Platelet Calcium Signaling Research Articles

Novel hemizygous CORO1A variant leads to combined immunodeficiency with defective platelet calcium signaling and cell mobility

To date, fewer than 20 patients have been identified as having germline biallelic mutations in the coronin-1A gene (CORO1A) and its protein with clinical features of combined immunodeficiency characterized by T-cell lymphopenia ranging from the severe phenotype to the mild phenotype, recurrent infections, and lymphoproliferative disorders. However, the effects of CORO1A protein disruption on actin-dependent functions in primary cells have not been fully delineated. We sought to characterize the underlying defects of actin-dependent cellular functions in a female patient with combined immunodeficiency caused by a novel missense variant in the CORO1A gene in combination with a de novo heterozygous microdeletion of chromosome 16p11.2 and also to provide evidence of the pathogenicity of this gene mutation. To identify the genetic defect, next-generation sequencing followed by Sanger confirmation and array comparative genomic hybridization were performed. Western blot and quantitative PCR tests were used to assess the effects on the protein. Flow cytometry and live microscopy were performed to investigate cellular motility and immune cell counts and function. We demonstrated that the CORO1A hemizygous variant c.19C>T, p. A7C induces significant decreases in cellular levels of the CORO1A protein while leaving mRNA concentrations unaffected. The observed mutation resulted in impaired natural killer cell cytotoxicity and platelet calcium signaling. In addition, primary granulocytes and mesenchymal cells showed significant defects in motility. Collectively, we added new data about the CORO1A gene as a key player in actin cytoskeleton dynamics and cell signaling. Our findings expand the clinical spectrum regarding CORO1A protein deficiency and confirm the importance of a personalized therapeutic approach for each patient.

Development of a parallel multiscale 3D model for thrombus growth under flow

Thrombus growth is a complex and multiscale process involving interactions spanning length scales from individual micron-sized platelets to macroscopic clots at the millimeter scale. Here, we describe a 3D multiscale framework to simulate thrombus growth under flow comprising four individually parallelized and coupled modules: a data-driven Neural Network (NN) that accounts for platelet calcium signaling, a Lattice Kinetic Monte Carlo (LKMC) simulation for tracking platelet positions, a Finite Volume Method (FVM) simulator for solving convection-diffusion-reaction equations describing agonist release and transport, and a Lattice Boltzmann (LB) flow solver for computing the blood flow field over the growing thrombus. Parallelization was achieved by developing in-house parallel routines for NN and LKMC, while the open-source libraries OpenFOAM and Palabos were used for FVM and LB, respectively. Importantly, the parallel LKMC solver utilizes particle-based parallel decomposition allowing efficient use of cores over highly heterogeneous regions of the domain. The parallelized model was validated against a reference serial version for accuracy, demonstrating comparable results for both microfluidic and stenotic arterial clotting conditions. Moreover, the parallelized framework was shown to scale essentially linearly on up to 64 cores. Overall, the parallelized multiscale framework described here is demonstrated to be a promising approach for studying single-platelet resolved thrombosis at length scales that are sufficiently large to directly simulate coronary blood vessels.

Inhibition of platelet aggregation by activation of platelet intermediate conductance Ca2+‐activated potassium channels

BackgroundWithin the vasculature platelets and endothelial cells play crucial roles in hemostasis and thrombosis. Platelets, like endothelial cells, possess intermediate conductance Ca2+‐activated K+ (IKCa) channels and generate nitric oxide (NO). Although NO limits platelet aggregation, the role of IKCa channels in platelet function and NO generation has not yet been explored. ObjectivesWe investigated whether IKCa channel activation inhibits platelet aggregation, and per endothelial cells, enhances platelet NO production. MethodsPlatelets were isolated from human volunteers. Aggregometry, confocal microscopy, and a novel flow chamber model, the Quartz Crystal Microbalance (QCM) were used to assess platelet function. Flow cytometry was used to measure platelet NO production, calcium signaling, membrane potential, integrin αIIb/β3 activation, granule release, and procoagulant platelet formation. ResultsPlatelet IKCa channel activation with SKA‐31 inhibited aggregation in a concentration‐dependent manner, an effect reversed by the selective IKCa channel blocker TRAM‐34. The QCM model along with confocal microscopy demonstrated that SKA‐31 inhibited platelet aggregation under flow conditions. Surprisingly, IKCa activation by SKA‐31 inhibited platelet NO generation, but this could be explained by a concomitant reduction in platelet calcium signaling. IKCa activation by SKA‐31 also inhibited dense and alpha‐granule secretion and integrin αIIb/β3 activation, but maintained platelet phosphatidylserine surface exposure as a measure of procoagulant response. ConclusionsPlatelet IKCa channel activation inhibits aggregation by reducing calcium‐signaling and granule secretion, but not by enhancing platelet NO generation. IKCa channels may be novel targets for the development of antiplatelet drugs that limit atherothrombosis, but not coagulation.

