Platelet Signaling Research Articles

Reduced Proteolytic Cleavage of von Willebrand Factor Leads to Aortic Valve Stenosis and Load-Dependent Ventricular Remodeling

We hypothesized that excess endothelial-associated von Willebrand factor (vWF) and secondary platelet adhesion contribute to aortic valve stenosis (AS). We studied hyperlipidemic mice lacking ADAMTS13 (LDLR -/- AD13 -/- ), which cleaves endothelial-associated vWF multimers. On echocardiography and molecular imaging, LDLR -/- AD13 -/- compared with control strains had increased aortic endothelial vWF and platelet adhesion and developed hemodynamically significant AS, arterial stiffening, high valvulo-aortic impedance, and secondary load-dependent reduction in LV systolic function. Histology revealed leaflet thickening and calcification with valve interstitial cell myofibroblastic and osteogenic transformation, and evidence for TGFβ1 pathway activation. We conclude that valve leaflet endothelial vWF-platelet interactions promote AS through juxtacrine platelet signaling.

Characterization of Platelet Function-Related Gene Predicting Survival and Immunotherapy Efficacy in Gastric Cancer.

Immunotherapy is widely used to treat various cancers, but patients with gastric cancer (GC), which has a high mortality rate, benefit relatively less from this therapy. Platelets are closely related to GC progression and metastasis. This study aimed to find novel potential biomarkers related to platelet function to predict GC and immunotherapy efficacy. First, based on platelet activation, signaling, and aggregation (abbreviation: function)-related genes (PFRGs), we used the least absolute shrinkage and selection operator (Lasso) regression method to construct a platelet-function-related genes prognostic score (PFRGPS). PRFGPS was verified in three independent external datasets (GSE26901, GSE15459, and GSE84437) for its robustness and strong prediction performance. Our results demonstrate that PRFGPS is an independent prognostic indicator for predicting overall survival in patients with GC. In addition, prognosis, potential pathogenesis mechanisms, and the response to immunotherapy were defined via gene set enrichment analysis, tumor mutational burden, tumor microenvironment, tumor immune dysfunction and exclusion (TIDE), microsatellite instability, and immune checkpoint inhibitors. We found that the high-PRFGPS subgroup had a cancer-friendly immune microenvironment, a high TIDE score, a low tumor mutational burden, and relatively low microsatellite instability. In the immunophenoscore model, the therapeutic effect on anti-PD-1 and anti-CTLA-4 in the high-PRFGPS subgroup was relatively low. In conclusion, PRFGPS could be used as a reference index for GC prognosis to develop more successful immunotherapy strategies.

Phosphorylation on Syk Y342 is important for both ITAM and hemITAM signaling in platelets

Immune cells express receptors bearing an immune tyrosine activation motif (ITAM) containing two YXXL motifs or hemITAMs containing only one YXXL motif. Phosphorylation of the ITAM/hemITAM is mediated by Src family kinases allowing for the binding and activation of spleen tyrosine kinase (Syk). It is believed that Syk must be phosphorylated on tyrosine residues for activation, and Tyr342, а conserved tyrosine in the interdomain B region, has been shown to be critical for regulating Syk in FcεR1-activated mast cells. Syk is a key mediator of signaling pathways downstream of several platelet pathways including the ITAM bearing glycoprotein VI (GPVI)/Fc receptor gamma chain collagen receptor and the hemITAM containing C-type lectin-like receptor-2 (CLEC-2). Since platelet activation is a crucial step in both hemostasis and thrombosis, we evaluated the importance of Syk Y342 in these processes by producing an Syk Y342F knock-in mouse. When using a CLEC-2 antibody as an agonist, reduced aggregation and secretion were observed in Syk Y342F mouse platelets when compared with control mouse platelets. Platelet reactivity was also reduced in response to the GPVI agonist collagen-related peptide. Signaling initiated by either GPVI or CLEC-2 was also greatly inhibited, including Syk Y519/520 phosphorylation. Hemostasis, as measured by tail bleeding time, was not altered in Syk Y342F mice, but thrombus formation in response to FeCl3 injury was prolonged in Syk Y342F mice. These data demonstrate that phosphorylation of Y342 on Syk following stimulation of either GPVI or CLEC-2 receptors is important for the ability of Syk to transduce a signal.

