We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca ATP-sensitive purinergic receptors comprise a network of cell-surface receptors that activate upon ATP binding, allowing them to transmit information in a tissue- and context-dependent manner. In bone, mechanically stimulated osteoblasts release ATP that stimulates low- and high-affinity P2 receptors in neighboring cellular populations, inducing appropriate physiological responses. P2 receptor signaling is characterized by elevations in intracellular calcium levels. When simultaneously stimulated by their common ligand, ATP, the contribution of each P2 receptor subtype gives rise to a complex calcium response, exhibiting oscillatory characteristics and biphasic dose-dependent behaviours. Here we used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts. The latter was done by developing a mathematical model that was comprised of a subset of low- (P2X7) and high- (P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models. We demonstrated that this model recapitulates experimental recordings of ATP-induced calcium signaling in osteoblasts and describes the dynamic interplay between P2Y2 and P2X7 receptors in the P2 receptor network. Extracellular ATP has long been implicated in diverse physiological functions , includi−8 M to 10−2 M) i) is one of the hallmarks of ATP-induced signaling in many cell types, including bone-forming osteoblasts [2+]i differs: P2XR activation increases Ca2+ influx across the plasma membrane i were recorded using live cell fluorescent microscopy . Qualitatively, the recorded Ca2+ response time-series signatures demonstrated a general trend of exhibiting transient single-peaked responses at low [ATP], multi-peaked oscillatory responses at mid-range [ATP], and relatively sustained single/multi-peaked response at high [ATP] . The Ca2+ responses at each [ATP] were analysed by quantifying several parameters, including overall response magnitudes and activation times, as well as oscillatory fractions, magnitudes, periods and peaks (see S1 Table for definitions) [S1 Fig). Such alignment allowed us to account for (i) inconsistencies in ATP solution preparations between experiments and (ii) varied dose-sensitivities across cell lines. Calcium responses induced by low [ATP] (<10−7 M) were consistently associated with low response magnitudes and slow activation kinetics , with little to no oscillatory component . Increasing [ATP] further induced responses with faster activation kinetics and higher magnitudes . This also coincided with the emergence of high frequency oscillations  which peaked at ~10−5 M ATP stimulation. Notably, the oscillatory peak did not coincide with the peak magnitude. Instead, as cells were stimulated with higher [ATP], the oscillatory component began to diminish, exhibiting lower frequency oscillations and fewer oscillatory peaks, while response magnitude continued to increase, peaking at ~2×10−4 M [ATP] . For [ATP] >2×10−4 M, the response magnitude decreased with increasing [ATP] in all osteoblast lines. Thus, in all osteoblast models, the intracellular Ca2+ response to ATP shifts with increase in [ATP] from a transient with a single narrow peak, to oscillatory and back to transient with a pronounced wide peak.ATP-stimulated P2R Canitions) . Similarnitions) , we harm2+ responses, we first examined their expression in osteoblastic cells of different origin. Among the P2Y family, P2ry2, P2ry4, P2ry12, and P2ry14 transcripts were detected in all osteoblastic cells by RT-qPCR . Among the P2X family, P2rx4 and P2rx7 transcripts were the most abundantly expressed in all osteoblastic cells . These expression profiles suggest that P2Y2, 4, 12, 14 and P2X4, 7 are the predominant P2 receptor subtypes expressed in osteoblastic cells, among which P2Y2 and P2X7 were the most abundant transcripts. To confirm that P2Y2 and P2X7 receptors are functional, we stimulated Fura2-loaded C2-OB cells with ATP and receptor-specific agonists: the P2Y2-agonist UTP and P2X7-agonist bzATP . Consistent with previously characterized P2 receptor sensitivities , respectively . Using CRISPR-Cas9 double-nickase constructs, we generated clonal C2-OB cells harboring mutations in P2ry2 (P2ry2Δ) or P2rx7 (P2rx7Δ)  to further investigate the independent contribution of P2Y2 and P2X7 to the P2-mediated Ca2+ responses (presented in subsequent sections).To examine which P2 receptors contribute to the ATP-induced Ca2+ responses in osteoblasts, we developed a mathematical model that integrates P2Y2R-mediated Ca2+ release, as described by the Li-Rinzel model of the IP3R ER and [Ca2+]i, respectively. The detailed description of the model is given in Methods. Briefly, the model describes (i) Ca2+ mobilization across cell membrane, including Ca2+ influx through P2X7R receptor channels (JP2X7) and the constant inward leak (JINleak), as well as Ca2+ efflux through the plasma-membrane-Ca2+-ATPase (PMCA) pumps (JPMCA); and (ii) Ca2+ mobilization across ER membrane, including P2Y2R-mediated Ca2+-induced Ca2+-release (CICR) through IP3R (JIP3R), Ca2+ leak across the ER membrane (JERleak), and Ca2+ uptake through the sarco/endoplasmic Ca2+ ATPase (SERCA) pumps (JSERCA). The reduced two-dimensional Li-Rinzel model for IP3R-mediated CICR was chosen for its simplicity and ability to produce transitions between the desired modes of activity; it follows the Hodgkin-Huxley gating formalism (see Methods), with two fast activation variables and one slow inactivation variable that depend on [IP3] and [Ca2+]i, producing an open probability profile for CICR that is biphasic with respect to [Ca2+]i. Given that P2Ys modulate intracellular Ca2+ responses indirectly by stimulating IP3 production, an equation describing [ATP]-dependent IP3 production was added to the P2Y2R submodel.To decipher the underlying biophysical mechanisms governing the ATP-stimulated Cathe IP3R , with a the IP3R  .2+ flux through P2X7R, JP2X7, on the other hand, was determined by the Ca2+ current (IP2X7) through the receptor channels generated by a 12-state Markov P2X7R sub-model   in three specific cases: (i) in naïve cells expressing both P2Y2R and P2X7R using the full model , (ii) in cells that do not express P2Y2R, using the submodel for P2X7 component only , and (iii) in cells that do not express P2X7R using the submodel for P2Y2 component only . The simulated Ca2+ responses were compared with those obtained experimentally in WT , P2ry2Δ  and P2rx7Δ  C2-OB cells. As shown, the responses to low [ATP] were predominantly P2Y2-mediated, while the response to high [ATP] were jointly mediated by P2Y2 and P2X7. Notably, the characteristic two-peaked response to 10−3 M observed in experimental recordings  was predicted by the full model  of WT cells, but was abolished in P2ry2Δ and P2rx7Δ recordings and in simulations of P2X7 and P2Y2 submodels. These data strongly support the interaction between P2Y2 and P2X7 receptors in generating this unique signaling feature.Using the model parameters provided in Fig 4 was the observation of oscillatory Ca2+ responses at intermediate [ATP], with transient responses at low and high [ATP], indicating that the mathematical model developed in this study recapitulates the characteristic Ca2+ signatures observed in C2-OB cells over a physiological range of [ATP].One interesting aspect of the recordings and simulations displayed in 3] and [Ca2+]ER are slow variables in the model defined by Eqs –3)3), we set JP2X7R = 0 and applied slow-fast analysis on the resulting P2Y2 receptor model by assuming that these two variables change slowly relative to other “fast” variables in the model. We set the two variables ([IP3] and [Ca2+]ER) as independent adjustable parameters in the P2Y2 model and investigated how the steady state dynamics of fast variables change when [IP3] and [Ca2+]ER are altered. The two-parameter bifurcation diagram  exhibited an oscillatory region in the “parameter” space formed by [IP3] and [Ca2+]ER within which [Ca2+]i is periodic . Outside this region, [Ca2+]i attained steady state values that are [IP3] and [Ca2+]ER-dependent. The boundary of this oscillatory region  defined the threshold for [Ca2+]i to transition between these two main patterns of activity: quiescence and oscillatory. Thus the application of increasing [ATP] in this model would induce an increase in IP3 (since [IP3]∝[ATP]) and a decrease in [Ca2+]ER, generating three possible scenarios for the time courses of [Ca2+]i. When low [ATP] is applied the trajectory stays to the left of the oscillatory region  because the ATP-induced IP3 increase is low (red curve) and [Ca2+]ER remains high , resulting in a low magnitude persistent transient response . Intermediate [ATP] results in a hybrid response that becomes periodic when the trajectory crosses the left boundary of the oscillatory regime , due to a higher increase in IP3 and a faster decrease in [Ca2+]ER , leading to a response characterized by damped oscillations . When high [ATP] is applied, the trajectory crosses the oscillatory region very briefly  because even though the ATP-induced IP3 increase is higher , the [Ca2+]ER does not decrease as fast  due to the biphasic nature of IP3Rs incorporated in the P2Y2 sub-model and the interaction of SERCA pumps with IP3Rs  observed experimentally  may be due to variations in the initial conditions, such as the expression levels of PMCA/SERCA pumps and IP3R, potentially leading to differences in the initial [IP3] and [Ca2+]ER. These simulations demonstrate that the oscillatory response strongly depends on the initial [IP3] and [Ca2+]ER, and that when increasing doses of [ATP] are applied, P2Y2-mediated changes in [IP3] and [Ca2+]ER result in different patterns of Ca2+ responses.Since the oscillatory component required P2Y2 activity in both experimental and simulated responses to ATP, we next investigated how the transition