproteomic analysis of a pair of cancerous and its adjacent normal tissues revealed site-specific elevated phosphorylated proteins/peptides in NSCLC. Through sequence motif-analysis and literature mining among the 198 most dramatically up-regulated phosphopeptides, HDGF was chosen as a model protein for the electrochemical studies. A peptide derived from the HDGF phosphorylation site was coupled to a gold electrode and electrochemical impedance spectroscopy was utilized to probe its phosphorylation by ERK2 and dephosphorylation by AP. The unification of proteomics and electrochemistry enabled us both to identify the cancer-related target sequence of the kinase and to detect the kinase levels at low concentrations. The developed method is general and could be applied to large-scale biomarker discovery and validation for other diseases such as other cancer types or neurodegenerative diseases.Here we report an integrated platform combining quantitative tissue phosphoproteomics for kinase–substrate biomarker discovery and electrochemical kinase detection method for ultra-sensitive kinase–substrate biomarker validation. This can be used as a step towards individualized cancer diagnostics. Phosphoproteomic approaches have demonstrated effective identification of the abnormal protein kinase activity involved in cancer.25 The phosphopeptides enriched from the IMAC were analyzed by LC-MS/MS (Triple-TOF 5600) for phosphoproteomic analysis. A total of 1451 phosphopeptides (derived from 540 phosphoproteins) were confidently identified by the Mascot search engine and quantified by the IDEAL-Q software.26 To identify kinase/substrate pairs that are over-activated and can serve as biomarkers involved in NSCLC, the extent of over-phosphorylation of every phosphopeptide in the tumor tissue was quantitatively compared by defining: T/N ratio = amount of phosphorylation in tumor tissue/amount of phosphorylation in adjacent normal tissue. The specific target phosphoprotein was selected by a series of steps  website-based software.31 ERK1/2 were shown to be potential kinases that are able to phosphorylate HDGF on serine 165  value (607.62 for charge of 3+). The serial of y-fragment ions (y10–y14) retaining the phosphate group in the MS/MS spectrum shown in in vitro kinase reaction at the protein level. The ERK kinase reaction for the dephosphorylated HDGF protein showed the same phosphorylation site at serine 165  increased over time and was at a plateau after 16 hours. We concluded that 16 hours of incubation is the optimal time for the adsorption of the peptide. The data was transferred to surface coverage (θ) using the expression:with Z′ being the real impedance at each point, Z′initial – the value of the clean gold and Z′final – the final value at the plateau. The adsorption isotherm was fit to the equation:34(1 – θ) = αek1t[C]– + (1 – α)ek2t[C]–where θ is the fractional surface coverage, k1 and k2 are the rate constants that were found to be k1 = 55.5 ± 0.6 M–1 s–1 and k2 = 0.858 ± 0.004 M–1 s–1, C – the initial concentration of the peptide solution (0.1 mM). The value of the coefficient α is 0.677 ± 0.001.The non-phosphorylated form of the selected HDGF peptide was synthesized with an N-terminal cysteine added for binding to gold electrodes  compared with the impedance of the bare gold electrode (380 Ω)  . The incRCT, showing that phosphorylation indeed took place (Rsol.(Q[RCTW])]] inset. TRCT decreased by 86% to 474 Ω. The reversibility of the phosphorylation reaction was examined by performing dephosphorylation with Alkaline Phosphatase (AP). Following dephosphorylation, the RCT increased back to 3250 Ω – almost to the starting value. A reversible change is evident also in α, the exponent, changing after phosphorylation from 0.94 to 0.90 and changing back to 0.94 after dephosphorylation.Following incubation with ERK2, the RCT decreased by only 12% from 4080 Ω to 3600 Ω. This shows that CaMK2 has much lower activity towards HDGF 160–174 and thus demonstrates the good selectivity of our assay.To verify the selectivity of the substrate-dependent kinase assay, the same process of phosphorylation and dephosphorylation on HDGF 160–174-covered electrodes was tested with CaMK2, which is not known to phosphorylate the HDGF 160–174 sequence . After i2). The orientation of most facets on the grains reveals the (111) plane of the fcc structure and have a triangular shape.35 Each triangle is an atomically flat surface. AFM characterization of annealed gold covered with the HDGF 160–174 peptide revealed a very smooth and homogeneous surface  roughness was calculated on small areas, 10 000–100 000 nm38 and so were detection methods for specific kinases.17 We combine the two aspects and show a direct path from the tissue level to the kinase detection stage. Specifically, we show that the over-phosphorylation level of proteins in tumour samples can be determined by proteomic tools. The identification of over-phosphorylated residues in a target protein leads to kinase identification and the use of a peptide containing the phosphorylation site in an electrochemical assay provides a powerful tool for kinase detection. Our detection method is unique, because it is label-free and very simple – the