ITRAQ-based quantitative proteomics reveals biomarkers/pathways in psoriasis that can predict the efficacy of methotrexate.

Methotrexate (MTX) is the first-line medicine to treat psoriasis. So far, there has been less research on protein biomarkers to predict its efficacy by the proteomic technique. To evaluate differentially expressed proteins in peripheral mononuclear cells (PBMCs) between good responders (GRs) and non-responders (NRs) after MTX treatment, compared with normal controls (NCs). We quantified protein expression of PBMCs with four GRs and four NRs to MTX and four NCs by isobaric tags for relative and absolute quantification (iTRAQ), analysing and identifying proteins related to efficacy of MTX in 18 psoriatic patients. A total of 3177 proteins had quantitative information, and 403 differentially expressed proteins (fold change ≥1.2, P < 0.05) were identified. Compared to NCs, upregulated proteins (ANXA6, RPS27A, EZR, XRCC6), participating in the activation of NF-κB, the JAK-STAT pathway and neutrophil degranulation were detected in GRs. The proteins (GPV, FN1, STOM), involving platelet activation, signalling and aggregation as well as neutrophil degranulation were significantly downregulated in GRs. These proteins returned to normal levels after MTX treatment. Furthermore, Western blotting identified the expression of ANXA6 and STAT1 in PBMCs, which were significantly downregulated in GRs, but not in NRs. We identified seven differentially expressed and regulated proteins (ANXA6, GPV, FN1, XRCC6, STOM, RPS27A and EZR) as biomarkers to predict MTX efficacy in NF-κB signalling, JAK-STAT pathways, neutrophil degranulation, platelet activation, signalling and aggregation.

GRK2 regulates ADP signaling in platelets via P2Y1 and P2Y12

AbstractThe critical role of G protein–coupled receptor kinase 2 (GRK2) in regulating cardiac function has been well documented for >3 decades. Targeting GRK2 has therefore been extensively studied as a novel approach to treating cardiovascular disease. However, little is known about its role in hemostasis and thrombosis. We provide here the first evidence that GRK2 limits platelet activation and regulates the hemostatic response to injury. Deletion of GRK2 in mouse platelets causes increased platelet accumulation after laser-induced injury in the cremaster muscle arterioles, shortens tail bleeding time, and enhances thrombosis in adenosine 5′-diphosphate (ADP)-induced pulmonary thromboembolism and in FeCl3-induced carotid injury. GRK2−/− platelets have increased integrin activation, P-selectin exposure, and platelet aggregation in response to ADP stimulation. Furthermore, GRK2−/− platelets retain the ability to aggregate in response to ADP restimulation, indicating that GRK2 contributes to ADP receptor desensitization. Underlying these changes in GRK2−/− platelets is an increase in Ca2+ mobilization, RAS-related protein 1 activation, and Akt phosphorylation stimulated by ADP, as well as an attenuated rise of cyclic adenosine monophosphate levels in response to ADP in the presence of prostaglandin I2. P2Y12 antagonist treatment eliminates the phenotypic difference in platelet accumulation between wild-type and GRK2−/− mice at the site of injury. Pharmacologic inhibition of GRK2 activity in human platelets increases platelet activation in response to ADP. Finally, we show that GRK2 binds to endogenous Gβγ subunits during platelet activation. Collectively, these results show that GRK2 regulates ADP signaling via P2Y1 and P2Y12, interacts with Gβγ, and functions as a signaling hub in platelets for modulating the hemostatic response to injury.