between transient and oscillatory responses is achieved by the P2Y2 receptor. Given that  ranging between 10−8 to 10−2 M ATP (in 10−8 M [ATP] increments), and computed the maximum [Ca2+]i reached within 120 s . The full model  recapitulated the experimental magnitude dose-response profile , including the troughs at moderate  and elevated  [ATP] within the plateau component of the response. This dose-dependency became monotonic in experimental recordings of Fura2-loaded P2rx7Δ C2-OB cells stimulated with ATP  and in the model lacking P2X7Rs , strongly implicating P2X7 in this phenomenon. Therefore, we next plotted the total Ca2+ flux through P2X7Rs, estimated as the area under the P2X7R flux curve, as well as the maximal Ca2+ fluxes through P2X7Rs and IP3Rs predicted by the model . When P2X7R-mediated Ca2+ entry became evident at 10−5 M ATP , the maximum flux through IP3Rs in the full model  dropped below that of the P2X7R-lacking submodel  due to the biphasic dependence of IP3Rs on [Ca2+]i, resulting in the first trough in the dose-response . At high 10−2 M [ATP], on the other hand, the time required for the P2X7R flux to decay to half of its maximum (t1/2) decreased . As a result, despite the maximum P2X7R flux continually increasing , the Ca2+ entering through P2X7R began to decrease at elevated [ATP] , resulting in the second trough in the dose-response . Taken together, these simulations indicate that the non-monotonic Ca2+ dose-response to ATP is driven by an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs.We next investigated the non-monotonic [ATP]-dependent dose-response profile for the magnitude of Ca2+ response to high [ATP] is dramatically affected in P2ry2Δ cells . We examined the magnitudes of Ca2+ responses to 10−2 M [ATP] in WT P2ry2Δ cells, and found that in the absence of P2Y2R, response magnitudes exhibited a distinct bimodal distribution with one cluster of responses similar to those in WT, and another one with much higher response magnitudes . We hypothesized that the bimodality in the P2X7R-mediated responses is due to P2X7Rs being in the naïve or sensitized initial state -dependent dose-response curve for [Ca2+]i generated from these time series simulations revealed that P2X7R responses initiated from C1 produced a dose-response curve that plateaued at a depressed level of [Ca2+]i , close to the mean of the left mode of the distribution . In contrast, P2X7R responses initiated from C4 state produced a dose-response curve that plateaued at an elevated [Ca2+]i , closely matching the mean of the right mode of the distribution . These data suggest that P2Y2 activation may alter the kinetics of P2X7 towards favouring naïve state activation.Next, we examined why the Caal state . To veriprimary” osteoblast with a glass micropipette leads to the release of 10−5 to 10−4 M ATP into the pericellular space, which then diffuses to stimulate neighbouring non-mechanically perturbed “secondary” cells i responses in the primary and secondary cells . We found that while in P2rx7Δ cells the response was qualitatively similar to WT, in P2ry2Δ cells secondary responses were abolished . Quantitatively, the primary response was unaffected in P2ry2Δ cells, but exhibited higher magnitude and faster decay in P2rx7Δ cells . The suppression of the secondary response in P2ry2Δ cells was evident by the reduced signaling radius (p = 9×10−3), fractions of responding cells (p = 5×10−11), response magnitudes (p = 2×10−4) and areas under the curves  . In contrast, in P2rx7Δ cells, the signaling radius and fraction of responding secondary cells was unaffected; however, the response magnitudes and areas under the curves of secondary cells were significantly higher in P2rx7Δ cells compared to parental C2-OB cells . These data demonstrate that P2Y2 receptor is critical for the secondary responses, consistent with its high sensitivity to ATP. In addition, the contribution of P2X7 to Ca2+ responses is evident even though extracellular ATP in these experiments remained below [ATP] required to stimulate P2X7. Taken together, these data strongly support the importance of an interplay between P2Y2R and P2X7R.To investigate the potential functional consequences of the complex interactions between P2Y2 and P2X7, we examined how the absence of each of these receptors affects ATP-mediated mechanotransduction. We have previously shown that mechanical stimulation of a single “y” cells ,12. Here2+ responses to ATP are conserved across three independent murine osteoblast models and identified the P2Y2 and P2X7 receptors as the dominant P2 receptors contributing to these responses. Based on previous experimental evidence highlighting the role of IP3 in regulating calcium responses [2+ signals induced by the activation of high affinity P2Y2R and low affinity P2X7R. Model predictions were validated by comparing the results of simulations to experimental data of ATP-generated Ca2+ signaling in parental and CRISPR/Cas9 -generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. We demonstrated that activation of P2Y2R by progressively increasing [ATP] induces a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3R activation/inactivation kinetics and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R was found to modulate the peak response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, our study suggests that P2Y2 activity may also alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated that functional consequences of lacking P2Y2 or P2X7 are evident beyond the absence of a signal at the expected range of ATP concentrations. Taken together, these findings support a model in which the response to ATP is not a simple superposition of individual P2 receptors, but a complex context-specific functional response build through interactions of multiple P2 receptors.In this study, we demonstrated that the patterns of P2R-mediated Caesponses , we cons2+ responses, including non-trivial features such as the transition of response from transient to oscillatory and back to transient when increasing [ATP], and the presence of two troughs in the plateau phase of the magnitude dose-response curve at high [ATP]. These finding suggest that P2 receptors contributing to these responses are also conserved. P2 receptors are ubiquitously expressed in every mammalian cell, with cell- and tissue-type specific patterns of expression i signaling. Thus, P2Y2 and P2X7 receptors play critical roles in mediating the osteoblast response to ATP, particularly in the context of mechanotransducive signaling in bone.Our experimental studies focused on characterizing ATP-mediated calcium responses in osteoblasts. In investigating these calcium responses, we chose to stimulate cells with a range of physiologically-relevant ATP concentrations (10 nM to 10 mM), such that 10–100 nM ATP represented small fluxes above baseline extracellular ATP concentrations, 1–100 μM ATP represented concentrations achieved following mechanical stimulation, and 1–10 mM ATP represented intracellular [ATP] that has the capacity to spill into the extracellular space and stimulate P2 receptor signalling following traumatic tissue injury ,7,11. Wepression  Consistepression ,26, we d loading ,28, and esponses . In our 2+]i response is prevalent. The oscillatory behaviour was abolished in P2ry2Δ cells, but preserved in P2rx7Δ cells, strongly implicating P2Y2 as a mediator of oscillations. Using the mathematical model of P2Y2-induced changes in [IP3] and [Ca2+]ER allowed us to examine the mechanism of transition between oscillatory and non-oscillatory (transient) responses. We found that moderate IP3 production evokes an oscillatory response because of two factors: i) CICR by the IP3Rs that exerts negative feedback on the receptors and inhibits them, and ii) the interaction of IP3Rs with SERCA that pumps Ca2+ back into the ER. In contrast, at low [ATP], the Ca2+ released by IP3Rs is insufficient to feedback and inhibit the receptors, whereas at high [ATP], IP3Rs become constitutively activated, making CICR larger than that in the moderate case but no longer able to inactivate the receptors; this produces, as a result, transient Ca2+ responses in both cases. While this model prediction is interesting, its validation is limited by difficulties in experimentally measuring osteoblastic IP3 dynamics. Specifically, little is known about the basal [IP3] (assumed to be 0 μM in our model), which plays a significant role in whether solutions will pass through the oscillatory region obtained in the [IP3] and [Ca2+]ER space. Furthermore, the ability of the model to predict some of the experimental response profiles observed at elevated [ATP] is limited by the chosen P2Y2R-IP3R sub-model, which is unable to slowly decay after a rapid increase in [Ca2+]i  over which oscillatory [Ca [Ca2+]i . Given t−4 M and 10−2 M ATP and a peak at 10-3M ATP was reported in MC3T3-E1 osteoblasts i. As a result, the peak [Ca2+]i response, which is predominantly mediated by IP3R activity, decreases, creating the first trough in the dose-response curve at 10−4 M [ATP]. With further increase in [ATP], the activation of P2X7Rs, known to monotonically increase with [ATP], starts to outweigh the reduced IP3R activity, causing the peak [Ca2+]i response to rise, reaching a global maximum around 10−3 M ATP. After that, the rate of P2X7R desensitization (that increases with [ATP]) becomes large enough to impede Ca2+ influx through the receptors, resulting in a decrease in the peak [Ca2+]i response (now predominantly mediated by P2X7Rs) which creates the second trough in the dose-response curve at 10−2 M ATP. These data demonstrate how activation of low affinity P2X7 may affect the responses mediated by the high affinity P2Y2. Our study also suggests the reciprocal effect of P2Y2 on the function of P2X7 through facilitating the naïve state activation of P2X7. Indeed, previous studies have documented such an effect through the allosteric regulation of P2XR (including P2X7R) by Ca2+ i transient characterization were performed in R. Statistical significance was assessed by one- or two-way ANOVA (as specified) and post-hoc two-way unpaired Students t-tests were adjusted using the Bonferroni correction. Significance levels were reported as single symbol (*p<0.05), double symbol (**p<0.01) or triple symbol (***p<0.001).Data are presented as representative images, means ± standard error (SE) or means ± 95% confidence intervals (CI), as specified in each figure panel along with sample sizes Eqs –3)3). The individual terms JINleak, JIPR, JERleak, JPMCA, JSERCA and JP2X7, listed in Eqs  and , were the key Ca2+ fluxes considered in this model, as described below.The mathematical model consisted mainly of (1) Plasma Membrane Ca2+Leak (JINleak). A constant inward leak across cell membrane to ensure that total [Ca2+] within the cell remained positive. It was assumed to be constant (see Table 1).