Discovery of 2,3-Dihydro[1,4]dioxino[2,3-g]benzofuran Derivatives as Protease Activated Receptor 4 (PAR4) Antagonists with Potent Antiplatelet Aggregation Activity and Low Bleeding Tendency.

Patients with arterial embolic disease have benefited greatly from antiplatelet therapy. However, hemorrhage risk of antiplatelet agents cannot be ignored. Herein, we describe the discovery of 2,3-dihydro[1,4]dioxino[2,3-g]benzofuran compounds as novel PAR4 antagonists. Notably, the isomers 36 and 37 with the chemotype of phenoxyl methylene substituted on the 2,3-dihydro-1,4-dioxine ring exhibited potent in vitro antiplatelet activity (IC50 = 26.13 nM for 36 and 14.26 nM for 37) and significantly improved metabolic stability in human liver microsomes (T1/2 = 97.6 min for 36 and 11.1 min for BMS-986120). 36 also displayed good oral PK profiles (mice: T1/2 = 7.32 h and F = 45.11%). Both of them showed overall potent ex vivo antiplatelet activity at concentrations of 6 and 12 mg/kg, with no impact on the coagulation system and low bleeding liability. Our work will facilitate development of novel PAR4 antagonists as a safer therapeutic option for arterial embolism.

Antiplatelet effects of the CEACAM1-derived peptide QDTT.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) restricts platelet activation via platelet collagen receptor GPVI/FcRγ-chain. In this study, screening against collagen-induced platelet aggregation was performed to identify functional CEACAM1 extracellular domain fragments. CEACAM1 fragments, including Ala-substituted peptides, were synthesized. Platelet assays were conducted on healthy donor samples for aggregation, cytotoxicity, adhesion, spreading, and secretion. Mice were used for tail bleeding and FeCl3-induced thrombosis experiments. Clot retraction was assessed using platelet-rich plasma. Extracellular segments of CEACAM1 and A1 domain-derived peptide QDTT were identified, while N, A2, and B domains showed no involvement. QDTT inhibited platelet aggregation. Ala substitution for essential amino acids (Asp139, Thr141, Tyr142, Trp144, and Trp145) in the QDTT sequence abrogated collagen-induced aggregation inhibition. QDTT also suppressed platelet secretion and "inside-out" GP IIb/IIIa activation by convulxin, along with inhibiting PI3K/Akt pathways. QDTT curtailed FeCl3-induced mesenteric thrombosis without significantly prolonging bleeding time, implying the potential of CEACAM1 A1 domain against platelet activation without raising bleeding risk, thus paving the way for novel antiplatelet drugs.

What is the context? The study focuses on Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) and its role in platelet activation, particularly through the GPVI/FcRγ-chain pathway.The research aims to identify specific fragments of CEACAM1's extracellular domain that could restrict platelet activation, without increasing bleeding risk.What is new? The researchers identified a peptide called QDTT derived from the A1 domain of CEACAM1's extracellular segment. This peptide demonstrated the ability to inhibit platelet aggregation, secretion, and GP IIb/IIIa activation.The study also revealed that specific amino acids within the QDTT sequence were essential for its inhibitory effects on collagen-induced aggregation.What is the impact? The findings suggest that the A1 domain-derived peptide QDTT from CEACAM1 could serve as a potential basis for developing novel antiplatelet drugs. This peptide effectively limits platelet activation and aggregation without significantly prolonging bleeding time, indicating a promising approach to managing thrombosis and related disorders while minimizing bleeding risks.

Raman spectra simulation of antiplatelet drug-platelet interaction using DFT.

The paper reflects the results of molecular docking and mathematical DFT simulation for antiplatelet drugs and the target platelet receptor/ferment interaction in the limited area. The results of Raman spectra simulation are implemented and obtained from the interaction of the clopidogrel metabolite of the P2Y12 receptor. The interaction of aspirin with the COX-1 enzyme was also investigated. As a result, theoretical Raman spectra of the drug-receptor area were obtained. The theoretical data were compared with the experimental SERS results. The characteristic bands corresponding to metabolite/ferment and antiplatelet drug vibrations were clarified. The prospects of obtaining results for pathologies based on platelet conformations during cardiovascular diseases have been demonstrated.