Noncanonical Sonic Hedgehog signaling amplifies platelet reactivity and thrombogenicity

AbstractSonic Hedgehog (Shh) is a morphogen in vertebrate embryos that is also associated with organ homeostasis in adults. We report here that human platelets, though enucleate, synthesize Shh from preexisting mRNAs upon agonist stimulation, and mobilize it for surface expression and release on extracellular vesicles, thus alluding to its putative role in platelet activation. Shh, in turn, induced a wave of noncanonical signaling in platelets leading to activation of small GTPase Ras homolog family member A and phosphorylation of myosin light chain in activated protein kinase-dependent manner. Remarkably, agonist-induced thrombogenic responses in platelets, which include platelet aggregation, granule secretion, and spreading on immobilized fibrinogen, were significantly attenuated by inhibition of Hedgehog signaling, thus, implicating inputs from Shh in potentiation of agonist-mediated platelet activation. In consistence, inhibition of the Shh pathway significantly impaired arterial thrombosis in mice. Taken together, the above observations strongly support a feed-forward loop of platelet stimulation triggered locally by Shh, similar to ADP and thromboxane A2, that contributes significantly to the stability of occlusive arterial thrombus and that can be investigated as a potential therapeutic target in thrombotic disorders.

Fast clearance of platelets in a commonly used mouse model for GPIbα is impeded by an anti‐GPIbβ antibody derivative

BackgroundGlycoprotein (GP)Ibα plays a critical role in regulating platelet clearance. Recently, we identified the mechanosensory domain (MSD) in GPIbα and reported evidence to suggest that unfolding of the GPIbα MSD induces exposure of the Trigger sequence therein and subsequent GPIb‐IX signaling that accelerates platelet clearance. In a commonly used transgenic mouse model, IL4R‐IbαTg, where the Trigger sequence is constitutively exposed, constitutive GPIb‐IX‐mediated cellular signals are present. Clearance of their platelets is also significantly faster than that of wild‐type mice. Previously, an anti‐GPIbβ antibody RAM.1 was developed. RAM.1 inhibits GPIbα‐dependent platelet signaling and activation. Further, RAM.1 also inhibits anti‐GPIbα antibody‐mediated filopodia formation. ObjectiveTo investigate whether RAM.1 can ameliorate trigger sequence exposure‐mediated platelet clearance. MethodsSpontaneous filopodia were measured by confocal microscopy. Other platelet signaling events were measured by flow cytometry. Endogenous platelet life span was tracked by Alexa 488‐labeled anti‐mouse GPIX antibody. ResultTransfected Chinese hamster ovary cells stably expressing the same chimeric IL4R‐Ibα protein complex as in IL4R‐IbαTg mice also constitutively exhibit filopodia, and that such filopodia could be abolished by treatment of RAM.1. Further, transfusion of a recombinant RAM.1 derivative that is devoid of its Fc portion significantly extends the endogenous life span of IL4R‐IbαTg platelets. ConclusionThese results provide the key evidence supporting the causative link of Trigger sequence exposure to accelerated platelet clearance, and suggest that a RAM.1 derivative may be therapeutically developed to treat GPIb‐IX‐mediated thrombocytopenia.

PAR4 Primarily Mediates Thrombin‐induced Morphological Changes in MEG‐01 Cells through PI3K/Akt and RhoA/ROCK Signaling Pathways

Platelet activation is critical in hemostasis and thrombosis. Thrombin most potently activates platelets via two protease‐activated receptors (PARs): PAR1 and PAR4. While PAR1 activation results in a rapid and transient signal required for the initiation phase of platelet aggregation, PAR4 activation induces a sustained signal associated with later phases responsible for stable thrombus formation. The present study focused on the differential signaling pathways by PAR1 and PAR4 involved in thrombin‐induced intracellular events in human megakaryoblastic leukemia cell line, MEG‐01. Interestingly, we found that thrombin triggers not only Gq/PLC‐mediated calcium signaling, but also G12/13/RhoA/ROCK‐mediated morphological changes in MEG‐01 cells via PAR1 and PAR4, which showed close similarity to those observed in platelets. We developed a new image‐based assay to quantify the morphological changes in living cells and extensively investigated on the underlying mechanism for thrombin‐induced morphological changes in MEG‐01 cells. Selective blockade of PAR1 and PAR4 by vorapaxar and BMS‐986120, respectively, indicated that PAR4 activation primarily mediates thrombin‐induced morphological changes in MEG‐01 cells. Moreover, treatment of a set of kinase inhibitors showed that the PAR4‐mediated morphological changes were predominantly mediated via PI3K/Akt and RhoA/ROCK signaling pathways. As in the platelets, PAR4‐mediated signaling pathways are significant in thrombin‐induced morphological changes in MEG‐01 cells. Based on the close similarity to thrombin‐induced signaling in platelets, MEG‐01 cell line is a useful complementary in vitro model that can be used in research on platelets and thrombosis.