(2) IP3R Ca2+Flux (JIP3R): The Ca2+ flux through IP3Rs, given byvIP3R is the maximum rate of Ca2+ release by the IP3R and OIP3R is the IP3R open probability, defined by3 (defined by m∞) and [Ca2+]i (defined by n∞) through binding to the receptor were assumed to be instantaneous, given by2+]i (defined by the gating variable h), also through binding, was assumed to occur at a much slower time scale governed dynamically byd Keizer  and lated Keizer . In thisdi  were fit to experimental data [OIP3R on [Ca2+]i in Eq  ER Ca2+Leak (JERleak). A small leak across the ER membrane  (the [IP3] in the absence of extracellular ATP) was assumed to be zero. P2Y2R and P2X7R knockout recordings were simulated by setting JP2X7 and JIP3R (Eqs  and 12)12)) to zero, respectively. These simulations were then used to compute [ATP]-dependent dose-response curves of [Ca2+]i by evaluating the maximum Ca2+ response at each ATP dose in MATLAB. The bifurcation analysis ( was performed using XPPAUT (a freeware available online at http://www.math.pitt.edu/~bard/xpp/xpp.html). To facilitate reproduction of results, the codes used to perform simulations of the model can be obtained online  interval . Curves: Loess curves; Markers: Response means .((TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


An aberrant elevation in endothelial can indeed lead to severe cardiovascular disorders. Likewise, moderate amounts of reactive oxygen species (ROS) induce intracellular Ca2+ signals to regulate vascular functions, while excessive ROS production may exploit dysregulated Ca2+ dynamics to induce endothelial injury. Herein, we survey how ROS induce endothelial Ca2+ signals to regulate vascular functions and, vice versa, how aberrant ROS generation may exploit the Ca2+ handling machinery to promote endothelial dysfunction. ROS elicit endothelial Ca2+ signals by regulating inositol-1,4,5-trisphosphate receptors, sarco-endoplasmic reticulum Ca2+-ATPase 2B, two-pore channels, store-operated Ca2+ entry (SOCE), and multiple isoforms of transient receptor potential (TRP) channels. ROS-induced endothelial Ca2+ signals regulate endothelial permeability, angiogenesis, and generation of vasorelaxing mediators and can be exploited to induce therapeutic angiogenesis, rescue neurovascular coupling, and induce cancer regression. However, an increase in endothelial [Ca2+]i induced by aberrant ROS formation may result in endothelial dysfunction, inflammatory diseases, metabolic disorders, and pulmonary artery hypertension. This information could pave the way to design alternative treatments to interfere with the life-threatening interconnection between endothelial ROS and Ca2+ signaling under multiple pathological conditions.An increase in intracellular Ca Like Car system ,57,58,59 [Ca2+]i ,60,61,62othelium ,64. Here2•−), hydroxyl anion (OH•), and hydrogen peroxide (H2O2) (2•− is rapidly transformed in H2O2 either spontaneously or by superoxide dismutase (SOD), and it is featured by a low selectivity toward molecular targets i .,69.2- an thought  and does thought . In cont insulin , whereasin [2+]i . Howeverin [2+]i . Finallyin [2+]i ,76. Finaxidation ,77. Intrxidation ,78,79.2O2 and O2•− as secondary byproducts  exists in two interconvertible isoforms, i.e., xanthine oxidase (XO) and xanthine dehydrogenase (XDH) . XOR is products  81]. Mo. Mo2O2 aand H2O2 . Thus, tand H2O2 . XDH is and H2O2 ,82. For and H2O2 , XDH is and H2O2 . Furthering H2O2 ,77.4, L-arginine, and COQ10) to maintain the monomeric structure that is necessary to produce NO. NOSs serve as oxidoreductases that catalyze flavin-dependent electron transfer from the COOH-terminal bound NADPH to the heme iron and BH4 that are located on the NH2 terminus, thereby oxidizing L-arginine to L-citrulline and forming NO (coupled NOS) i i i ,116. Furor MAMs)  and inhit manner . Ryanodie (CICR) ,120 or u (cADPr) . As revi (cADPr) , endotheng ECFCs . Therefo [Ca2+]i . The EL and TPC2 , whereasand TPC2 . In accoand TPC2 ,125,126, lineage .3R channel activity and that H2O2 also controls SERCA-mediated ER Ca2+ sequestration.In the following Sections, we focus on the wide literature supporting the notion that ROS stimulate InsP2+]i xanthine (H)X/XO i resulting from InsP3-induced ER Ca2+ release and extracellular Ca2+ entry  in vascular endothelial cells [2•− could either sensitize InsP3Rs to mobilize ER Ca2+ and thereby engage the SOCE pathway in response to agonist stimulation [2+]i X/XO ,129, thee (G/GO) ,129, witGO) 2O2 ,131, wit, wit2+i GO) 2O2 ,116, wit, wit2+i GO) . T. T2+]i X/XO . At concentrations ≥100 µM, H2O2 induced repetitive oscillations in [Ca2+]i, which overlapped a gradual elevation in [Ca2+]i and then merged into a sustained plateau phase i well above prestimulation levels [2O2 caused a massive reduction in ER Ca2+ concentration ([Ca2+]ER) following InsP3R stimulation in human umbilical vein endothelial cells (HUVECs) i ER presents intraluminal gradients i, acute oxidant signaling via either H2O2 [2•− [2+ response to InsP3-producing autacoids (3]) around InsP3Rs that dictates their ROS sensitivity. Additional mechanisms that may underlie the differential effects of H2O2 and O2•− on the endothelial Ca2+ toolkit could depend on the vascular bed . I. I2O2 co(HUVECs)  and calf(HUVECs) . This mimulation . H2O2 cong PLCγ1 ,142 and/Cγ1 i ,116. Altications , a preli changes . A recenl domain . Disulfi protein . The ER 2 levels  and, thenal loop ,116. Accactivity ,146. Int, and pH , while tradients . Therefoorted in ,138,150.2O2  and promoting InsP3-induced ER Ca2+ mobilization [2+ oscillations evoked in HAECs by histamine [2+ signaling to reduce endothelial permeability and facilitate leukocyte transendothelial migration [2+]i and von Willebrand factor (vWF) secretion in HUVECs i that results in the activation of the Ca2+/CaM-sensitive NOX5 isoform. For instance, bradykinin-dependent ROS production in PAECs requires InsP3-dependent ER Ca2+ release, whereas SOCE is ineffective at engaging NOX5 i over time, and recruit a plethora of Ca2+-dependent decoders. Thus, SOCE regulates most of endothelial functions, ranging from NO release and vWF secretion to the control of endothelial permeability and proliferation [2+ signaling in circulating ECFCs recruited to ischemic tissues to participate in vascular regrowth [2+ entry in the endothelial lineage. Three independent studies reported that SOCE is mediated by the physical interaction between stromal interaction molecule 1 (STIM1) and Orai1 channels in HUVECs [2+]ER due to its low affinity for Ca2+ (≈200 µM). STIM1 is activated by a large reduction in [Ca2+]ER and is thereafter prompted to undergo a conformational remodeling and translocate to close (10-20 nm) junctions between ER and plasma membrane, known as puncta. Herein, STIM1 physically interacts with and gates Orai1, which provides the pore forming subunit of a store-operated channel termed the Ca2+ release-activated Ca2+ (CRAC) channel. STIM1 and Orai1 were also shown to mediate SOCE in HAECs ER ER . Althouge [2+]ER ,186, thee [2+]ER ,186, whie [2+]ER . A recentivation . On the tivation ,181, in al tails . Orai1 aal tails ,186. Bog T cells ,186. Cys T cells . Convers T cells . Intrigui by activating InsP3Rs and/or SOCE i controls proliferation, migration, tube formation, barrier permeability, and generation of chemical  and electrical  vasorelaxing signals . Meability  236]. T. Trev) oeability  237]. T. Trev) oD5 cells .2+-activated, Ca2+-impermeable nonselective cation channel that presents a PCa/PNa of 0.09 i in the adjoining cells, thus initiating a spreading intercellular Ca2+ wave that impinges on Ca2+-dependent Panx1 activation and paracrine ATP signaling i is transformed into a hyperpolarizing electrical signal, i.e., EDH, by the Ca2+-dependent recruitment of IKCa/SKCa channels, thereby vasodilating the upstream intraparenchymal arterioles and causing a local increase in CBF . A . A 2+ th glucose ,25. An ieability  and releeability . Thakoreeability  or astroeability ,246. TRPignaling . Once the in CBF . The rede in CBF . Indeed,terioles . Therefoterioles ,247.2+ signals regulate a variety of endothelial functions, which may be hampered when ROS overproduction overwhelms the intrinsic antioxidant capacity of vascular endothelial cells. In this conclusive Section, we first discuss the evidence in favor of the therapeutic applications of ROS-dependent endothelial Ca2+ signaling to rescue vascular functions. Then, we describe how aberrant and/or chronic oxidant stress may result in an exaggerated increase in endothelial [Ca2+]i that may severely compromise vascular signaling.As inferred by the evidence described above, ROS-induced intracellular Ca2+]i sustains endothelial cell proliferation, migration, and tube formation . T. T2+]i s2+]i may result in a cytotoxic effect by stimulating several