Cell Surface Platelet Tissue Factor Expression: Regulation by P2Y12 and Link to Residual Platelet Reactivity.

BACKGROUND: ADP-induced platelet activation leads to cell surface expression of several proteins, including TF (tissue factor). The role of ADP receptors in platelet TF modulation is still unknown. We aimed to assess the (1) involvement of P2Y1 and P2Y12 receptors in ADP-induced TF exposure; (2) modulation of TFpos-platelets in anti-P2Y12-treated patients with coronary artery disease. Based on the obtained results, we revisited the intracellular localization of TF in platelets. METHODS: The effects of P2Y1 or P2Y12 antagonists on ADP-induced TF expression and activity were analyzed in vitro by flow cytometry and thrombin generation assay in blood from healthy subjects, P2Y12-/-, and patients with gray platelet syndrome. Ex vivo, P2Y12 inhibition of TF expression by clopidogrel/prasugrel/ticagrelor, assessed by VASP (vasodilator-stimulated phosphoprotein) platelet reactivity index, was investigated in coronary artery disease (n=238). Inhibition of open canalicular system externalization and electron microscopy (TEM) were used for TF localization. RESULTS: In blood from healthy subjects, stimulated in vitro by ADP, the percentage of TFpos-platelets (17.3±5.5%) was significantly reduced in a concentration-dependent manner by P2Y12 inhibition only (-81.7±9.5% with 100 nM AR-C69931MX). In coronary artery disease, inhibition of P2Y12 is paralleled by reduction of ADP-induced platelet TF expression (VASP platelet reactivity index: 17.9±11%, 20.9±11.3%, 40.3±13%; TFpos-platelets: 10.5±4.8%, 9.8±5.9%, 13.6±6.3%, in prasugrel/ticagrelor/clopidogrel-treated patients, respectively). Despite this, 15% of clopidogrel good responders had a level of TFpos-platelets similar to the poor-responder group. Indeed, a stronger P2Y12 inhibition (130-fold) is required to inhibit TF than VASP. Thus, a VASP platelet reactivity index <20% (as in prasugrel/ticagrelor-treated patients) identifies patients with TFpos-platelets <20% (92% sensitivity). Finally, colchicine impaired in vitro ADP-induced TF expression but not α-granule release, suggesting that TF is open canalicular system stored as confirmed by TEM and platelet analysis of patients with gray platelet syndrome. CONCLUSIONS: Data show that TF expression is regulated by P2Y12 and not P2Y1; P2Y12 antagonists downregulate the percentage of TFpos-platelets. In clopidogrel good-responder patients, assessment of TFpos-platelets highlights those with residual platelet reactivity. TF is stored in open canalicular system, and its membrane exposure upon activation is prevented by colchicine.

Response of human blood platelets to preparations from leaves of Paulownia Clon in Vitro 112.

Paulownia Clon in Vitro 112, also called the Oxytree, is a fast-growing hybrid of two trees belonging to the Paulowniaceae family - P. elongata and P. fortunei. It demonstrates a wide range of biological effects (including antioxidant, anti-inflammatory, antibacterial, and neuroprotective) due to the high concentration of secondary metabolites. Our previous results showed an in vitro antioxidant and antiplatelet activity of the extract and four fractions (A-D) from the leaves of Paulownia Clon in Vitro 112 in human plasma and washed blood platelets. Here, we used a microchip flow chamber-based thrombus formation analysis system (T-TAS) and flow cytometry to assess the anticoagulant and antiplatelet activity of the extract and four fractions with different chemical content (A-D) from Paulownia Clon in Vitro 112 leaves in human whole blood. Two tested fractions: fraction C and D (at the concentrations of 5 and 50 µg/mL) inhibited the exposition of the active form of GPIIb/IIIa (integrin αIIbß3) on the surface of blood platelets stimulated by ADP and collagen. The antiplatelet activity of fraction C is likely due to its high verbascoside content and the presence of apigenin's derivatives. Fraction D contains triterpenoids, including ursolic, pomoleic, and maslinic acid, which could be responsible for decreased activation of ADP- and collagen-stimulated blood platelets. These results suggest that fractions C and D might be promising sources of phytochemicals with antiplatelet activity, which are important for prophylaxis and treatment of cardiovascular diseases associated with hyperactivation of blood platelets. However, further research is needed to ascertain which exact compounds and mechanisms are responsible for this phenomenon.