OC-06: Identification of predictive hemostatic biomarkers for cancer diagnosis: results from the HYPERCAN study

ABL1 kinase T315I positive chronic myelogenous leukemia (CML) is treated with the tyrosine kinase inhibitor (TKI) ponatinib. However, its use is associated with a high incidence (29%) of arterial occlusive events.Determine the molecular basis of ponatinib-induced thrombosis.A C57BL/6 mouse model of ponatinib-induced rose bengal thrombosis was created. We characterized ponatinib or combined ponatinib with pioglitazone and measured aortic ROS and apoptosis with antibodies to nitrotyrosine and caspase 3, respectively, using immunohistoperoxidase. The influence of ponatinib influence on collagen-rich peptide (CRP)- and thrombin-induced JON/A and P-selectin expression and ADP-induced fibrinogen binding was examined. Aorta RNAseq studies were performed.Treatment of 20-week old mice with >3 mg/kg oral ponatinib daily for 14 days shortened carotid artery thrombosis times (18.7+3.7 min) compared to control (32.3+4.8 min, p<0.0001) (see Blood Adv. 3 (2019) 2312). Ponatinib-treated mice have normal CBCs, PT, aPTT, and contact- and tissue factor-induced TGT. Aortic adventitia of ponatinib-treated mice express increased nitrotyrosine and caspase 3. Treated mice have shortened tail bleeding times (55+12 min) versus untreated mice (102+9.3 min). CRP- or thrombin-induced JON/A and P-selectin occurred at lower threshold concentrations in ponatinib-treated mice. Concurrent pioglitazone treatment corrected ponatinib-induced thrombosis, aortic ROS and caspase 3 expression, and CRP-induced platelet hyper-reactivity. The mechanism for ponatinib-induced thrombosis and its correction by pioglitazone was sought. Aorta RNAseq data was examined on Reactome’s Hemostasis Enrichment Gene Set. Ponatinib-treated mice have 293/488 up-regulated gene sets (see Fig. 1). The most up-regulated aortic gene is F5. Other up-regulated aortic genes with ponatinib treatment include F8, F2, F10, and F7 and APBB1P, SERPINB2 (PAI-3), and TIMP. Additionally, related integrins (ITGB2, ITGA2B, ITGAL, are ITGAX), receptors (MPL, GP5, GP9), and adhesive proteins (SELL and AMICA1) also are increased. Further, the kinases of the GPVI activation cascade (LCK, LAT, SYK, LYN, FYN) were elevated. Combining pioglitazone with ponatinib treatment reduced the upregulation of 100 gene sets seen with ponatinib alone. Pioglitazone corrected the ponatinib-induced prothrombotic phenotype. F8, F2 and the genes of and Lyn were knocked off the list of top genes expressed.These investigations indicate that ponatinib treatment increases gene expression of aortic prothrombotic and platelet signalling genes that promote GPVI activation. Addition of pioglitazone reduces the prothrombotic characteristics conferred by ponatinib treatment. These data indicate that cancer-associated thrombosis can be managed by non-anticoagulant pharmacologic agents.

MicroRNA-26b Attenuates Platelet Adhesion and Aggregation in Mice.

Platelets are key regulators of haemostasis, making platelet dysfunction a major driver of thrombosis. Numerous processes that determine platelet function are influenced by microRNAs (miRs). MiR-26b is one of the highest-expressed miRs in healthy platelets, and its expression in platelets is changed in a diseased state. However, the exact effect of this miR on platelet function has not been studied yet. In this study, we made use of a whole-body knockout of miR-26b in ApoE-deficient mice in order to determine its impact on platelet function, thrombus formation and platelet signalling both ex vivo and in vivo. We show that a whole-body deficiency of miR-26b exacerbated platelet adhesion and aggregation ex vivo. Additionally, in vivo, platelets adhered faster, and larger thrombi were formed in mice lacking miR-26b. Moreover, isolated platelets from miR-26b-deficient mice showed a hyperactivated Src and EGFR signalling. Taken together, we show here for the first time that miR-26b attenuates platelet adhesion and aggregation, possibly through Src and EGFR signalling.