Ca2+-dependent modes of cell death, including necrosis and apoptosis [2+]i [2O2-releasing nanomaterials can exert an anticancer effect by increasing the already high extent of oxidant stress imposed to cancer cells by tumor microenvironment [2+]i. For instance, a recent transcriptional analysis revealed that TRPA1 is upregulated in prostate-cancer-derived endothelial cells (PCECs), but not in those harvested from breast and kidney cancer i ,259,260.ironment ,262. Intironment ,263 and ma cells . As revima cells , the H2O therapy ,266 and aterials ,268,269,er cells ,271, the2+]i sustains ROS-induced endothelial injury i that causes endothelial cell contraction . A. A2+ entaspase-3  58]. In. In2+ enter (PM) ,291. PM-sruption . More reine, H5V  248]. T. T2+ entpoptosis  [248]. Lothelium . In addiothelium , brain mothelium ,41. Iadedementia , may indoverload . In accoal cells  38]. O2. O22+ enarteries . Furtherfunction . Furtherfunction . METH and of ZO1 . The notd of ZO1 . A recenicroglia . Of noteothelium . Future za virus . H9N2 vipoptosis .2+ entry triggered endothelial barrier dysfunction and favored PMN transendothelial migration through the disassembly of VE-cadherin  as compared to those fed with low-fat chow diet (LFD)  cascade, which inhibits insulin-induced eNOS activation, NO production, and aortic vasorelaxation , to promote mitochondrial fission and, therefore, compromise mitochondrial functioning [TRPM2 may also sustain endothelial damage during acute lung injury (ALI)  and metacadherin  36]. Mo. Mo2+ enet (LFD)  54]. Pa. Pa2+ enlaxation  [54]. Inby TRPM2 . This ROctioning , which ictioning ,228. A cctioning .+ entry, resulted in cytosolic Ca2+ overload, and promoted endothelial cell death i in vascular endothelial cells i and ROS production in vascular endothelial cells have been widely investigated, the complex interplay between such two highly versatile signaling pathways is far from being fully dissected. A large body of investigations was devoted to ascertaining the effect of ROS on endothelial TRP channels, while it is still unclear whether ROS engage SOCE in vascular endothelium. Since SOCE plays a pivotal role in the regulation of endothelial Ca2+ homeostasis by reloading the ER with Ca2+ and maintaining long-lasting Ca2+ signals, assessing this issue is of compelling relevance. Similarly, a thorough investigation is necessary to understand the molecular mechanisms whereby ROS  control endothelial InsP3Rs and whether this mode of regulation changes across the vascular beds or in the presence of pathological conditions enhancing the oxidative stress imposed on the endothelial monolayer. Future work is also necessary to assess whether and which NOX isoform contributes  to trigger the Ca2+ response to extracellular stimuli by providing the surge of ROS that sensitize InsP3Rs to the accompanying increase in cytosolic InsP3 levels and/or to ambient Ca2+. Finally, the pathophysiological role of ROS-induced Ca2+ signals in circulating ECFCs is still largely unclear and deserves to be more deeply unraveled due to the reduction in ECFCs’ proangiogenic activity in cardiovascular disorders associated to oxidative stress. This wealth of information could pave the way to design alternative treatments to interfere with the life-threatening interconnection between endothelial ROS and Ca2+ signaling under multiple pathological conditions.While the mechanisms shaping the increase in [Ca


Using genetic algorithms, we fitted the myofibroblast model parameters to the existing in vitro data. In silico experiments showed that Ca2+ currents can explain the experimentally observed variability regarding the myofibroblast resting membrane potential. The presence of an L-type Ca2+ current can trigger automaticity in the myofibroblast with a cycle length of 799.9 ms. Myocyte action potentials were prolonged when coupled to myofibroblasts with Ca2+ handling machinery. Different spatial myofibroblast distribution patterns increased the vulnerable window to induce arrhythmia from 12 ms in non-fibrotic tissue to 22 ± 2.5 ms and altered the reentry dynamics. Our findings suggest that Ca2+ handling can considerably affect myofibroblast electrophysiology and alter the electrical propagation in atrial tissue composed of myocytes coupled with myofibroblasts. These findings can inform experimental validation experiments to further elucidate the role of myofibroblast Ca2+ handling in atrial arrhythmogenesis.During atrial fibrillation, cardiac tissue undergoes different remodeling processes at different scales from the molecular level to the tissue level. One central player that contributes to both electrical and structural remodeling is the myofibroblast. Based on recent experimental evidence on myofibroblasts’ ability to contract, we extended a biophysical myofibroblast model with Ca Non-myocyte cells are the major population in cardiac tissue (60–70% of cells). In healthy and diseased myocardium, this population is predominantly comprised of cardiac fibroblasts . Once fiV1.5 sodium channel protein expression i is the intracellular potassium concentration, and [Ca2+]i is the calcium intracellular concentration.+]i, and [Ca2+]i. Otherwise, the algorithm continued by selecting the fittest parameter sets and allowing a 0.8 fraction of crossover and mutation rate (following a Gaussian distribution with 0 mean) between the genes of the selected individuals to create the next generation. The process continued until the convergence condition was met.If the sum of the weighted absolute error was less than 0.1, the algorithm was terminated, providing the parameters of the fittest model. Weights  were the same for transmembrane potential, [KCaLI and the Ca2+ handling system. Different numbers of myofibroblasts  were coupled to a single myocyte [90) was measured in an S1–S2 dynamic pacing protocol to obtain the restitution curve.In silico experiments of isolated cells were carried out in order to quantify the effect of the electrical coupling between myocytes and myofibroblasts with  myocyte ,52,53. T myocyte ,40. The H is the entropy of the central fibrotic area, k is the number of levels (0(myocyte) or 1 (myofibroblast)), and p is the probability associated with said binary level.Tissue simulations were carried in a patch of 50 mm × 50 mm × 3 mm with a central fibrotic area (radius 10 mm) featuring 11 different fibrosis patterns. The patterns were generated as Perlin noise , which iTissue conduction velocity was set to 43.39 cm/s ,59 to reAll meshes had an average edge length of 100 µm to study the electrical dynamics due to the hetero cellular coupling. The tissue was stimulated from one side to simulate a planar wave propagation during sinus rhythm with 10 pulses at a basic cycle length of 1000 ms to reach a limit cycle. Additionally, a second stimulus was introduced to induce an arrhythmia via a cross-field protocol and quantify the tissue’s vulnerable window. The vulnerable window was defined as the total time when a reentry activity was initiated by the cross-field stimulus and maintained for more than 2 s of simulated time. Fibrotic clusters were defined as the areas where all elements of fibrosis were connected. The Euclidean distance between the cluster centroids was measured. Monodomain simulations were performed using a time step of 1 µs to account for the stiff differential equations of the ionic models. All simulations were performed using openCARP. .NaI [CaLI [The genetic algorithm fitted the 11 parameters of the ionic model  in orderINa  and ICaLNa [ICaL ,9,12  was 62.60 mV/s. When in addition to CaLI an intracellular Ca2+ handling system was added, the automaticity of the cell ceased and a stable resting membrane potential of –46 mV was reached.An in silico patch-clamp experiment was conducted in order to measure the maxi mal current at different potentials. The fitted e et al. , as depiCaLI and the intracellular Ca2+ handling system), the myocyte action potential duration was affected. Control myocyte APD90 was shortened from 180 ms and when coupling to 3, 6, or 9 myofibroblasts, 110 ms, 70 ms, and 50 ms, respectively. Myocyte APD90 with AF electrical remodeling was 130 ms. When coupled to 3, 6, or 9 myofibrob-lasts, it was prolonged to 163 ms, 168 ms, and 185 ms, respectively (max), for control myocytes, was increased from 119.3 mV/s (non-coupled) to 120.1 mV/s when coupled to 9 myofibroblasts. AF-remodeled myocyte dV/dtmax was slightly reduced from 111.5 mV/s (non-coupled) to 110.9 mV/s when coupled to 9 myofibroblasts.When a single myocyte was coupled to different numbers of myofibroblasts  when coupled to 9 myofibroblasts. Additionally, APD90 restitution curve maximum slope was increased for 1, 3, and 6 coupled myofibroblasts  in comparison to the slope of the uncoupled AF-remodeled myocyte (2.97). However, the slope was flattened when coupled to 9 myofibroblasts (slope of 1.83) b.Tissue-level simulations revealed that myofibroblasts change reentry dynamics depending on their density and pattern. Fibrosis patterns with high entropy  increased the tissue vulnerability to reentrant activity  last rowTissue vulnerability mainly depended on the myofibroblast density independent from the infiltration pattern. Increasing myofibroblast density increased the vulnerable window : from 122+ and an intracellular Ca2+ handling system. This model is based on in vitro data and assumptions following Occam’s razor when no specific data were available. In silico experiments using this new model can help to explore and potentially understand the electrophysiology of the human atrial myofibroblast. We showed that intracellular calcium handling can tip the scales of cellular automaticity and that myofibroblast infiltration patterns can promote and maintain reentrant activity.In this work, we have introduced a human atrial myofibroblast model, which includes the