Tspan5 promotes the EMT process to regulate the syncytialization of trophoblast cells by activating Notch signalling.

Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. ß-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

Dual antiplatelet therapy inhibits neutrophil extracellular traps to reduce liver micrometastases of intrahepatic cholangiocarcinoma.

The involvement of neutrophil extracellular traps (NETs) in cancer metastasis is being clarified, but the relationship between intrahepatic cholangiocarcinoma (iCCA) and NETs remains unclear. The presence of NETs was verified by multiple fluorescence staining in clinically resected specimens of iCCA. Human neutrophils were co-cultured with iCCA cells to observe NET induction and changes in cellular characteristics. Binding of platelets to iCCA cells and its mechanism were also examined, and their effects on NETs were analyzed in vitro and in in vivo mouse models. NETs were present in the tumor periphery of resected iCCAs. NETs promoted the motility and migration ability of iCCA cells in vitro. Although iCCA cells alone had a weak NET-inducing ability, the binding of platelets to iCCA cells via P-selectin promoted NET induction. Based on these results, antiplatelet drugs were applied to these cocultures in vitro and inhibited the binding of platelets to iCCA cells and the induction of NETs. Fluorescently labeled iCCA cells were injected into the spleen of mice, resulting in the formation of liver micrometastases coexisting with platelets and NETs. These mice were treated with dual antiplatelet therapy (DAPT) consisting of aspirin and ticagrelor, which dramatically reduced micrometastases. These results suggest that potent antiplatelet therapy prevents micrometastases of iCCA cells by inhibiting platelet activation and NET production, and it may contribute to a novel therapeutic strategy.

Glucosamine effects on platelet aggregation of type 2 diabetes mellitus patients: in vitro assays.

Hyperglycemia, insulin resistance, and endothelium dysfunction are related to platelet hyperactivity in type 2 diabetes mellitus (T2D) patients. Glucosamine (GlcN) has inhibitory effects on platelets of animals and healthy donors, but this role in platelets from T2D patients is unknown. The aim of this study was to evaluate the GlcN in vitro effects on platelet aggregation in T2D patients and healthy donors. Donors´ and T2D patients' samples were analyzed through flow cytometry, Western blot, and platelet aggregometry. Platelet aggregation was induced using ADP and thrombin, with or without GlcN, N-Acetyl-glucosamine, galactose, or fucose. GlcN inhibited ADP and thrombin-induced platelet aggregation, while the other carbohydrates did not. GlcN suppressed the second wave of ADP-induced platelet aggregation. No differences in the percent of inhibition of ADP-induced platelet aggregation by GlcN were found between donors and T2D patients, but this effect was significantly higher in healthy donors using thrombin as an agonist. In addition, GlcN increased protein O-GlcNAcylation (O-GlcNAc) in the platelets from T2D patients but not in healthy donors. In conclusion, GlcN inhibited the platelet aggregation induced by ADP and thrombin for both study groups and increased O-GlcNAc in platelets from T2D patients. Further studies are required to evaluate the possible use of GlcN as an antiplatelet agent.

Inhibitory Effects of Aspirin and Cilostazol on Intracellular Ca2+ Mobilization and Aggregation in Thrombin-activated Human Platelets.