biosensor is composed of only the peptide and a simple gold macro electrode. This is unlike other electrochemical kinase detection methods that utilize a variety of labels and amplification methods.42In this work we present a novel approach for kinase related cancer research. We show that discovery and detection of over-phosphorylated proteins and the relative kinases can be done in a relatively simple manner, which is very versatile. Methods for detection of phosphorylated proteins in tissues had been suggested before36 Phosphoproteomics has also been applied to directly identify kinase inhibitor targets in cancer cell lines and mouse xenografts.43 Though these studies show the promise of phosphoproteomics to provide new targets for designing anticancer therapies, most current approaches are not accessible to individualized human tissue analysis yet. This advantage of our approach is critical since it enables to reflect the human microenvironment as well as the difference between individual patients.Protein kinases are highly attractive targets for therapeutic intervention. Therefore, identification of their pathway-associated phosphorylation substrates may generate biomarkers to aid either the disease detection or the drug development process. The selection of the kinase/substrate target is the most crucial aspect of drug target identification. Proteomics technology is a powerful tool for discovering aberrant protein expression and post-translational modification associated with disease. The polyMAC has been applied to tissue phosphoproteomics in transgenic mouse model of HER2 positive breast cancer and identified potential therapeutic targets.To our knowledge, our study is the first report of an individualized phosphoproteomic analysis on a pair of cancerous and adjacent normal tissues from the same patient. By our sensitive label-free quantitative phosphoproteomic strategy, the results revealed identification of over one thousand phosphorylation substrates in the NSCLC tumour tissue. Hundreds of abnormally activated phosphorylation substrates enriched in human lung cancer tissue served as starting point to search for their putative kinases by motif-based informatics analysis. By comparing to adjacent normal tissues, we could largely reduce the individual difference between samples. Through the logical dissection of the phosphoproteomics data by bioinformatics tools, the HDGF peptide and the corresponding ERK2 kinase were studied by the electrochemical impedance spectroscopy method. In addition to HDGF, dozens of up-regulated phosphoproteins identified by our platform had dramatic change in expression in the tumour tissue compared to the normal tissue and also passed our criteria since their function is related to cancer. With the added advantage of multiplexed phosphopeptide enrichment by automatic IMAC protocol, the phosphoproteomic method described herein is an efficient strategy for identifying the full panel of substrate–kinase biomarker pairs and can be applied in a large cohort of clinical samples.RCT values obtained compared with other studies.16 Previously, high initial RCT gave us better sensitivity in this system. The introduction of the negatively charged phosphate groups into a denser monolayer causes a more prominent change, resulting in a larger change in the RCT following phosphorylation. This is evident in RCT after phosphorylation.To provide a facile and sensitive assay to monitor the substrate-dependent kinase activity, we have devised an electrochemical assay using peptide-coated gold electrodes. The peptide assembly on the electrode is carried out in two steps – a fast adsorption process, beginning right at the moment of peptide injection, followed by a second, slower process. The rate constants show that the first adsorption process lasts until about two thirds of the adsorption sites are bound. Then the second process, which is likely to be an interaction between the peptides in the solution and the adsorbed peptide layers, takes place and slows down the adsorption of more peptides onto the surface. We interpret this barrier in terms of increasing peptide–peptide electrostatic and steric repulsion with increasing coverage. After the adsorption, the resulting monolayer was dense, as observed by the high RCT can be caused by many reasons, among them detachment of the peptides from the monolayer. We showed that the process is reversible, and harmless to the monolayer, by performing the dephosphorylation reaction with AP. AP removes the phosphate groups from the peptide, resulting in free hydroxyl groups. We observed that following dephosphorylation, the RCT of the peptide monolayer increased again, practically to the same starting value as of the native peptide. This reversible change may also indicate a change in the properties of the monolayer. Upon phosphorylation, the order of the monolayer is disrupted and more water molecules penetrate into the monolayer, facilitating the passage of the redox species and making it more conductive. The larger hydration layer of the phosphate group relative to the hydroxyl group most likely also influences the dielectric properties of the monolayer and therefore the diffuse layer of the electrode.A