L-type CaOur fitting methodology yielded a myofibroblast model with long-term stability that is able to reproduce patch-clamp experiment recordings from in vitro data. Despite the lack of data, the model yielded physiological transmembrane potential courses in accordance with different experimental works ,61.2+ signaling has been described as a pathway of fibroblast proliferation and differentiation [2+ channels [2+ ion channels and intracellular Ca2+ handling plays an essential role in the electrophysiology of myofibroblasts and the development of cardiac fibrosis. Nevertheless, the specific effect of voltage-gated Ca2+ channels and their impact on myofibroblast electrophysiology has not yet been fully described. In our study, using in silico experiments and based on the notion that myofibroblasts can contract, we were able to study the effect of the expression of L-type Ca2+ voltage-channel and the intracellular Ca2+ handling system on myofibroblast electrophysiology. We observed two different behaviors: first, the presence of L-type Ca2+ current triggers automatic activity in isolated myofibroblasts. Second, the presence of the intracellular Ca2+ handling system, which was composed of the sarcoplasmic reticulum and the Ca2+ SERCA pump, stops this automaticity and leads to a steady-state with a resting membrane potential of –46 mV, which is in accordance with experimentally reported values [2+ channel had a DDR100 of 62.60 mV/s and a cycle length of 799.9 ms, which could potentially trigger ectopic activity [2+ current and intracellular Ca2+ handling system to the myofibroblast model showed a different effect when coupled to a single myocyte. A previous study [2+ for the function of the atrial cardiomyocytes and its relation to AF. In this study, we present how Ca2+ can alter the electrophysiology of the human atrial myofibroblast. In particular, myofibroblast automaticity was only observed in the absence of Ca2+ handling machinery. While the relevance of myofibroblast automaticity for atrial ectopy is unclear [2+ handling could be a contributor to arrhythmia triggers and help to understand arrhythmogenicity and may hold potential for future therapeutic approaches [Cantiation . In vitrchannels ,64,65. Td values ,38,53,61d values ,67,68. Tactivity . Automatactivity . In cardactivity . The obsus study  showed tus study  reviewed unclear , abnormaproaches .Tissue simulations showed that not only could the myofibroblast infiltration affect the arrhythmia vulnerability ,74, but Our results show that patterns that resemble low degree patchy fibrosis with low entropy did not markedly affect the tissue vulnerability . However2+ entry via the L-type Ca2+ channel to the intracellular medium and the intracellular Ca2+ handling. Nevertheless, Ca2+ signaling in the myo-/fibroblast has been shown to be mostly mediated by TRP channels. Different studies suggest that under the presence of TGF-β1, myofibroblast TRPM7 channels are activated and contribute to Ca2+ influx [2+ concentration. However, the role of low Mg2+ in patients with AF has not yet been clarified [2+ current as the primary contributor to Ca2+ influx and did not consider any changes in the Mg2+ concentration. Another possible Ca2+ entry could be via CRAC channels (Orai/STIM), which are essential regulators of intracellular Ca2+ homeostasis in different cells of the cardiovascular system [2+ entry may be mediated via the activation of angiotensin II-induced signaling cascades in cardiac fibroblasts but have not reported the effect on the myofibroblast electrophysiology or the effect on electrical propagation. These findings are a motivation to further study the influence of Ca2+ in the differentiation of fibroblasts in the cardiac tissue and their importance for cardiac arrhythmias. The scarcity of experimental data is the main limitation of this work, rendering independent validation of the myofibroblast electrophysiology impossible at the current stage. As such, this study should be considered hypothesis-generating, aims to stimulate discussion in the scientific community, and hopes to inform future experimental work.Based on reports that myofibroblasts can contract ,31,32 an+ influx ,26,84. Dlarified ,86. In tr system . Camachor system  reportedWe did not consider paracrine effects ,14,79, w2+ handling in the myofibroblast alters the cellular electrophysiology and prolongs the action potential when coupled to an atrial myocyte. If only the L-Type Ca2+ membrane channel is added, myofibroblasts exhibit sustained automaticity. Tissue simulations show that myofibroblast infiltration increases the vulnerability of the tissue to arrhythmia and that different fibrosis patterns change the dynamics of the reentrant activity. Future experiments based on these findings will further elucidate the role of calcium in the myofibroblast and the electromechanical effects of coupling between myofibroblasts and myocytes.In silico models can integrate the available in vitro data on myofibroblast elec- trophysiology and provide additional insight based on them. Ca


Ca2+ is a versatile signalling messenger that regulates functions that prime and drive oncogenesis, favouring metabolic reprogramming and gene expression. Ca2+ is present in cell compartments, between which it is trafficked through a network of transporters and exchangers, known as the Ca2+ transportome. The latter regulates and controls Ca2+ dynamics and tonicity. In HCC, the deregulation of the Ca2+ transportome contributes to tumorigenesis, the formation of metastasizing cells, and evasion of cell death. In this review, we reflect on these aspects by summarizing the current knowledge of the Ca2+ transportome and overviewing its composition in the plasma membrane, endoplasmic reticulum, and the mitochondria.Hepatocellular carcinoma (HCC) is a type of liver cancer with a poor prognosis for survival given the complications it bears on the patient. Though damages to the liver are acknowledged prodromic factors, the precise molecular aetiology remains ill-defined. However, many genes coding for proteins involved in calcium (Ca Among2+ is a versatile signalling messenger that regulates vital as well as lethal cellular functions, spanning gene expression, metabolism, apoptosis, muscle excitation-contraction, and neurotransmission [2+, both at the cellular and intraorganellar level, depend on many ion transporters and exchangers that are collectively referred to as the Ca2+ transportome. The Ca2+ transportome is finely regulated [2+ homeostasis, thus contributing to tumour proliferation [2+ is well known as a mediator of interorganelle communications and most important metabolic processes, including the production of reactive oxygen species (ROS), ATP, oncoproteins, and oncometabolites [Casmission . The infegulated , and itsferation . Finallyabolites .2+ transportome has been actively studied in recent years, showing its contribution to tumor development in several cancers. Thus, in oncogenesis, remodelling Ca2+ transportome activity is required to shift the balance between cell life and death towards the development of many cancer types, including HCC [2+ entry (SOCE) mechanism, are generally downregulated to avoid cytosolic Ca2+ overload, thus evading cell death and promoting tumor proliferation [3) receptor (IP3R)-mediated endoplasmic reticulum–mitochondria crosstalk is also involved in the apoptosis resistance of glioblastoma, thus revealing the role of ER and mitochondrial calcium in tumorigenesis [2+ uniporter (MCU) can regulate tumor progression in breast cancer and colorectal cancer growth [2+ uptake 1 (MICU1) protein drives chemoresistance in ovarian cancer [The deregulation of the Cading HCC . In mostferation ,19. Moreferation ,21,22. Pferation ,24; alteferation . The inoigenesis . At the r growth ,28, whern cancer .2+ transportome composition and, secondly, the impact of its deregulation on HCC, via interorganelle communication, based on the latest studies. We will discuss recent evidence revealing that some members of the Ca2+ transportome , TRPC6, and STIM1) notably impact intracellular Ca2+ level, mitochondrial ROS (mtROS) production, and transcriptomic profiles, and how these deregulations specifically promote HCC progression.Here, we provide an overview of the Ca2+ signalling controls crucial cellular functions, basal Ca2+ concentration must be tightly regulated and maintained at very low levels [2+ between the extracellular environment and the cytosol are mediated by the PM Ca2+-permeable ion channels and energy-dependent channels described below.The plasma membrane (PM) defines the boundary separating the intracellular environment and the extracellular space. Following the fluid mosaic model , the PM w levels . Thus, t2+-permeable ion channels are passive transporters, since the Ca2+ flux is electrochemical gradient-dependent and does not consume energy. There are seven subclasses of PM Ca2+ permeable channels in both excitable and non-excitable human cells, including: (1) voltage-gated Ca2+ channels (VGCC or Cav), (2) ligand-gated Ca2+ channels (LGC), (3) store-operated channels (SOC), (4) transient receptor potential (TRP) channels, (5) second messenger-operated channels (SMOC), (6) acid-sensing ion channels (ASIC), and (7) mechano-gated channels [2+ transport , and is also the most well-known channel to contribute to the SOCE mechanism [2+. The SOCE mechanism requires the physical interaction between the C-terminal of the Orai proteins with the ER STIM1 (described below), thus inducing the conformational change of the Orai proteins and allowing Ca2+ influx when the ER Ca2+ level is depleted [2+ influx is mainly mediated by the SOCE mechanism via SOCs, represented by Orai proteins. In HCC cell lines , TRP channels can also interact with Orai1 and STIM1, thus contributing to SOCE regulation and promoting tumour proliferation [The TRP channel family is a large family of conserved cation-permeable channels that sense exogenous and endogenous stimuli  to mediate Ca2+ entry . Many TR2+ entry . In HCC,2+ entry ,36. SOCsechanism ,38,39. Oechanism . The chadepleted ,42. In nferation . This clferation .To date, there has been no observation of functional VGCCs in normal hepatocytes, since VGCCs are present mostly in excitable cells  . Surpris2+ extrusion include the high-affinity, low-capacity Ca2+-ATPase, also known as the PM Ca2+ pump (PMCA), and the low-affinity, high-capacity Na+/Ca2+ exchanger (NCX) [2+ levels [Two systems of Caer (NCX)   that are important for its regulation [NCX1, NCX2, and NCX3 [2+ influx and Na+ efflux [2+ level can affect the growth of cancer cells as efficiently as it affects normal cell growth. Unfortunately, the contribution of NCX members to HCC remains under-studied [NCX mediates Cagradient . Its strgulation ,63. The and NCX3 . Accordi+ efflux . Among t-studied .2+ signalling), and transitional ER  [2+ is stored, its Ca2+ content depending on the cooperation of various channels belonging to the ER Ca2+ transportome, such as the ryanodine receptor (RyR) and the IP3R as Ca2+ release channels, and the SERCA family for ER Ca2+ accumulation (The endoplasmic reticulum (ER) was described by K.R. Porter in 1945 as a cytoplasmic “lace-like reticulum” structure connected to the nuclear envelope . Differeication) . The ER ication) . It is tmulation .2+ fluxes [2+ (~100 µM), whereas the cytosolic Ca2+ concentration is in the 50–100 nM concentration range [2+ between the ER and the other compartments is strictly regulated, as leakage of ER luminal Ca2+ can cause cell death by mediating ER stress and activating unfolded protein responses (UPR) [2+ into the cytosol is crucial to mediate Ca2+-dependent signalling pathways; however, its over-release can induce ER Ca2+ depletion [2+ depletion, the SOCE mechanism is activated to enhance the influx of cytosolic Ca2+ through the PM via STIM proteins and PM Orai proteins [2+ transport is crucial to visualize the impact of the ER on the regulation of cytosolic Ca2+ levels and Ca2+ signalling pathways in HCC.The ER forms membrane contact sites (MCS) with the mitochondria, PM, and endosomes to mediate Ca+ fluxes ,71,72,73on range . The traes (UPR) . The relproteins . Thus, a2+-permeable channels are categorized into two types according to two mechanisms: (1) Ca2+-induced Ca2+ release (CICR), and (2) agonist-induced G protein-coupled receptor (GPCR)-dependent release [ER Ca release   family is known to comprise the major Ca process . Three i process . The maiss known . Nicola nsporter . MoreoveR2 genes .2+ release also regulates intracellular Ca2+ homeostasis in hepatocytes. IP3Rs are known to be involved in this process. There are three isoforms of IP3R, named IP3R1 (314 kDa), IP3R2 (308 kDa), and IP3R3 (304 kDa), encoded by homologous genes. To activate IP3Rs, phospholipase C (PLC) breaks down phospholipid phosphatidylinositol 4,5-biphosphate (PIP gm) to generate diacylglycerol and InsP3  [3R3 was found to be absent or poorly expressed in normal hepatocytes, but was overexpressed in HCC patients and HCC cell line models. This overexpression enhances the Ca2+ signalling in HCC, and prevents apoptosis [3R mediates the Ca2+ transport from the ER into mitochondria via the chaperone glucose-regulated protein 75 (Grp75) and voltage-dependent anion channel (VDAC), creating the so-called ER–mitochondrial contact sites [Adjacent to CICR, agonist-induced GPCR-dependent Carelease) . Among tpoptosis . Moreovect sites .2+-ATPases (SERCAs) are ER transmembrane proteins, encoded by ATP2A1, ATP2A2, and ATP2A3 genes, represented, respectively, by three isoforms SERCA1 (110 kDa), SERCA2 (114 kDa), and SERCA3 (109 kDa) [2+ into the ER by transporting Ca2+ ions against the concentration gradient, as described for the first time by Toyoshima and colleagues [2+ concentration balance between the ER and cytosol, as shown by the use of SERCA inhibitors, such as thapsigargin [ATP2A1, ATP2A2, and ATP2A3 genes resulted in over 70 SERCA-derived isoforms, which contribute differently to various types of cancer cells [2+ homeostasis, SERCAs also regulate cell survival and the ER stress signalling pathway [2+ overload [Sarco/ER Ca109 kDa) . SERCAs lleagues ,86. Thissigargin . In facter cells . Additio pathway ,89. For overload . On the overload .2+ concentration is low, in order to refill the Ca2+ stock and promote the Ca2+ signalling pathways. SOCE mainly involves two actors: the STIM and the Orai channel [Drosophila melanogaster and in humans in 2005, with two isoforms reported, STIM1 and STIM2 [2+ influx when the ER Ca2+ concentration is decreased [2+ level is high, STIM1 is inactive. In contrast, when the ER Ca2+ level is low, STIM1 is translocated within the ER to PM proximal sites to stimulate PM Ca2+ entry [2+ channel of STIM1 activation is named Orai1; STIM1 and Orai1 physically interact with each other and promote the SOCE-mediated PM Ca2+ influx [2+ level changes in the ER, thus keeping STIM1 in an inactive state when the ER Ca2+ level is high, and in an active state when the ER Ca2+ level is low. Recently, many groups have investigated the deregulation of STIMs and Orai proteins in a wide range of cancers. Interestingly, STIM1 is overexpressed in several types of tumours, and is therefore acknowledged to be a promoter of tumour migration and invasion, thus standing as an attractive target for controlling tumorigenesis [The SOCE mechanism is present only in non-excitable cells. The channels that participate in this mechanism only open when the ER Ca channel . STIM prnd STIM2 . It has ecreased . When th2+ entry . The tar+ influx . The strigenesis .2+ regulation at a spatio–temporal level. Alterations in the mitochondrial Ca2+ concentration directly impact OXPHOS function, ATP production, as well as cell death execution. Thus, cytosolic Ca2+ propagates into the mitochondria and leads to an enhanced respiration rate, H+ extrusion, ATP synthesis, ultimately changing the whole dynamic of mitochondrial metabolism and energy production. Cancer cells have the tendency to remodel mitochondrial Ca2+ homeostasis to facilitate tumour development.The mitochondria were described for the first time in the 1840s; later, in 1912, Otto Heinrich and Warburg linked this organelle to cellular respiration . The con2+ uptake is conducted via VDAC in the outer mitochondrial membrane (OMM), and via the MCU in the inner mitochondrial membrane (IMM) . Indeed, mitochondria were found to be positioned at the cytosolic edge facing the ER, and in actual contact with ER Ca2+ transporters [2+ signalling due to the plausible uptake of ER-released Ca2+ through the MCU, possibly via IP3R and MCU-associated proteins [Mitochondrial Cane (IMM) . Interessporters . The MCSproteins ,99.In the IMM, the MCU channel functions as a heteromeric protein complex of ~450–800 kDa that includes the ion-conducting core MCU protein and several MCU-associated regulatory proteins, including MCU dominant negative beta subunit (MCUb), essential MCU regulator (EMRE), the MICU family , and MCUR1. Together, they constitute the mitochondrial uptake machinery (MCUT-M) ,101.2+. Recently, the structure of the entire 480 kDa supercomplex indicates the main molecular interactions between the four major components: MCU–EMRE–MICU1–MICU2 [2+ concentrations, the MICU1–MICU2 heterodimer blocks the MCU channel, thus Ca2+ cannot accumulate inside the matrix. When Ca2+ concentration is increased, the EF-hands of MICU1 and MICU2 sense the change in Ca2+ level in the intermembrane space. These MICU proteins undergo conformational changes that dissociate the MCU holocomplex, thus allowing Ca2+ to enter the mitochondrial matrix [The MCU was identified by two leading studies in 2011 ,103. TheU1–MICU2 . At low l matrix .2+ signalling. Subsequently, MCU deficiency and overexpression were studied by many independent research groups to determine its role in evolution and pathophysiology. For instance, Huang et al., demonstrated that the loss of MCU decreased ATP production, whereas MCU overexpression increased mtROS production and apoptotic rate in Trypanosoma brucei [Genetic manipulations of MCU revealed its crucial role in the regulation of mitochondrial Caa brucei . In adul2+ transport, cell junctions, the and electron transport chain [MCU knockout mice displayed a smaller body size, with no changes in overall body composition. Mitochondria isolated from MCU knockout mice were not able to uptake Ca2+; strikingly, no basal metabolic processes were altered in the absence of MCU [Transcriptomic analysis of MCU mutant zebrafish showed the deregulated expression of genes that are involved in potassium transport activity, Cart chain . Moreovee of MCU .CCDC19, a gene expressed exclusively in vertebrates, plays the role of a negative regulator of MCU [2+ homeostasis.MCU function can be regulated by several proteins. Thus, MCUb (39 kDa) encoded by r of MCU , the ratCBARA1, establishes the threshold for MCU activity via its EF-hand Ca2+ binding domains [2+ uptake. This was demonstrated notably by the knockdown of MICU1 in HeLa cells, which have an increased Ca2+ accumulation, mtROS production, and apoptosis rate [MICU1 in mice induced Ca2+ accumulation and altered mitochondrial morphology, resulting in decreased ATP production [2+ overload, thus preventing cells from bioenergetic crises and programmed cell death [In addition, in the intermembrane space, MICU1 (54 kDa), encoded by  domains . MICU1 isis rate . Moreoveoduction . In mousoduction . Interesll death .2+ binding sites. MICU2 also acts as a gatekeeper, keeping the channel closed at low cytosolic Ca2+ levels [2+ crosstalk between IP3R in the ER and MCU in the mitochondria [MICU2 is a 45 kDa protein that is ubiquitously expressed in mammalian cells. It