Reduction in SOCE and Associated Aggregation in Platelets from Mice with Platelet-Specific Deletion of Orai1.

Calcium signalling in platelets through store operated Ca2+ entry (SOCE) or receptor-operated Ca2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet’s SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1flox/flox; Pf4-Cre termed as Orai1Plt-KO) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca2+-entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

On calcium fluorophore’s impact in platelet signaling studies

Observation of calcium signaling in platelets - blood cells designed to be involved in stopping bleeding and forming blood clots - is an important part of fundamental research in hemostasis. However, such a study is possible only with the use of calcium fluorophores - small molecules that penetrate the platelet membrane due to their hydrophobic -AM part, which is then hydrolyzed by cytosol esterases. In this work, we consider the phenomenon of inhomogeneous loading of calcium fluorophores into platelets.We used platelets from healthy adult donors loaded with various fluorescent probes (CalBryte590, DiOC6 (3), Fura Red, Fluo-4 and CellTracker Violet BMQC) and immobilized on antibodies to CD31 in parallel plane flow chambers. Total internal reflection fluorescence (TIRF) microscopy was used for observations.We demonstrated that all studied probes are loaded heterogeneously, with 30% platelets being loaded with a probe 2-6 times higher than the population median value. Using the CalBryte590 probe as an example, we have shown that a decrease in the incubation temperature, the addition of Pluronic 127 to the incubation medium, or membrane cholesterol depletion significantly reduces the heterogeneity of the probe distribution in the population. By looking at platelet activation from the surface, we have shown that the probability of experiencing strong activation, as measured by the intensity of calcium oscillations, correlates with the amount of probe in the platelet.Thus, we conclude that the type of fluorophore used and the conditions of its loading into platelets can significantly affect the results of experiments on the observation of calcium signaling in platelets.

BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation.

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Platelet dysfunction during trauma involves diverse signaling pathways and an inhibitory activity in patient-derived plasma.

Trauma-induced coagulopathy occurs in about 25% of injured patients and accounts for about 10% of deaths worldwide. Upon injury, hemostatic function may decline due to vascular dysfunction, clotting factor deficiencies, hyperfibrinolysis, and/or platelet dysfunction. We investigated agonist-induced calcium signaling in platelets obtained over time from trauma patients. Platelets from trauma patients and healthy donors were monitored via intracellular calcium mobilization and flow cytometry markers (α2bβ3 activation, P-selectin display, and phosphatidylserine exposure) following stimulation with a panel of agonists (adenosine 5'-diphosphate sodium salt, U46619, convulxin, PAR-1/4 activating peptides, iloprost) used in isolation or in pairwise tests. Furthermore, healthy donor platelets were tested in heterologous plasma isolated from healthy subjects and trauma patients. When exposed to agonists over the first 24 hours postinjury, trauma patient platelets mobilized less calcium in comparison to healthy platelets. Partial recovery of platelet activity was observed in about a third of patients after 120 hours, although not fully obtaining healthy baseline function. Flow cytometry markers of trauma platelets were similar to healthy platelets prior to stimulation, but were depressed in trauma platelets stimulated with adenosine 5'-diphosphate sodium salt or convulxin. Also, washed healthy platelets showed a significant reduction in calcium mobilization when reconstituted in plasma from trauma patients, relative to healthy plasma, at all plasma doses tested. Platelet dysfunction in trauma patients included poor response to multiple agonists relevant to hemostatic function. Furthermore, the inhibitor effect of patient plasma on healthy platelets suggests that soluble plasma species may downregulate endogenous or transfused platelets during trauma.