Rac Inhibition Causes Impaired GPVI Signalling in Human Platelets through GPVI Shedding and Reduction in PLCγ2 Phosphorylation.

Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.

Calcium- and Integrin-Binding Protein 2 (CIB2) in Physiology and Disease: Bright and Dark Sides.

Calcium- and integrin-binding protein 2 (CIB2) is a small EF-hand protein capable of binding Mg2+ and Ca2+ ions. While its biological function remains largely unclear, an increasing number of studies have shown that CIB2 is an essential component of the mechano-transduction machinery that operates in cochlear hair cells. Mutations in the gene encoding CIB2 have been associated with non-syndromic deafness. In addition to playing an important role in the physiology of hearing, CIB2 has been implicated in a multitude of very different processes, ranging from integrin signaling in platelets and skeletal muscle to autophagy, suggesting extensive functional plasticity. In this review, we summarize the current understanding of biochemical and biophysical properties of CIB2 and the biological roles that have been proposed for the protein in a variety of processes. We also highlight the many molecular aspects that remain unclarified and deserve further investigation.

The effect of Bruton's tyrosine kinase inhibitor ibrutinib on atherothrombus formation under stenotic flow conditions

BackgroundBruton's kinase (Btk) is critical for collagen-triggered platelet signal transduction. The Btk inhibitor ibrutinib has been shown to selectively block platelet adhesion to atherosclerotic plaque material under laminar arterial flow. However, this has not been studied under a shear gradient, which is characteristic for atherothrombosis. ObjectiveTo determine the effect of ibrutinib treatment on in vitro thrombus formation on collagen and atherosclerotic plaque material in the absence or presence of a shear gradient. MethodsBlood was obtained from patients with chronic lymphocytic leukemia, mantle-cell lymphoma and Waldenström macroglobulinemia with and without ibrutinib treatment and perfused through a microfluidic channel with(out) 60% stenosis over Horm type I collagen or human atherosclerotic plaque homogenate. ResultsAt a constant shear rate of 1500 s−1, platelet deposition was significantly decreased in blood from haematological malignancy patients treated with ibrutinib as compared to untreated patients, on atherosclerotic plaque material but not on collagen. However, thrombus size, stability, and height, were reduced on both plaque material and collagen. An increase in shear rate up to 3900 s−1, as induced by 60% stenosis, resulted in decreased platelet deposition and thrombus parameters on plaque material but not on collagen when compared to a laminar shear of 1500 s−1. Ibrutinib treatment decreased platelet deposition and thrombus parameters even further around the stenosis. ConclusionTreatment of patients with haematological disorders with the Btk inhibitor ibrutinib reduces in vitro platelet deposition, thrombus size and contraction on human atherosclerotic plaque around a stenosis when compared to patients not receiving ibrutinib.

Upregulation of cytokine signalling in platelets increases risk of thrombophilia in severe COVID-19 patients

Abnormal coagulation dynamics, including disseminated intravascular coagulopathy, pulmonary embolism, venous thromboembolism and risk of thrombosis are often associated with the severity of COVID-19. However, very little is known about the contribution of platelets in above pathogenesis. In order to decipher the pathophysiology of thrombophilia in COVID-19, we recruited severely ill patients from ICU, based on the above symptoms and higher D-dimer levels, and compared these parameters with their asymptomatic counterparts. Elevated levels of platelet-derived microparticles and platelet-leukocyte aggregates suggested the hyperactivation of platelets in ICU patients. Strikingly, platelet transcriptome analysis showed a greater association of IL-6 and TNF signalling pathways in ICU patients along with higher plasma levels of IL-6 and TNFα. In addition, upregulation of pathways like blood coagulation and hemostasis, as well as inflammation coexisted in platelets of these patients. Further, the increment of necrotic pathway and ROS-metabolic processes in platelets was suggestive of its procoagulant phenotype in ICU patients. This study suggests that higher plasma IL-6 and TNFα may trigger platelet activation and coagulation, and in turn aggravate thrombosis and hypercoagulation in severe COVID-19 patients. Therefore, the elevated IL-6 and TNFα, may serve as potential risk factors for platelet activation and thrombophilia in these patients.