decrease in α, the exponent of the constant phase element, indicates a change in the homogeneity of the surface.45 The values of the parameter α are usually between 0–1, 1 indicating a perfect capacitor, and 0 – a perfect resistor. For flat metal electrode the value is close to 1. The decrease in α indicates roughening of the surface upon phosphorylation. This is in agreement with our model and the AFM results, showing the reversible formation of pinholes upon phosphorylation of the monolayer.Fitting the plots to the equivalent circuit shown in 46 is a secreted protein47 that stimulates growth of several cell types including smooth muscle cells,48 endothelial cells49 and lung epithelial cells.51 HDGF was previously linked to lung cancer and specifically to poor clinical outcome. It was shown to cause tumorigenesis in vivo.52 Exogenously supplied HDGF was found to enhance the invasiveness of breast cancer.47 Inhibition of HDGF using antibodies showed anti-tumor activity.50 The observation that HDGF is over-phosphorylated in NSCLC tissues is in agreement with previous studies, which showed that the S165A mutation in HDGF prevented the secretion of HDGF. The S165D mutation, which introduces a negative charge as the phosphorylation does, caused secretion of HDGF.46 Therefore, the phosphorylation on HDGF S165 is a valid target for cancer therapy based on kinase inhibitors.HDGF5 ERK1 and ERK2 share 83% amino acid identity, but despite their high similarity, they have distinct biological roles.55 They promote cell proliferation and metastasis.2 Over-activation of ERK1/2 is common in cancer, including NSCLC,56 prostate and breast cancers.58 Overexpression of HDGF causes activation of ERK1/2.59 Residues 8–17 on HDGF are a possible MAPK docking motif with 100% similarity to the consensus sequence.46 We show that HDGF S165 is a likely substrate of ERK2, and therefore inhibition of ERK2 could possibly inhibit the secretion of the phosphorylated HDGF, thus decreasing its tumorigenic activity.ERK1/2, also known as Mitogen Activated Protein Kinases 3/1 (MAPK3/1), are part of the MAPK/ERK pathway that communicates signals from the cell surface receptors to the nucleus.in vitro and MS/MS sequencing to confirm the phosphorylation site. The developed label-free electrochemical assay then provides rapid and sensitive quantification of the substrate-dependent kinase activity without the requirement for specific antibodies or electrochemical labels. Therefore our methodology is general and could be applied for a panel of substrate–kinase biomarkers. Integrated approaches for the mapping of phosphorylation sites and prediction of their corresponding kinase have been previously suggested.60 Our method shows, however, a simpler implementation from the patient to the detection stage. It can be used for finding new drug targets, for mapping over-phosphorylation in specific cancer types and matching specific conditions for a specific overactive kinase, thus facilitating the design of specific kinase inhibitors as personalized drugs.In this study we combined mapping of over-phosphorylated proteins in tumor tissues, quantification of the extent of over-phosphorylation that points to abnormal kinase activity in the tissue and the detection of a kinase that is likely to phosphorylate the substrate peptide sequence on the selected target protein. Using the model system of NSCLC, we demonstrated a sensitive integrated approach for targeted substrate–kinase pair selection based on differential phosphoproteomic profiling between cancer and adjacent normal tissues. This provides the advantage of individualized analysis as well as a generic platform to be applied to a specific type of cancer. The phosphorylating kinase can be predicted by bioinformatics tools and verified by phosphorylation iii) chloride (FeCl3), formic acid, acetic acid, HPLC-grade acetonitrile (ACN) and all of the other chemical reagents were obtained from Sigma-Aldrich . The bicinchoninic acid assay (BCA) protein assay kit was obtained from Pierce . Modified sequencing-grade trypsin was purchased from Promega . Ammonium persulfate (APS) and N,N,N′,N′-tetramethylenediamine (TEMED) were purchased from Amersham Pharmacia . Polypropylene frits disk was purchased from Agilent . Ni-NTA silica resin was purchased from Qiagen . SDB-XC Empore disks were obtained from 3 M . Water was obtained from a Millipore Milli-Q system . Cell lysis buffer was obtained from Cell Signaling Technology, Inc.Triethylammonium bicarbonate (TEABC), iron-. By bead mill homogenizer , the pre-cleaned 50 mg tissue samples were put into one tube contain 10 beads in 200 μL lysis buffer. The tissues were crashed in 20 seconds at 5500 rpm under 4 °C. The protein concentration was determined by BCA protein assay kit.We received the tissue from National Taiwan University Hospital and the approved IRB case number was 201104061RC . Frozen tissues were thawed rapidly at 37 °C, cut into small pieces, and washed by PBS to remove surface blood from tissues. The pre-cleaned tissues were homogenized in Triton cell lysis buffer containing 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na23 The protein sample was fixed into a gel directly in the Eppendorf vial with