Computational biology analysis of platelet signaling reveals roles of feedbacks through phospholipase C and inositol 1,4,5-trisphosphate 3-kinase in controlling amplitude and duration of calcium oscillations

Blood platelet activation is required to allow their participation in hemostasis and thrombosis. It is regulated by a complicated signaling network, whose functioning has been recently attracting attention for basic research and pharmacological purposes. Phospholipase С (PLC) is an enzyme playing an important role in platelet calcium signaling and responsible for release of inositol triphosphate (IP3) into platelet cytoplasm thus controlling intracellular calcium concentration. Using a comprehensive computational model of platelet calcium signaling, we studied the influence of the positive feedback executed by cytosolic calcium on the PLC isoform β2 during platelet activation. With the positive feedback, the model predicted hyperintensive response to platelet activation by thrombin, where non-physiologically high calcium concentrations arose. However, if one took into account a negative feedback determined by IP3 3-kinase (IP3K), combination of the feedback resulted in the formation of a stepped response (with a stable oscillation amplitude and activation-dependent duration). Stochastic simulations confirmed that PLC and IP3K should act in pair to ensure platelet's “all-or-none” response to activation, when the activation level sets the probability of platelet activation, but not its intensity.

Compartmentalized calcium signaling triggers subpopulation formation upon platelet activation through PAR1.

Blood platelets need to undergo activation to carry out their function of stopping bleeding. Different activation degrees lead to a stepped hierarchy of responses: ability to aggregate, granule release, and, in a fraction of platelets, phosphatidylserine (PS) exposure. This suggests the existence of decision-making mechanisms in the platelet intracellular signaling network. To identify and investigate them, we developed a computational model of PAR1-stimulated platelet signal transduction that included a minimal set of major players in the calcium signaling network. The model comprised three intracellular compartments: cytosol, dense tubular system (DTS) and mitochondria and extracellular space. Computer simulations showed that the stable resting state of platelets is maintained via a balance between calcium pumps and leaks through the DTS and plasma membranes. Stimulation of PAR1 induced oscillations in the cytosolic calcium concentrations, in good agreement with experimental observations. Further increase in the agonist level activated the mitochondrial uniporter leading to calcium uptake by mitochondria, which caused the collapse of mitochondrial membrane potential in a fraction of platelets leading to the PS exposure. The formation of this subpopulation was shown to be a stochastic process determined by the small number of activated PAR1 receptors and by heterogeneity in the number of ion pumps. These results demonstrate how a gradual increase of the activation degree can be converted into a stepped response hierarchy ultimately leading to formation of two distinct subpopulations from an initially homogeneous population.

Platelet Expression of a Genetically Encoded Calcium Indicator Reveals Platelet Activation Gradients in Real Time during Thrombus Formation in Vivo

Introduction: Recent studies have demonstrated that platelet activation during hemostatic thrombus formation in vivo is heterogeneous in time and space. We have previously shown that in response to a penetrating injury in the microcirculation platelet accumulation and activation are driven by gradients of soluble agonists emanating from the site of injury. These gradients result in a characteristic thrombus architecture in which a core of fully activated platelets that have released their alpha granules is overlaid by a shell of less activated platelets that frequently embolize. While dependent on ADP/P2Y12 signaling, the extent and dynamics of platelet activation in the shell region remain poorly understood. Here, we used intravital imaging of platelet cytosolic calcium concentration to examine platelet activation in real time following vascular injury in vivo.Methods: We generated transgenic mice expressing a genetically encoded calcium indicator (GCaMP3) specifically in megakaryocytes and platelets by crossing PF4-Cre mice with Ai38 mice carrying the GCaMP3 transgene. GCaMP3 fluorescence was visualized in platelets following laser-induced injury in mouse cremaster arterioles using spinning disk confocal intravital microscopy.Results: Rapid and dynamic changes in GCaMP3 fluorescence were observed during platelet accumulation following vascular injury. Platelets immediately adjacent to the site of injury exhibited a rapid and sustained increase in cytosolic calcium. Peak cytosolic calcium levels in the core region occurred within the first minute post-injury, prior to peak P-selectin expression. Platelets in the shell region were characterized by dynamic changes in cytosolic calcium at the level of individual platelets, with the appearance of transient “waves” of calcium signaling propagating among groups of platelets. At the population level, cytosolic calcium concentration was substantially lower in the shell region as compared to the core.Conclusions: Expression of a genetically encoded calcium indicator in platelets allows for the visualization of platelet activation events in real time in vivo. Platelets within the core region of a thrombus exhibit a rapid and sustained increase in cytosolic calcium concentration, indicating robust activation. In contrast, platelets in the shell region exhibit transient increases in cytosolic calcium indicating weak activation, consistent with their increased likelihood of embolization. The examination of platelet calcium signaling in vivo provides a sensitive readout of platelet activation that will shed new light on mechanisms regulating hemostasis and thrombosis, and may be useful in assessing the impact of anti-thrombotic agents on platelet function. Research support was provided by the American Heart Association and National Heart, Lung and Blood Institute. DisclosuresStalker:Medicines Company: Research Funding. Brass:Medicines Company: Research Funding.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) influences platelet calcium signaling and function by regulation of Orai1 expression in megakaryocytes