is also located in the mitochondria intermembrane space, and physically interacts with MCU and MICU1 . Sequenc+ levels . MICU2 achondria .SMDT1-encoded 10 kDa protein EMRE [EMRE are like that of knockout MCU in human embryonic kidney (HEK)-293T cells [Another direct activator of MCU is ein EMRE . Withoutein EMRE . Phenoty3T cells . Neutral3T cells .CCDC90A gene encodes a 35 kDa MCUR1 protein required for MCU-mediated mitochondrial Ca2+ uptake as a scaffold factor for MCU channel function. Its role in the assembly of the MCU supercomplex is also important [2+ threshold [m).Finally, the mportant . The exahreshold , whereashreshold . The los2+ transportome to guarantee proper mitochondrial function.Altogether, this emphasizes the importance of the correct assembly and coordination of the Ca2+ via the VDAC, and Ca2+ itself plays a role as a negative feedback regulator of the VDAC [2+ [The voltage-dependent anion channel (VDAC) located at the OMM is responsible for the flux of several metabolites and small cations into the mitochondria, serving as the mitochondrial gateway, and ensuring the crosstalk between mitochondria and other cellular compartments . OMM is the VDAC . The opeVDAC [2+ . The VDAVDAC [2+ . Among iVDAC [2+ . VDAC1 dVDAC [2+ .2+-dependent; furthermore, they are involved in several metabolic processes and are identified as potential biomarkers of various cancers [2+ solute carrier SLC25A23 is an adenine nucleotide transporter that interacts with the MCU and MICU1 and enhances mitochondrial Ca2+ uptake. SLC25A23 knockdown reduces mitochondrial Ca2+ uptake rate and protects cells from oxidative stress due to mtROS production, as demonstrated in a HeLa cell line model [The solute carrier protein family 25 (SLC25) members are located at the IMM, and are responsible for transporting metabolites, nucleotides, and cofactors across the non-permeable IMM. SLC25 proteins contain EF-hands at the N-terminal domain, and are Ca cancers . For exane model .+/Ca2+/Li+ exchanger (NCLX) is responsible for Ca2+ extrusion from the mitochondrial matrix into the intermembrane space (IMS). Each exported Ca2+ ion is exchanged with three imported Na+ [2+ uptake) and the NCLX (Ca2+ extrusion) regulates Ca2+ homeostasis, thus impacting cellular metabolism and Ca2+-related signalling pathways. By extruding Ca2+ from the mitochondria, NCLX can impact OXPHOS and mtROS production [2+ transportome, NCLX can also contribute to many metabolic processes, such as ATP, fatty acid, and nucleotide synthesis, etc., [2+ transportome, it has been shown that genetic loss of NCLX in colorectal cell lines (HCT116 and DLD1) could cause mitochondrial Ca2+ overload and inhibits proliferation and increases migration and chemoresistance by transcriptional reprogramming. Accordingly, low NCLX mRNA levels are correlated with advanced tumour stages in colorectal cancer [Moreover, the mitochondrial Narted Na+ . The baloduction . Similars, etc., . Althougl cancer .2+ fluxes regulate tumour proliferation, invasion, metastasis, programmed cell death resistance, etc., [2+ behaves as a signalling messenger between the mitochondria, ER, cytosol, and the nucleus, participating in tumour metabolic adaptation. Particularly in HCC, many Ca2+ transporters and exchangers are modulated, and can be classified according to their localization and expression level /superoxide dismutase 2 (SOD2) pathway. MCU overexpression in MHCC97H cells increased the mitochondrial uptake Ca2+ under histamine stimulation, and significantly increased the enzymatic activities of several TCA enzymes, such as PDH pyruvate dehydrogenase (PDH), alpha ketoglutarate dehydrogenase (α-KGDH), and isocitrate dehydrogenase (IDH), and decreased the NAD+/NADH ratio. In contrast, the MCU downregulated SMMC-7721 cells showed opposite effects [To elucidate the molecular role of the MCU and the consequences of its deregulation on HCC, the hepatocarcinoma cell lines SMMC-7721 and MHCC97H were used as models . These m effects . This in effects ,149.MCU overexpression in MHCC97H cells greatly increases phosphorylated JNK (p-JNK) levels but has no impact on the levels of p38 or ERK. Moreover, JNK downstream actors, such as p-Paxillin and matrix metalloproteinase 2 (MMP2), are also upregulated in MCU-overexpressed MHCC97H cells. In contrast, MCU downregulated SMMC-7721 showed opposite effects. P-Paxillin is implicated in the lamellipodia formation and focal adhesion turnover that facilitate tumour migration [2+ in HCC metastasis was confirmed in an orthotopical nude mice model. MCU overexpressed mice exhibited a higher metastatic capacity, with a decreased NAD+/NADH ratio and an increased p-JNK level, in vivo.In addition, the mitogen-activated protein kinase (MAPK) pathway appeared to be crucial for HCC cell migration and invasion by activating cJun NH2-terminal kinase (JNK), p38, and/or extracellular signal-regulated kinase (ERK) . MCU oveigration , whereasigration . Futherm2+ excessively increases mtROS production via the NAD+/ SIRT3/ SOD2 pathway, thus promoting HCC invasion, as well as migration via the mtROS/JNK pathway  and metastatic capacity via the ROS/nuclear factor erythroid 2-related factor 2 (Nrf2)/Notch1 pathway in HCC, in both in vitro and in vivo models [MCUR1 overexpressed MHCC97L cell lines displayed a significant elevation in mitochondrial Ca2+ and mtROS production, and facilitated the EMT, coinciding with a decreased expression of epithelial markers (E-cadherin and ZO-1) and an increased expression of mesenchymal markers (N-cadherin and vimentin), both in vitro and in an orthotopic transplantation model. MCUR1 downregulated BEL7402 displayed the complete opposite effects [Snail expression was significantly decreased by MCUR1 knockdown in HCC cell lines. A significant reduction in cytoplasmic Notch1 and nuclear NICD1 protein levels in the MCUR1 knockdown HCC cell line was also observed. The simultaneous inhibition of the Snail-related EMT and ROS/Nrf2/Notch pathway, via Nrf2 knockdown and using the Notch1 inhibitor DAPT, decreased MCUR1-induced EMT [2+ in facilitating HCC EMT by overproducing mtROS, and thus mediates Nrf2 translocation and activates Snail-related EMT via Notch1 and its active form NICD1 [Recently, Jin and colleagues observed that MCUR1 was upregulated in metastatic HCC, by comparing 63 metastatic patient tissues with 74 non-metastatic patient tissues . MCUR1 uo models . Moreove effects . The epi effects . Indeed,uced EMT . These rrm NICD1  ) induces the intrahepatic metastasis of HCC; alternatively, knockdown of TRPC6 or NCX1 inhibits this metastatic development. Interestingly, after 24 h of TGFβ stimulation, EMT was observed with a decreased expression of epithelial marker E-cadherin and an increased expression of mesenchymal vimentin. Thus, both MCRU1 and the TRPC6/NCX1 complex are involved in the EMT mechanism [In HCC cell lines (HepG2 and Huh7) and an HCC mouse model, transforming growth factor beta (TGFβ) stimulation  mechanisms were demonstrated to be cytosolic Caependent ,157,158.ependent . Using tR in HCC . TRPC6/Cor STAT3 . Indeed,or STAT3 .2+ in TGFβ-driven EMT via the Smad2-dependent pathway, and in the MDR-related mechanism via the pSTAT3-dependent pathway. Further studies need to be conducted to verify the exact roles and molecular mechanisms of the TRPC6/NCX1 complex in different HCC stages, and TRPC6/Ca2+-dependent MDR mechanisms  and p300 [In hypoxic HCC cells, hypoxia-inducible factor-1 alpha (HIF-1α) is a transcription factor that plays important roles in hypoxic hepatocarcinogenesis. An analysis of 10 HCC patients revealed that the protein expression of STIM1 and HIF-1α was positively correlated in patient tissues and was also associated with the tumour size in a xenograft mouse model . HIF-1α and p300 ,161. TogSTIM1 knockout cell lines (SMMC7721 and HepG2), tumour invasion and proliferation were significantly inhibited. On the other hand, the resistance of anoikis that support HCC cell survival during metastasis was significantly increased in this model, suggesting the participation of STIM1-mediated cytosolic Ca2+ in anoikis [In metastatic HCC cells, immunohistochemical analysis of 12 HCC patients demonstrated that STIM1 protein level was downregulated and correlated with a lower overall survival of HCC patients . In two  anoikis .STIM1 was knocked out in SMMC7721 and HepG2 cells. Remarkably, downregulation of glycolysis-involved genes  and de novo lipogenesis-involved genes  were observed. In contrast, fatty acid oxidation-involved genes  were upregulated [Metastasis requires the metabolic switch from anabolism (ATP production by glycolysis and lipogenesis) to catabolism (fatty acid oxidation) to protect tumour cells against starvation and anoikis . To examegulated . This fi2+ entry by the SOCE mechanism stabilizes HIF-1α via CaMKII-mediated p300 activation, thus promoting tumour proliferation. In contrast, STIM1 protein level is downregulated in metastatic HCC cells. This leads to a shift of the metabolic balance from anabolism to catabolism, and contributes to metastasis and anoikis resitance.Taken together, under hypoxic conditions, STIM1 is upregulated and mediates the SOCE mechanism. STIM1-mediated cytosolic Ca2+ transportome and its components in cancer, with an emphasis on HCC. Based on its various cellular locations , it is indisputable that the Ca2+ transportome influences interorganelle communication and can play a major role in cancer cell proliferation, metastasis, and drug resistance.This review describes some key roles of the Ca2+ transportome deregulation is increasingly expanding with regards to HCC initiation as well as progression [2+ transportome have been identified in almost all stages of HCC development. Thus, in the future, deeper study of the MCU, MCUR1, STIM1, and TRPC6 in hepatocarcinogenesis may provide important information for the development of diagnostics and therapeutic approaches required to fight this aggressive and frequently relapsing cancer.The understanding of Cagression . Indeed,2+, and lead to a better understanding of the oncogenic deregulation of Ca2+-mediated interorganelle communication in HCC.Finally, it can be speculated that future works will identify new components of Ca