acrylamide/bisacrylamide solution , 10% (w/v) ammonium persulfate, 100% N,N,N′,N′-tetramethylenediamine by a 14 : 5 : 0.7 : 0.3 ratio (v/v). The gel was cut into small pieces and washed several times with 25 mM TEABC containing 50% (v/v) ACN. The gel samples were further dehydrated with 100% ACN and then completely dried by vacuum centrifugation. Trypsin was then added into gel for proteolytic digestion  in 25 mM TEABC with incubation overnight at 37 °C. Digested peptides were extracted three times with 5% (v/v) FA in 50% (v/v) ACN for 30 min, dried completely by vacuum centrifugation at room temperature.The protein samples from tissue samples were subjected to gel-assisted digestion.25 The in-house-constructed IMAC tip was capped at one end with a 20 μm polypropylene frits disk enclosed in a tip-end fitting. The tip was packed with 20 mg of Ni-NTA silica resin. First, Ni2+ ions were removed with 50 mM EDTA in 1 M NaCl. The tip was then activated with 100 mM FeCl3 and equilibrated with loading buffer (6% (v/v) acetic acid (AA) at pH 3.0) prior to sample loading. Tryptic peptides were reconstituted in loading buffer and loaded onto the IMAC tip. After successive washes with 6% (v/v) AA, 25% ACN, and 6% (v/v) AA, the bound peptides were eluted with 200 mM NH4H2PO4. The eluted peptides were desalted using reverse phase-Stage Tips(SDB-XC).61Phosphopeptide purification was performed using an IMAC protocol.2O) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The TripleTOF 5600 system  equipped with a nanoACQUITY UPLC  was employed for nanoLC-MS/MS analysis. The samples were loaded and separated on a 15 cm column with 100 μm inner diameters, packed in-house with 3 μm C18 particles . The LC mobile phase system consists of water with 0.1% acetic acid (buffer A) and acetonitrile with 0.1% acetic acid (buffer B). The separation gradient of buffer B was up to 80% in 120 min with flow rate of 500 nL min–1 in nanoACQUITY UPLC system. The TripleTOF 5600 was fitted with a Nanospray III source. Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 20 PSI, nebulizer gas of 15 PSI, and an interface heater temperature of 150 °C. For information dependent acquisition (IDA), MS survey scan range was m/z 300–1500 acquired in 250 ms. The top 10 precursor ions were selected if exceeding a threshold of 100 counts per second (counts per s) in each MS survey scan, and subsequent 10 MS/MS scans were performed for 200 ms each. Collision energy setting of 40 ± 15 eV was applied to all precursor ions for collision-induced dissociation. To minimize repeated scan, dynamic exclusion was set for 6 s and then the precursor was refreshed off the exclusion list.Purified phosphopeptides were reconstituted in buffer A . The mgf-files were searched using Mascot against the Swissprot database (Homo_sapiens) with the following settings: the fragment ion mass tolerance was set at 10 ppm, and the parent ion tolerance at 0.1 Da; for phosphopeptide search, phosphorylation (STY) and oxidation (M) were specified as variable modifications; for protein search, only oxidation (M) were specified as variable modifications. Peptides were considered identified when their Mascot Significance threshold is set as 62 If the delta score is greater than 5, the top ranked phosphorylated site is considered confidently determined. For delta score less than 5, the phosphorylated site assignment is considered ambiguous.To evaluate the protein identification false discovery rate (FDR), we repeated the searches using identical search parameters and validation criteria against a randomized decoy database created by Mascot. For phosphorylation site determination, we reported the difference in Mascot scores of peptides with top two assignments on different phosphorylated sites (delta score).26 The raw data files from the TripleTOF 5600 were transformed to mzML format using the AB SCIEX MS Data Converter. The search results in MASCOT were exported in eXtensive Markup Language data (.XML) format. After data conversion, the confident peptide identification results (p < 0.05) from each LC-MS/MS run were loaded and merged to establish a global peptide information list . Alignment of elution time was then performed based on the peptide information list using linear regression in different LC-MS/MS runs followed by correction of aberrational chromatographic shift across fragmental elution-time domains. To increase correct assignment, the detected peptide peaks were validated by the SCI validation using the three criteria: (a) signal-to-noise (S/N) ratio > 3, (b) accurate charge state and (c) correct isotope pattern. To calculate relative peptide abundance, the tool performs reconstruction of extracted ion chromatography (XIC), and calculation of XIC area. The fold-change of a given phosphopeptide was calculated by the ratio of relative peptide abundance between different samples.The quantitative analysis of phosphopeptide was performed by IDEAL-Q.2, 0.4 mg mL–1, 0.2 mM DTT), 5 μL of 250 μM ATP solution (Promega), and 4 μL of ERK2 as kinase . The mixture was allowed to react for 4 hours at 30 °C under constant shaking. Afterwards, the reaction was stopped by cooling on ice and acidifying the solution with TFA . The mixture was desalted using