AbstractPlatelets are activated on increase of cytosolic Ca2+ activity ([Ca2+]i), accomplished by store-operated Ca2+ entry (SOCE) involving the pore-forming ion channel subunit Orai1. Here, we show, for the first time, that the serum- and glucocorticoid-inducible kinase 1 (SGK1) is expressed in platelets and megakaryocytes. SOCE and agonist-induced [Ca2+]i increase are significantly blunted in platelets from SGK1 knockout mice (sgk1−/−). Similarly, Ca2+-dependent degranulation, integrin αIIbβ3 activation, phosphatidylserine exposure, aggregation, and in vitro thrombus formation were significantly impaired in sgk1−/− platelets, whereas tail bleeding time was not significantly enhanced. Platelet and megakaryocyte Orai1 transcript levels and membrane protein abundance were significantly reduced in sgk1−/− mice. In human megakaryoblastic cells (MEG-01), transfection with constitutively active S422DSGK1 but not with inactive K127NSGK1 significantly enhanced Orai1 expression and SOCE, while effects reversed by the SGK1 inhibitor GSK650394 (1μM). Transfection of MEG-01 cells with S422DSGK1 significantly increased phosphorylation of IκB kinase α/β and IκBα resulting in nuclear translocation of NF-κB subunit p65. Treatment of S422DSGK1-transfected MEG-01 cells with the IκB kinase inhibitor BMS-345541 (10μM) abolished SGK1-induced increase of Orai1 expression and SOCE. The present observations unravel SGK1 as novel regulator of platelet function, effective at least in part by NF-κB–dependent transcriptional up-regulation of Orai1 in megakaryocytes and increasing platelet SOCE.

Potential Role of Recombinant Erythropoietin and Intravenous Iron Preparations in Target Vessel Re-Stenosis Following Coronary Stint Insertions in Hemodialysis Patients