CRACking the Molecular Regulatory Mechanism of SOCE during Platelet Activation in Thrombo-Occlusive Diseases.

Thrombo-occlusive diseases such as myocardial infarction, ischemic stroke and deep vein thrombosis with subsequent pulmonary embolism still represent a major health burden worldwide. Besides the cells of the vasculature or other hematopoietic cells, platelets are primarily responsible for the development and progression of an occluding thrombus. The activation and function of platelets crucially depend on free cytosolic calcium (Ca2+) as second messenger, which modulates platelet secretion, aggregation and thrombus formation. Ca2+ is elevated upon platelet activation by release of Ca2+ from intracellular stores thus triggering of the subsequent store-operated Ca2+ entry (SOCE), which is facilitated by Ca2+ release-activated channels (CRACs). In general, CRACs are assembled by the pore-forming unit Orai in the plasma membrane and the Ca2+-sensing stromal interaction molecule (STIM) in the endoplasmic reticulum after the depletion of internal Ca2+ stores. In the last few years, there is a growing body of the literature demonstrating the importance of STIM and Orai-mediated mechanism in thrombo-occlusive disorders. Thus, this review provides an overview of the recent understanding of STIM and Orai signaling in platelet function and its implication in the development and progression of ischemic thrombo-occlusive disorders. Moreover, potential pharmacological implications of STIM and Orai signaling in platelets are anticipated and discussed in the end.

Genetic deletion of platelet PAR4 results in reduced thrombosis and impaired hemostatic plug stability

BackgroundProtease‐activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis. ObjectiveWe aimed to generate a mouse line with a megakaryocyte/platelet‐specific deletion of PAR4 (PAR4fl/fl;PF4Cre+) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice. MethodsPlatelets from PAR4fl/fl;PF4Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet‐specific deletion of PAR4 or wild‐type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors. ResultsPAR4fl/fl;PF4Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4−/− and PAR4fl/fl;PF4Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4−/− mice and PAR4fl/fl;PF4Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild‐type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability. ConclusionWe generated a novel PAR4fl/fl;PF4Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability.

SIGNALING ROLE OF REACTIVE OXYGEN SPECIES IN PLATELETS

Abstract: Review. Reactive oxygen species are formed as a result of redox reactions and are involved in signaling associated with cell growth, differentiation, and death. Platelets are both a source and a target of reactive oxygen species. The review regards the molecular mechanisms of the reactive oxygen species formation under platelet activation conditions and effects of altered redox balance on platelet function. The main platelet sources of reactive oxygen species are isoforms of NADPH oxidases, cyclooxygenase, xanthine oxidase, and mitochondrial electron transport chain. Reactive oxygen species produced during platelet activation by agonists act as secondary messengers and can interfere with signaling pathways that enhance agonist-induced platelet activation. The signaling pathway mediated by glycoprotein VI, the main platelet receptor for collagen, leading mainly to the generation of intracellular reactive oxygen species, as well as the signaling pathway activated by thrombin, leading to the production of extracellular reactive oxygen species are considered. Extracellular reactive oxygen species enhance initial signaling and support platelet recruitment and activation. Activated platelets are involved in the coupling of thrombosis, inflammation, and innate immune responses. The platelet antioxidant systems play an important role in preventing the cytotoxic effects of reactive oxygen species, as well as in the regulation of platelet signaling pathways. The authors of the review and other researchers have shown that the redox status of platelets can serve as a biomarker in the studies on prevention and treatment of diseases involving oxidative stress and can be used for objective assessments of the patients' condition severity (including depression and schizophrenia, in which the authors found a decrease in the activity of platelet glutathione -dependent antioxidant enzymes).

A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution.