reverse phase-Stage Tips (SDB-XC)61 and the phosphopeptides were enriched using a Fe-IMAC protocol previously described.255 μL of 0.11 mM HDGF 160–174 solution (1 μg) were mixed with 5 μL of 2× kinase reaction buffer from Promega ADP Glo kinase assay . These electrodes were hand-polished on micro-cloth pads  with de-agglomerated alumina suspension (Buehler) of decreasing particle size (1.0 and 0.05 μm) using a homemade polisher. After polishing the electrodes were sonicated in triple distilled water (TDW) for 15 min followed by additional sonication in EtOH solution (50% v/v) for 10 min. The washed gold electrodes were then cleaned with warm HNO3 (70%) for 20 min. The electrodes were then rinsed with TDW and cycled from –0.4 to 1.6 V in 0.5 M H2SO4 at 0.1 V s–1 until stable reproducible cyclic voltammograms were obtained representing the electrochemical reduction of an AuO monolayer.Polycrystalline bulk gold electrodes with a surface area of 4 mmThe peptide used in the monolayer experiments was HDGF 160–174 with an added C-terminal cysteine for attachment to the gold surface (CDLLEDSPKRPKEAEN) . Treated gold macroelectrodes were immersed into prepared HDGF 160–174 peptide solution (0.1 mM) and were kept for 16 hours at 25 °C. After removal, the electrodes were rinsed three times in TDW and dried under nitrogen gas stream. Electrochemical characterization was preformed directly after the electrodes were washed. The peptide solution was prepared by dissolving the peptide in weakly basic TDW.2, 0.2 mg mL–1 BSA and 100 μM ATP. The reaction was initiated with the addition of the freshly prepared protein kinase solution and stopped by rinsing, after 30 min. Electrodes were washed multiple times in TDW, followed by a brief sonication in TDW.Peptide modified gold electrodes were reacted with Glutathione S-transferase (GST) – tagged ERK2 (New England Biolabs). Kinase mediated phosphorylation was performed by dripping of kinase solution on modified electrodes surfaces at 37 °C. Reaction medium  80 mM Tris–HCl pH = 7.5, 40 mM MgCl2, 0.2 mg mL–1 BSA, 2.4 μM calmodulin, 4 μM CaCl2 and 100 μM ATP.Phosphorylation with CaMK2 was done with CaMK2 (New England Biolabs) in a reaction medium containing 3 μM kinase in a final reaction volume of 40 μl) including 80 mM Tris–HCl pH = 7.5, 40 mM MgCl2. (One unit (U) is defined as the amount of enzyme that can hydrolyze 1 μmol of phosphorylated peptide in a total reaction volume of 1 ml in 1 min at 37 °C).Hydrolysis of surface grafted phosphorylated peptides was performed in the presence of AP, 0.1 Units in a final reaction volume of 40 μl, for 30 minutes at 37 °C. 1 Unit AP was dissolved in a 1 ml of tris borate buffer, (pH 8.5), containing 10 mM MgCl3[Fe(CN6)], 5.0 Mm K4[Fe(CN6)], and 0.1 M of KCl as supporting electrolyte. The spectra were recorded at a frequency range of 0.1 Hz–10 kHz with an amplitude of 10 mV, at the formal potential of the redox couple vs. Ag/AgCl electrode. The peptide adsorption was done over 15 hours. A three electrode cell, containing a clean gold electrode, was filled with a solution of 0.10 M phosphate buffer, pH = 8, that was also used as the supporting electrolyte, with added redox species: 5.0 mM K3Fe(CN6) and 5.0 mM K4Fe(CN6). The measurement was started, and after it reached a stable signal, a concentrated peptide solution was injected into the cell, to a final peptide concentration of 0.1 mM. Data points were measured every 25 s, at 100 Hz. The data was fit to an appropriate equivalent circuit using NOVA software.Electrochemical measurements were taken following each modification step. EIS measurements were performed using an Autolab PGSTAT12 digital potentiostat  connected to NOVA software. A conventional three-electrode cell was employed, with a peptide modified gold electrode as working electrode, and a standard Ag/AgCl reference electrode (Metrohm) with a KCl concentration of 3 M. The EIS measurements solution contained 5.0 mM K35 A HDGF 160–174 peptide layer was deposited by submerging the substrate immediately after annealing in peptide solution (0.1 nM) overnight. The samples were then washed with TDW and dried in a flow of nitrogen gas. AFM scans were performed with a Smart-AIST system  using Olympus tips  with cantilever force constant of 0.76 N m–1 and tip radius of 15 nm. To reduce surface damage, the scanning mode was true non-contact – verified by using tip-surface distance which was larger than the oscillation amplitude and with positive phase shift. Several macroscopically distant locations on each sample were scanned to eliminate “local effects” and at least five images of 1 μm × 1 μm were taken at each position. Scratches were done with 200 nN force. The scanning resolution was 1 pixel per nm. Image processing and analysis were done using WSxM 5.0 develop 7.0 software.63The substrates for AFM scans were freshly prepared annealed gold with atomically flat terraces.