LETTERS TO THE EDITOR Potential Role of Recombinant Erythropoietin and Intravenous Iron Preparations in Target Ves- sel Re-Stenosis Following Coronary Stint Inser- tions in Hemodialysis Patients Key words: ESRD, atherosclerosis, cardiovascular disease, anemia, oxidative stress, inflammation (Intern Med 50: 1449-1450, 2011) (DOI: 10.2169/internalmedicine.50.5173) To the Editor In an article published in the November is- sue of the Journal, Dr. Takeuchi and colleagues (1) reported on the incidence of target lesion re-stenosis in a cohort of 165 consecutive patients with coronary artery disease who had undergone 231 paclitaxel-eluting stent placements in their facility between May 2007 and August 2009. All pa- tients had been treated with two types of anti-platelet agents (aspirin and Clopidogrel or Ticlopidine) and 90% of the subjects had received follow-up coronary angiogram 6 months later. The underlying diseases in the study popula- tion included hypertension in 75%, hyperlipidemia in 78%, diabetes mellitus in 60% and end-stage renal disease treated with intermittent hemodialysis in 14%. Overall target lesion revascularization rate within 6 months following stent place- ment was 14.6%. The rate of target lesion re-stenosis in hemodialysis patients (43%) was far greater (p=0.0001, odds ratio: 6.61, 95% C.I.: 2.34-18.6) than in those with hyper- tension (15.0%), hyperlipidemia (12.4%), and diabetes mel- litus, treated with either oral medications (12.5%) or insulin (12.0%). Based on these observations the authors concluded that hemodialysis confers the greatest risk of target lesion re-stenosis after a coronary artery drug-eluting stint place- ment procedure. In fact comparison of outcomes among pa- tients with chronic kidney disease undergoing coronary ar- tery stint placement has revealed a significantly greater inci- dence of target vessel revascularization in the dialysis than in non-dialysis patients (2-5). The increased risk of target le- sion re-stenosis in hemodialysis patients has been attributed to chronic vascular inflammation and poor vessel dila- tion (6) due to medial calcification commonly present in these patients (7-9). It is noteworthy however, that using in- travascular ultrasonography or angiography Takeuchi et al (1) found no difference in the minimal stent area or mini- mal stent lumen diameter between patients who did and those who did not experience target lesion re-stenosis, thus excluding technical factors such as poor dilation as the cause of increased risk of stint re-stenosis in their hemodialysis-dependent patients. Exposure of blood to artifi- cial surfaces such as tubing and dialyzer membrane, me- chanical stress induced by the roller pump and influx of im- purities from the dialysate compartment are known to result in activation of leukocytes and release of pro-inflammatory mediators in patients undergoing hemodialysis. The recur- rent hemodialysis-induced inflammatory response is thought to, in part, contribute to target vessel re-stenosis following stent placement by stimulating neo-intimal expansion in hemodialysis-dependent patients (10). The author wishes to suggest that two additional factors can, in part, contribute to the high incidence of target vessel re-stenosis in patients maintained on chronic hemodialysis treatment. These include recombinant erythropoietin prod- ucts collectively referred to as erythropoiesis-stimulating agents (ESAs) and intravenous iron preparations which are used to treat anemia in nearly all dialysis-dependent patients with end-stage renal disease. ESAs have been shown to cause vasoconstriction-dependent hypertension and vascular smooth muscle cell proliferation, increase endothelin, thromboxane and reactive oxygen species (ROS) production, reduce prostacyclin and nitric oxide generation and up- regulate vascular tissue rennin-angiotensin system (reviewed in Ref. (11, 12)). These effects of ESAs particularly when administered in high doses to overcome ESA-resistant ane- mia in highly inflamed patients can contribute to target ves- sel re-stenosis following coronary stint placement. In addi- tion, by stimulating platelet production, increasing platelet activity (via augmentation of platelet calcium signaling), raising E-selectin and P selectin expression and release of plasminogen activator inhibitor-1 and von Willebrand factor, ESAs can promote thrombosis (reviewed in Ref. (11-14)). Together via these non-erythropoietic actions, the use of ESAs in dialysis patients can, at least, in part, account for the observed high incidence of target vessel re-stenosis and thrombosis in this population. In fact, the use of these agents has been linked to vascular access stenosis and thrombosis in dialysis patients and to accelerated tumor growth and thrombotic complications in cancer pa- tients (15, 16). In addition to ESAs, intravenous iron preparations which are commonly used to replenish iron stores in hemodialysis patients can intensify oxidative stress, stimulate monocyte activation and adhesion and promote vascular remodel- ing (17, 18), events that can potentially facilitate target ves- sel re-stenosis following stint placement. Given the universal use of ESAs and intravenous iron preparations in dialysis-dependent ESRD patients and the well-known effects of these agents on vascular tissue and the blood coagulation system, careful assessment of the po- Division of Nephrology and Hypertension, University of California Irvine, USA Received for publication January 17, 2011; Accepted for publication March 23, 2011 Correspondence to Dr. Nosratola D. Vaziri, ndvaziri@uci.edu

Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Novel molecules in calcium signaling in platelets.

A rise in the intracellular calcium (Ca2+) concentration is a major component of the signaling mechanisms regulating platelet function in thrombosis and hemostasis. Previous studies, however, failed to identify many key molecules regulating Ca2+ signaling in platelets. Here, we review recent findings, which identified CalDAG-GEFI as a critical Ca2+ sensor that links increases in intracellular Ca2+ to integrin activation, TxA2 formation, and granule release in stimulated platelets. Furthermore, we summarize work that lead to the discovery of STIM1 and Orai1 as key regulators of store-operated calcium entry (SOCE) in platelets. A short discussion on the usefulness of each molecule as a potential new target for antiplatelet therapy is included.

Calcium signaling in platelets.