Procoagulant platelets are a subtype of activated platelets that sustains thrombin generation in order to consolidate the clot and stop bleeding. This aspect of platelet activation is gaining more and more recognition and interest. In fact, next to aggregating platelets, procoagulant platelets are key regulators of thrombus formation. Imbalance of both subpopulations can lead to undesired thrombotic or bleeding events. COAT platelets derive from a common pro-aggregatory phenotype in cells capable of accumulating enough cytosolic calcium to trigger specific pathways that mediate the loss of their aggregating properties and the development of new adhesive and procoagulant characteristics. Complex cascades of signaling events are involved and this may explain why an inter-individual variability exists in procoagulant potential. Nowadays, we know the key agonists and mediators underlying the generation of a procoagulant platelet response. However, we still lack insight into the actual mechanisms controlling this dichotomous pattern . In this review, we describe the phenotypic characteristics of procoagulant COAT platelets, we detail the current knowledge on the mechanisms of the procoagulant response, and discuss possible drivers of this dichotomous diversification, in particular addressing the impact of the platelet environment during in vivo thrombus formation. Procoagulant platelets are a subpopulation of platelets appearing upon strong activation, which localize and enhance thrombin generation at sites of vascular injury by expressing negatively charged phospholipids and sustaining the formation of the tenase and prothrombinase complexes on their surface . EventuaThis balance seems to be altered in bleeding diatheses ,7 and he2+) signaling in procoagulant platelets and how this may be targeted for a selective treatment. Similarly, the present review offers a comprehensive overview of the mechanisms underlying procoagulant platelet formation, which may be explored as possible druggable targets.Current antiplatelet therapies focus on inhibition of platelet aggregation . However2+]cyt), secretion of granule content and secondary agonists, and activation of the fibrinogen receptor cyt cyt ,26.This review will first describe the key features of procoagulant platelets focusing on their phenotype, machinery, and functions. Then, we will dissect the intracellular mechanisms underlying the dichotomous activation pattern between aggregating and procoagulant platelets. Finally, we will address the current and future research axes that could be useful to understand the origin of procoagulant platelets and the variability of their response.2+-induced platelets) cyt, this flippase activity is inhibited. Moreover, subsequent activation of the transmembrane 16 F (TMEM16F) scrambles PS/PE across the membrane cyt [GPIIb-IIIa may be inactivated by a calpain-2/TMEM16F mechanism following high and sustained [Ca2+]cyt . An alte2+]cyt . Accordi2+]cyt . Therefo2+]cyt to be activated cyt increases after activation through multiple Ca2+ channels. Mitochondria accumulate Ca2+ during activation [2+ accumulation. The future procoagulant platelets eventually reach supramaximal [Ca2+]cyt required for PS exposure cyt (>100 µM) cyt is a marker of platelet activation and mediator of the downstream signaling cyt efflux cyt necessary for procoagulant activity are reached through different receptor-operated (ROCE) and store-operated (SOCE) Ca2+ entry pathways. Of note, most of the Ca2+ influx pathways are considered working in parallel. Understanding the temporality and the hierarchy of these channels might help elucidating their implication in the procoagulant response.Very high and sustained cyt is referred as oscillatory because platelets can still manage Ca2+ efflux  and internalization in mitochondria to maintain their homeostasis [2+]cyt required to depolarize the membrane of mitochondria. Indeed, as the Ca2+ concentration rises in the cytosol, the mitochondria act normally as buffer accumulate and store Ca2+ through the MCU [2+]cyt see their mitochondria undergoing mitochondrial permeability transition pores (mPTP) opening [2+]mito in the extracellular space.During the initial Caeostasis ,105. Aft the MCU . Platele opening . This ev+ uptake following thrombin or collagen stimulation cyt mobilization was also impaired in presence of Cl- channel inhibitors or with platelet activation performed in Cl--free buffer. However, inhibitors of Cl- channels did not affect PS exposure mediated by Ca2+ ionophore. Since Cl- blockers (or absence of Cl- in the buffer) only affect agonist-dependent PS exposure and not ionophore-induced PS-exposure, Cl- entry is expected to play a role in the signaling—upstream of TMEM16F—to achieve a full procoagulant activity cyt mobilization and Ca2+/DAG-mediated guanine exchange factor I  signaling, while DAG activates protein kinase C (PKC). Both PKC and CalDAG-GEFI activate the small-GTPase Rap1 signaling, resulting in integrin activation and secretion of granule and soluble agonists cyt rise and CalDAG-GEFI signaling, and PKC activation, respectively. G12/13 trigger the reorganization of the actin cytoskeleton (shape change) mediated by RhoA pathway cyt cyt ,163,164.tivation . This istivation  or inhibtivation ,162.2+ by PLCβ-mediated cleavage of PIP2 in IP3 (Ca2+ mobilization) and DAG (shape change and granule release). This mechanism is redundant with thrombin signaling and might explain the low relevance of the P2Y1 receptor in the context of COAT platelets cyt efflux [2+ is expelled from the cell using the electrochemical gradient of Na+ with a ratio of 3Na+:1Ca2+ [2+]cyt. The reverse mode pumping Ca2+ in the cytosol while expelling exceeding Na+ promotes further Ca2+ entry and thus procoagulant signaling mito efflux is mediated by mitochondrial H+/Ca2+ and Na+/Ca2+ exchangers [2+]cyt required for PS exposure to occur are reached only after mPTP opening, releasing an important amount of [Ca2+]mito into the cytosolic space [2+]mito threshold depending on cyclophilin D (CypD) cyt of 1–10μM cyt in the presence of a low number of mitochondria. Therefore, mPTP opening as well as PS exposure would occur more rapidly in those cells.Wiskott–Aldrich syndrome, caused by mutations in the Wiskott–Aldrich syndrome protein (WASP) regulating actin activity, is characterized by increased clearance of platelets due to early PS exposure and lower mitochondrial count . In line2+]cyt required for PS exposure might be due to a two-step “decision-making” mechanism. First, the low (ca. 1000 per platelet) and highly variable surface density of the PAR1 receptor between individuals and allegedly between platelets correlates with the platelet response [2+-pumps may explain the variability of [Ca2+]cyt achieved after a similar stimulus and the fact that only a fraction of platelets becomes procoagulant cyt cyt . These sAging of the platelets is an intrinsic heterogeneity factor. Aged platelets expose PS through an apoptotic-like mechanism ,223. HowPlatelet aging induces ROS accumulation within the cell . In accoShedding of platelet receptor ecto-domains occurs with aging, both in vitro (storage lesion of platelet concentrates) and in vivo. The A disintegrin and metalloprotease 17 (ADAM17) constitutively cleaves GPIbα that becomes less available for the adhesion with in vivo aging ,230. GPV2+ induced by ionomycin and convulxin/thrombin [Acute GPIbα and GPVI shedding also occurs upon strong activation . GPVI isthrombin . Dichotothrombin .2+ entry thought to be necessary for mitochondria overload and mPTP formation eventually triggering PS exposure.As depicted in 2+]mito required to open mPTP cyt platelets, which still retains some pro-aggregatory properties and mitochondrial potential. They represented a maximum 15% of the total platelet population after thrombin activation. The functional procoagulant response of this population, which does not fit the consensus definition of COAT platelets, has not yet been studied.As proposed by Mischnik et al. , upon ouIn vitro experiments in suspension do not reflect the complexity of in vivo platelet activation with the whole spectrum of physiological agonists present at various concentrations in the growing thrombus. Indeed, clot architecture drives agonist concentrations, cell-to-cell interactions and therefore platelet responses. Intra-vital experiments with mice can represent more accurately how the phenotypical diversification occurs among the platelets.As presented below in 2+ accumulates within the thrombus and triggers a synchronous ballooning and micro-vesiculation of the platelets cyt to trigger specific key events such as mitochondria depolarization. The ensuing high and sustained [Ca2+]cyt triggers the functional response composed of PS exposure, formation of the tenase and prothrombinase complexes, coating with adhesive factors and FXIII, GPIIb-IIIa downregulation, and GPIb and GPVI shedding.Future procoagulant COAT platelets need to accumulate enough [Ca+ and in particular of the reverse mode of NCX for the formation of procoagulant platelets, the regulatory upstream mechanisms are not yet elucidated. Intrinsic platelet heterogeneity and extrinsic factors may explain the dichotomous procoagulant response and the high variability observed between individuals. Studies investigating the generation of procoagulant COAT platelets in three-dimensional models will contribute deciphering their role in vivo.While recent research has revealed the key role of cytosolic Na