Evidence has emerged to suggest that thrombi are dynamic structures with distinct areas of differing platelet activation. It was hypothesised that PGI2 could reverse platelet spreading by actin cytoskeletal modulation, leading to reduced capability of platelet aggregates to withstand a high shear environment. Our data demonstrates that post-flow of PGI2 over activated and spread platelets on fibrinogen, identified a significant reduction in platelet surface area under high shear. Exploration of the molecular mechanisms underpinning this effect revealed that PGI2 reversed stress fibre formation in adherent platelets, reduced platelet spreading, whilst simultaneously promoting actin nodule formation. The effects of PGI2 on stress fibres were mimicked by the adenylyl cyclase activator forskolin and prevented by inhibitors of protein kinase A (PKA). Stress fibre formation is a RhoA dependent process and we found that treatment of adherent platelets with PGI2 caused inhibitory phosphorylation of RhoA, reduced RhoA GTP-loading and reversal of myosin light chain phosphorylation. Phospho-RhoA was localised in actin nodules with PKA type II and a number of other phosphorylated PKA substrates. This study demonstrates that PGI2 can reverse key platelet functions after their initial activation and identifies a novel mechanism for controlling thrombosis.Prostacyclin (PGI However, upon vascular damage platelets overcome this inhibition allowing the formation of a thrombus. Within the thrombus, the activation status of the platelet is determined by the relative activatory and inhibitory signalling in the microenvironment of the vascular lesion and its location within the thrombus. Here, fully activated platelets are found within a core region, while those at the periphery are only weakly activated2. However, the diffusion of platelet agonists within the thrombus is not uniform3, and furthermore there is little known on the diffusion of the endogenous platelets inhibitors, NO and PGI2, within the thrombus. Furthermore as endothelial release of PGI2 is induced by the activity of thrombin4, this brings forward the idea that this induction of PGI2 by thrombin is not only limited to the inhibition of non-stimulated platelets, but also to reverse the activation of activated platelets within the thrombus in order to effectively control the extent of the thrombus formation induced by the injury.In healthy blood vessels platelets are exposed to endothelial derived nitric oxide (NO) and prostacyclin . Elevated cAMP activates PKA, the primary effector of cAMP signalling in platelets, leading to the inhibition of platelet functions in vitro and diminished platelet accrual at sites of vascular injury in vivo1. Once activated PKA isoforms blunt multiple aspects of platelet function including Ca2+ mobilisation, integrin activation, secretion, although knowledge of how these specific platelet functions are targeted by cAMP signalling remains unclear16. Proteomic studies have demonstrated that cAMP/PKA signalling targets a cluster of potential proteins associated with cytoskeletal reorganisation suggesting that the cytoskeleton is a major target in order to effectively mediate platelet regulation17. In this context, we have recently described a mechanism by which cAMP signalling prevented platelet shape change through inhibition of RhoA signalling and phosphorylation of myosin light chains (MLC)18. However, the mechanisms by which cyclic nucleotide signaling can return platelets to the resting state after activation, thereby preventing permanent attachment to the thrombus or at points distant to the injury, are unknown.Understanding the role of PGI2 can reverse platelet activation leading to alteration of the platelets function in a high shear environment.In the present study we examined the role of cAMP in modulating the function of activated platelets. We found that cAMP signaling can reverse stress fibre formation, significantly reduce the surface area coverage of the platelet, and induce actin nodule formation in a cAMP and PKA dependent manner. This reversal of stress fibre formation, and surface area coverage was associated with inhibitory phosphorylation and inactivation of both RhoA and the localisation of pRhoA to actin nodules. Furthermore these changes in the actin cytoskeleton were associated with a reduction in surface area coverage in platelets flowed over fibrinogen in high shear environment. This data identified that PGI2 (100 nM) can inhibit thrombus formation on fibrinogen19. However the effects of PGI2 on activated platelets has not been shown. Therefore, whole blood was flowed over fibrinogen (300 ug/ml) at arterial rates of shear (1000 s−1) resulting in the formation of small thrombi and a surface area coverage of 38.2 ± 6.8%  led to a significant reduction in surface area coverage to 24.4 ± 2.9% (p < 0.05). In contrast, perfusion of the thrombi with buffer alone had no significant effect on surface area coverage  was added to the spread platelets after 25 minutes, and spreading evaluated for a further 60 minutes  for 10 minutes. The platelets were then lysed and analysed for the content of filamentous actin. Control platelets underwent a 2.0 ± 0.2 fold increase in F-actin polymerisation, which was unaffected by stimulation with PGI2 (2.2 ± 0.3 fold increase), suggesting a targeted modulation of stress fibre formation   as a direct activator of adenylyl cyclase. The addition of Fsk (1 μM) to adherent platelets led to a significant and sustained reversal of stress fibre formation  and indomethacin (10 μM) prior to treatment with PGI2 (10 nM). The presence of apyrase and indomethacin reduced the number of adherent platelets, but did not influence surface area or actin structures of the adherent platelets. Importantly treatment of adherent platelets with PGI2 caused a reversal of stress fibre formation, production of actin nodules, and a reduction in surface area. This demonstrates that the effect of PGI2 on the actin cytoskeletal rearrangement is independent of ADP and TXA2 bioavailability  for 2 minutes led to a significant increase in cAMP from 152 ± 52 fmol/μg to 1576 ± 293 fmol/μg (p < 0.05) and was consistent with platelets in suspension stimulated with PGI2 for 1 minute25  and KT5720 (10 μM) were employed27. The presence of Rp-8cpt-cAMPs (500 μM) and KT5720 (10 μM) blocked the ability of PGI2 to reverse stress fibres, induce actin nodule formation, and reduce platelet spreading  or Fsk (1 μM) and the phosphorylation of key proteins evaluated. Confirmation of cAMP signalling in adherent platelets was illustrated by the sustained VASP-ser157 phosphorylation in response to PGI2 or Fsk  and Fsk (1 μM) stimulation, in a PKA dependent manner  in adherent platelets, but that addition of PGI2 significantly reduced the active RhoA present within the spread platelets  PGI2 can cause the reversal of surface area of platelet aggregates under high shear (ii) PGI2 can reverse platelet spreading via dissolution of stress fibres, and the formation of actin nodules in a cAMP/PKA dependent mechanism, (iii) PKA localises to the actin nodule, using it as a platform to mediate PKA mediated inhibitory phosporylation of RhoA. These data suggest that in addition to its already known roles in causing platelet inhibition prior to thrombus formation, cAMP signalling can control the size and stability of existing platelet aggregates via modulating the actin cytoskeleton, causing the aggregates to become vulnerable to high shear stress.Haemostasis relies on stable platelet adhesion for subsquent thrombus formation. The ability of platelets to undergo a rapid remodelling of the actin cytoskeleton is critical in order to withstand shear stress. Indeed an inability to form lamellipodia or stress fibres upon platelet activation and spreading is linked to thrombus instability and embolisation2 and the PKA signaling pathway have been implicated in actin cytoskeleton modulation in multiple cell types21. Although prior incubation of platelets with PGI2 and Fsk leads to inhibition of platelet adhesion, spreading and thrombus formation33, it is unclear if PGI2 can actively modulate a fully activated platelet. This is a critical idea as it demonstrates that PGI2 plays a dual role in the control of thrombus formation, both inhibition of platelets prior to activation, and reversal of platelets once they are already active. Both effects could then lead to reduction in thrombus formation. The reversal of platelet activation links into recent modelling of thrombus formation that suggests a dynamic structure with a core area, within which the platelets are fully activated and surrounded by a pheripheral area in which the activatory signals are much weaker2. The balance between the activatory and inhibitory signals acting on the platelets likely determines their activatory status. Therefore we set out to explore how PGI2 affected preexisting platelet aggregates. Using an in vitro flow assay we show for the first time that post perfusion of thrombi on fibrinogen with PGI2 caused a significant reduction in surface area. The ability of the platelet to withstand high shear is driven, at least in part, by the formation of remodelled actin structures within the platelet and we focussed our investigation on these processes. Platelet spreading was associated with progressive changes in actin structures including filopodia, actin nodules, lamellipodia and ultimately stress fibres. Addition of PGI2 to spread platelets induced cytoskeletal remodelling associated with stress fibre dissolution consistent with previous observations in other cell types34. The effects of PGI2 were mimicked by forskolin, but blocked by PKA inhibitors, indicating a central role for the cAMP-PKA signalling cascade.PGI8. Since the addition of PGI2 led to a loss of stress fibres, we speculated that PGI2 may target RhoA signalling in adherent platelets. Consistent with our previous studies in suspended platelets we found that PGI2 increased the inhibitory phosphorylation of RhoA at ser188 and reduced the GTP-loading of RhoA. Here we have extended our original findings to show that cAMP signalling can switch off activated RhoA in addition to blocking its activation. The inhibition of RhoA/ROCK signalling led to dephosphorylation of MLC, which likely occurs through the ability of cAMP signalling to induce disinhibition of MLCP. Moreover we show that the inhibitory phosphorylation of RhoA identified by immunoblotting is primarily located at the actin nodule. The actin nodule was identified as an F-actin rich structure that was formed in spreading platelets prior to lamellipodia formation9 that required both the Arp2/3 complex and WASp, and are sites of tyrosine kinase signalling activity35. Given that RhoA phosphorylation is mediated