Agonist-induced elevation in cytosolic Ca2+ concentrations is essential for platelet activation in hemostasis and thrombosis. It occurs through Ca2+ release from intracellular stores and Ca2+ entry through the plasma membrane (PM). Ca2+ store release is a well-established process involving phospholipase (PL)C-mediated production of inositol-1,4,5-trisphosphate (IP3), which in turn releases Ca2+ from the intracellular stores through IP3 receptor channels. In contrast, the mechanisms controlling Ca2+ entry and the significance of this process for platelet activation have been elucidated only very recently. In platelets, as in other non-excitable cells, the major way of Ca2+ entry involves the agonist-induced release of cytosolic sequestered Ca2+ followed by Ca2+ influx through the PM, a process referred to as store-operated calcium entry (SOCE). It is now clear that stromal interaction molecule 1 (STIM1), a Ca2+ sensor molecule in intracellular stores, and the four transmembrane channel protein Orai1 are the key players in platelet SOCE. The other major Ca2+ entry mechanism is mediated by the direct receptor-operated calcium (ROC) channel, P2X1. Besides these, canonical transient receptor potential channel (TRPC) 6 mediates Ca2+ entry through the PM. This review summarizes the current knowledge of platelet Ca2+ homeostasis with a focus on the newly identified Ca2+ entry mechanisms.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization

Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

Altered calcium signalling in platelets from bile-duct-ligated rats

In the present study, we have analysed the mechanisms of Ca(2+) entry and release in platelets obtained from BDL (bile-duct-ligated) rats, 11-13 days and 4 weeks after surgery. Platelets were washed and loaded with fura-2, and [Ca(2+)](i) (cytosolic Ca(2+) concentration) was determined in cell suspensions by means of fluorescence spectroscopy. Basal [Ca(2+)](i) was similar in platelets from BDL rats compared with those from their respective controls, both in the absence and presence of extracellular Ca(2+). Platelet stimulation with thrombin in the absence and presence of extracellular Ca(2+) induced a rapid rise in [Ca(2+)](i) that was of greater magnitude in platelets from BDL rats than in controls. Ca(2+) storage was significantly elevated in platelets from BDL rats, as well as the activity of SERCA (sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase). Capacitative Ca(2+) entry, as evaluated by inhibition of SERCA with thapsigargin, was also altered in platelets from BDL rats, having lower rates of Ca(2+) entry. In conclusion, chronic BDL alters intracellular Ca(2+) homoeostasis in platelets, such that an enhanced Ca(2+) release is evoked by thrombin, which may be due to an increased amount of Ca(2+) stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis has completely developed and occurs during the cholestasis phase.

Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader

Calcium signaling in platelets is an important physiological response to various aggregation stimuli. Loading platelets with various fluorescent dyes and measuring the change in calcium concentration using a spectrofluorometer has been the traditional approach to studying calcium signaling. This method suffers from the need for large platelet samples and a decrease in total fluorescence signal with time due to photobleaching. Therefore, it is rarely used to measure the quantitative effect of an agonist or antagonist on calcium signaling. Adaptation of these measurements to a fluorescent imaging plate reader (FLIPR) format allows the sample size to be reduced by 5- to 10-fold, and the microplate format allows a significant increase in throughput. Addition of the agonists to all wells simultaneously serves to normalize the total response. This article describes the first use of a FLIPR to study the calcium flux in human platelets. The IC 50 values showed a linear correlation with the K i for receptor binding in washed platelets. The generality of the methodology was shown by measuring EC 50 values for agonists and IC 50 values for antagonists of the platelet G protein-coupled receptor P2Y 1 and for the ion channel P2X 1.

Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats

BackgroundIn the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy.ResultsBasal calcium levels were always elevated in the platelets of the L-NAME-treated rats, both in the presence and in the absence of extracellular calcium. The administration of thrombin in the absence and in the presence of extracellular calcium induced important elevations in calcium levels that were always of greater magnitude in the platelets of the L-NAME-treated rats than in those of the controls. The addition of calcium to thapsigargin-treated platelets produced a massive elevation in calcium levels in both groups that was significantly greater in the platelets obtained from the hypertensive rats than in those of the controls.ConclusionsIt is concluded that the arterial hypertension induced by the reduction of nitric oxide alters the regulation of platelet calcium levels so that elevated baseline levels and calcium entry and mobilization are enhanced. This could be the result of direct or indirect effects of the lack of nitric oxide synthesis in platelets or in other tissues.