Platelets play an important role in physiological hemostatic mechanisms. In contrast, platelet activation has been implicated in pathological conditions, such as atherosclerosis, angiogenesis, and inflammation. Thrombin is considered to be of particular pathological importance as a platelet-activating substance, and thrombin-activated platelets are detected in the blood of patients with advanced occlusive arterial disease. Ca2+ acts as a second messenger in platelet activation, and the regulation of intracellular Ca2+ concentrations ([Ca2+]i) is important for controlling platelet functions. However, changes in [Ca2+]i by antiplatelet agents remain unclear. Therefore, we herein investigated the relationship between [Ca2+]i and the intensity of platelet aggregation after a thrombin stimulation, the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents, and the effects of antiplatelet agents on thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. Fura2-loaded platelets were treated with phosphate-buffered saline or a low concentration of thrombin (0.005 U/mL), followed by antiplatelet agents (aspirin or cilostazol), and changes in [Ca2+]i and the intensity of platelet aggregation by the thrombin stimulation were measured using fluorescence spectrophotometry. Changes in [Ca2+]i and the intensity of platelet aggregation after the thrombin stimulation as well as the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents indicated that cilostazol exerted stronger antiplatelet effects than aspirin and also that antiplatelet effects may be attenuated in thrombin-activated platelets. The present results also suggest the utility of thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. These results may contribute to future drug development for antiplatelet therapy. J. Med. Invest. 70 : 94-100, February, 2023.

CYP2C19 Polymorphism in Ischemic Heart Disease Patients Taking Clopidogrel After Percutaneous Coronary Intervention in Egypt.

BACKGROUND: Cardiovascular diseases (CVDs) are considered a leading cause of death worldwide. Allelic variation in the CYP2C19 gene leads to a dysfunctional enzyme, and patients with this loss-of-function allele will have an impaired clopidogrel metabolism, which eventually results in major adverse cardiovascular events (MACE). Ischemic heart disease patients (n = 102) who underwent percutaneous cardiac intervention (PCI) followed by clopidogrel were enrolled in the present study. METHODS: The genetic variations in the CYP2C19 gene were identified using the TaqMan chemistry-based qPCR technique. Patients were followed up for 1 year to monitor MACE, and the correlations between the allelic variations in CYP2C19 and MACE were recorded. RESULTS: During the follow-up, we reported 64 patients without MACE (29 with unstable angina (UA), 8 with myocadiac infarction (MI), 1 patient with non-STEMI, and 1 patient with ischemic dilated cardiomyopathy (IDC)). Genotyping of CYP2C19 in the patients who underwent PCI and were treated with clopidogrel revealed that 50 patients (49%) were normal metabolizers for clopidogrel with genotype CYP2C19*1/*1 and 52 patients (51%) were abnormal metabolizers, with genotypes CYP2C19*1/*2 (n = 15), CYP2C19*1/*3 (n = 1), CYP2C19*1/*17 (n = 35), and CYP2C19*2/*17 (n = 1). Demographic data indicated that age and residency were significantly associated with abnormal clopidogrel metabolism. Moreover, diabetes, hypertension, and cigarette smoking were significantly associated with the abnormal metabolism of clopidogrel. These data shed light on the inter-ethnic variation in metabolizing clopidogrel based on the CYP2C19 allelic distribution. CONCLUSION: This study, along with other studies that address genotype variation of clopidogrel-metabolizing enzymes, might pave the way for further understanding of the pharmacogenetic background of CVD-related drugs.

[Study on Platelet Adhesion and Aggregation Induced by Gradient Shear Stress Using Microfluidic Chip Technology].

OBJECTIVE: To study the effect of gradient shear stress on platelet aggregation by microfluidic chip Technology. METHODS: Microfluidic chip was used to simulate 80% fixed stenotic microchannel, and the hydrodynamic behavior of the stenotic microchannel model was analyzed by the finite element analysis module of sollidwork software. Microfluidic chip was used to analyze the adhesion and aggregation behavior of platelets in patients with different diseases, and flow cytometry was used to detect expression of the platelet activation marker CD62p. Aspirin, Tirofiban and protocatechuic acid were used to treat the blood, and the adhesion and aggregation of platelets were observed by fluorescence microscope. RESULTS: The gradient fluid shear rate produced by the stenosis model of microfluidic chip could induce platelet aggregation, and the degree of platelet adhesion and aggregation increased with the increase of shear rate within a certain range of shear rate. The effect of platelet aggregation in patients with arterial thrombotic diseases were significantly higher than normal group (P<0.05), and the effect of platelet aggregation in patients with myelodysplastic disease was lower than normal group (P<0.05). CONCLUSION: The microfluidic chip analysis technology can accurately analyze and evaluate the platelet adhesion and aggregation effects of various thrombotic diseases unde the environment of the shear rate, and is helpful for auxiliary diagnosis of clinical thrombotic diseases.