by PKA we also examined the presence of the kinase. cAMP signalling events are highly regulated and involve the selective coupling of cAMP signalling complexes to specific substrates or regions within the cell , Forskolin , Fibrinogen , Y-27632 , Rp-8CPT-cAMP , KT5720 , pRhoAser188 , pMLCser19 and pVASPser157 , GAPDH and Arp2/3 , PKA RI , PKARII , pPKA substrate , WASP , pTyrosine , RhoA pulldown kit , cAMP assay and ProLong Diamond Antifade Mountant , Flourescent secondary anti-mouse 800 and anti-rabbit 680 antibodies  All other chemicals were from Sigma Ltd  unless otherwise stated.PGI18. Platelets were allowed to rest for 30 minutes prior to experimentation. Written informed consent was acquired for the donation of blood. The work was conducted in accordance with the relevant guidelines and regulations and was completed under the ethical permission granted by the Hull York Medical School ethical committee, for “The study of platelet activation, signalling and metabolism”.Whole blood was mixed with acid-citrate dextrose   and platelets were isolated using low pH as previously described7/ml) were adhered to immobilised proteins for 25 minutes at 37 °C in the presence or absence of Apyrase (2 U/ml), Indomethacin (10 μM), or Rp-8-CPT-cAMP (500 μM) and KT5720 (10 μM). After 25 minutes the coverslips were washed twice with PBS to remove nonadhered platelets and then treated with PGI2 (1-1000 nM) or forskolin (0.1–100 μM) in the presence or absence of Apyrase (2 U/ml), Indomethacin (10 μM), or Rp-8-CPT-cAMP (500 μM) and KT5720 (10 μM). At the required timepoint the platelets were fixed with 4% paraformaldehyde for 10 minutes, lysed with 0.1% Triton X-100, stained with FITC-phalloidin and if required the relevant primary antibody , pRhoAser188, Arp2/3, and 4G10 (1:1000)), followed by the secondary antibody (1:200), mounted and visualized using a Zeiss Axio Observer  with a x63 oil immersion objective (1.4 NA) and Zen Pro software . Images of spread platelets were analysed using ImageJ software  to identify the surface area, the platelets containing either actin nodules or stress fibres, and platelet adhesion. In some cases platelets (2 × 107/ml) were preincubated with PGI2 (1–1000 nM) or Forskolin (0.1–100 μM), for 2 minutes prior to adhesion for 45 minutes.Coverslips were incubated with fibrinogen (100 μg/ml) overnight at 4 °C, washed with PBS and blocked with denatured fatty acid free BSA (5 mg/ml) for 1 hour at room temperature. Platelets (2 × 106 (10 μM) and flowed through the biochips for 2 minutes at a shear rate of 1000 s−1 at 37 °C. Biochips then underwent a post-flow with Tyrode’s buffer supplemented with or without PGI2  for 10 minutes, before fixation with 4% paraformaldehyde, staining overnight with DiOC6 (10 μM) and imaging using a Apotome.2 confocal unit on a Zeiss Axio Observer  with a x63 oil immersion objective (1.4 NA) and Zen Pro software . Analysis of the surface area coverage of each condition was performed using ImageJ software .Flow studies were performed with multichannel biochips . Biochips were coated with 300  μg/ml fibrinogen overnight at 4 °C and blocked with denatured BSA (5 mg/ml) for 1 hour. Whole blood was stained with DIOC8/ml) were adhered for 25 minutes before removal of non-adherent platelets. Adherent platelets were then treated with PGI2 (10 nM), FSK (1 μM), or Y27632 (10 μM) in the presence or absence of Rp-8CPT-cAMP (500 μM) and KT5720 (10 μM). Platelets were lysed with Laemelli buffer and the lysates were separated by SDS-PAGE, transferred and immunoblotted with the required antibodies (pVASPser157 (1:1000), pRhoAser188 (1:1000), pMLCser19 (1:1000), or GAPDH (1:6000)). The blots were imaged by Odyssey CLx Infrared Imaging system , and densitometry was analysed by Image Studio ver.5.2 .Six well plates were coated with fibrinogen as per platelet spreading. Platelets (2 × 108/ml) platelets were adhered for 25 minutes before being washed with PBS. The platelets were then treated with Tyrode’s buffer, PGI2 (10 nM) or FSK (1 μM) for 10 minutes. The samples were processed as previously described38. Aliquots of unstimulated platelets (1 × 108/ml to 8 × 108/ml) were used to calculate both the protein concentration and F-actin concentration at these platelet numbers as an unstimulated control.Six well plates were coated with fibrinogen as per platelet spreading. Platelets (2 × 108/ml) were spread for 25 minutes before washing, and treatment with Tyrodes buffer or PGI2 (10 nM) for 2 minutes. Platelets were lysed and cAMP concentrations measured using a commerical available assay kit18. For the RhoA pull down platelets the samples were prepared as above, but then lysed and lysates incubated for 90 minutes at 4 °C with Rhotekin-RBD-beads. Bead pellets were washed once and Laemmeli buffer was added prior to immunoblotting as previously published39.Six well plates were coated with fibrinogen as per platelet spreading. Platelets (2 × 10Results are shown as mean ± standard error of mean (SEM). Data was arcsin transformed as appropriate and then subject to statistical analysis. Statistical analysis was performed by using one-way ANOVA with a P value of < 0.05.Supplementary FigureSupplementary Video 1Supplementary Video 2Supplementary Video 3