Modeling thrombus growth in pathological flows allows evaluation of risk under patient-specific pharmacological, hematological, and hemodynamical conditions. We have developed a 3D multiscale framework for the prediction of thrombus growth under flow on a spatially resolved surface presenting collagen and tissue factor (TF). The multiscale framework is composed of four coupled modules: a Neural Network (NN) that accounts for platelet 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. A reduced model of the coagulation cascade was embedded into the framework to account for TF-driven thrombin production. The 3D model was first tested against in vitro microfluidics experiments of whole blood perfusion with various antiplatelet agents targeting COX-1, P2Y1, or the IP receptor. The model was able to accurately capture the evolution and morphology of the growing thrombus. Certain problems of 2D models for thrombus growth (artifactual dendritic growth) were naturally avoided with realistic trajectories of platelets in 3D flow. The generalizability of the 3D multiscale solver enabled simulations of important clinical situations, such as cylindrical blood vessels and acute flow narrowing (stenosis). Enhanced platelet-platelet bonding at pathologically high shear rates (e.g., von Willebrand factor unfolding) was required for accurately describing thrombus growth in stenotic flows. Overall, the approach allows consideration of patient-specific platelet signaling and vascular geometry for the prediction of thrombotic episodes.

Comprehensive Landscape of Immune Infiltration and Aberrant Pathway Activation in Ischemic Stroke.

Ischemic stroke (IS) is a multifactorial disease caused by the interaction of multiple environmental and genetic risk factors, and it is the most common cause of disability. The immune microenvironment and inflammatory response participate in the whole process of IS occurrence and development. Therefore, the rational use of relevant markers or characteristic pathways in the immune microenvironment will become one of the important therapeutic strategies for the treatment of IS. We collected peripheral blood samples from 10 patients diagnosed with IS at the First Affiliated Hospital of Gannan Medical University and First Affiliated Hospital, Jinan" University, and from 10 normal people. The GSE16561 dataset was downloaded from the Gene Expression Omnibus (GEO) database. xCell, gene set enrichment analysis (GSEA), single-sample GSEA (ssGSEA) and immune-related gene analysis were used to evaluate the differences in the immune microenvironment and characteristic pathways between the IS and control groups of the two datasets. xCell analysis showed that the IS-24h group had significantly reduced central memory CD8+ T cell, effector memory CD8+ T cell, B cell and Th1 cell scores and significantly increased M1 macrophage and macrophage scores. GSEA showed that the IS-24h group had significantly increased inflammation-related pathway activity(myeloid leukocyte activation, positive regulation of tumor necrosis factor biosynthetic process, myeloid leukocyte migration and leukocyte chemotaxis), platelet-related pathway activity(platelet activation, signaling and aggregation; protein polymerization; platelet degranulation; cell-cell contact zone) and pathology-related pathway activity (ERBB signaling pathway, positive regulation of ERK1 and ERK2 cascade, vascular endothelial growth factor receptor signaling pathway, and regulation of MAP kinase activity). Immune-related signature analysis showed that the macrophage signature, antigen presentation-related signature, cytotoxicity-related signature, B cell-related signature and inflammation-related signature were significantly lower in the IS-24h group than in the control group. In this study, we found that there were significant differences in the immune microenvironment between the peripheral blood of IS patients and control patients, as shown by the IS group having significantly reduced CD8+ Tcm, CD8+ Tem, B cell and Th1 cell scores and significantly increased macrophage and M1 macrophage scores. Additionally, inflammation-related, pathological, and platelet-related pathway activities were significantly higher in the IS group than in the control group.

PI3K Isoform Signalling in Platelets.

Platelets are unique anucleated blood cells that constantly patrol the vasculature to seal and prevent injuries in a process termed haemostasis. Thereby they rapidly adhere to the subendothelial matrix and recruit further platelets, resulting in platelet aggregates. Apart from their central role in haemostasis, they also kept some of their features inherited by their evolutionary ancestor-the haemocyte, which was also involved in immune defences. Together with leukocytes, platelets fight pathogenic invaders and guide many immune processes. In addition, they rely on several signalling pathways which are also relevant to immune cells. Among these, one of the central signalling hubs is the PI3K pathway. Signalling processes in platelets are unique as they lack a nucleus and therefore transcriptional regulation is absent. As a result, PI3K subclasses fulfil distinct roles in platelets compared to other cells. In contrast to leukocytes, the central PI3K subclass in platelet signalling is PI3K class Iβ, which underlines the uniqueness of this cell type and opens new ways for potential platelet-specific pharmacologic inhibition. An overview of platelet function and signalling with emphasis on PI3K subclasses and their respective inhibitors is given in this chapter.