Single-cell transcriptome analysis of NEUROG3+ cells during pancreatic endocrine differentiation with small molecules.

The efficiency of inducing human embryonic stem cells into NEUROG3+ pancreatic endocrine cells is a bottleneck in stem cell therapy for diabetes. To understand the cell properties and fate decisions during differentiation, we analyzed the modified induction method using single-cell transcriptome and found that DAPT combined with four factors (4FS): nicotinamide, dexamethasone, forskolin and Alk5 inhibitor II (DAPT + 4FS) increased the expression of NEUROG3 to approximately 34.3%. The increased NEUROG3+ cells were mainly concentrated in Insulin + Glucagon + (INS + GCG+) and SLAC18A1 + Chromogranin A+(SLAC18A1 + CHGA +) populations, indicating that the increased NEUROG3+ cells promoted the differentiation of pancreatic endocrine cells and enterochromaffin-like cells. Single-cell transcriptome analysis provided valuable clues for further screening of pancreatic endocrine cells and differentiation of pancreatic islet cells. The gene set enrichment analysis (GSEA) suggest that we can try to promote the expression of INS + GCG+ population by up-regulating G protein-coupled receptor (GPCR) and mitogen-activated protein kinase signals and down-regulating Wnt, NIK/NF-KappaB and cytokine-mediated signal pathways. We can also try to regulate GPCR signaling through PLCE1, so as to increase the proportion of NEUROG3+ cells in INS+GCG+ populations. To exclude non-pancreatic endocrine cells, ALCAMhigh CD9low could be used as a marker for endocrine populations, and ALCAMhigh CD9lowCDH1low could remove the SLC18A1 + CHGA+ population.

Notoginsenoside R1 Facilitates Cell Angiogenesis by Inactivating the Notch Signaling During Wound Healing.

The development of chronic, nonhealing wounds is a persistent medical problem that drives patient morbidity and increases healthcare costs. Angiogenesis is a critical accompanying activity in the proliferation stage during the wound healing process. Notoginsenoside R1 (NGR1) isolated from Radix notoginseng has been reported to alleviate diabetic ulcers by promoting angiogenesis and decreasing inflammatory responses and apoptosis. In the present study, we investigated the effect of NGR1 on angiogenesis and its therapeutic functions in cutaneous wound healing. For in vitro evaluation, cell counting kit-8 assays, migration assays, Matrigel-based angiogenic assays, and western blotting were conducted. The experimental results showed that NGR1 (10-50 µM) had no cytotoxicity to human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMEC), and NGR1 treatment facilitated the migration of HSFs and enhanced angiogenesis in HMECs. Mechanistically, NGR1 treatment inhibited the activation of Notch signaling in HMECs. For in vivo analysis, hematoxylin-eosin staining, immunostaining, and Masson's trichrome staining were performed, and we found that NGR1 treatment promoted angiogenesis, reduced wound widths, and facilitated wound healing. Furthermore, HMECs were treated with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT; a Notch inhibitor), and DAPT treatment was found to exert pro-angiogenic effects. Simultaneously, DAPT was administrated into experimental cutaneous wound healing model, and we found that DAPT administration prevented the development of cutaneous wounds. Collectively, NGR1 promotes angiogenesis and wound repair via activation of the Notch pathway and exhibits therapeutic effects on cutaneous wound healing.

Notch Pathways Regulate Expression of Angiostatic Factor Activin A in Endothelial-Pericyte-Like Mesenchymal Stromal Cell Interactions.

Vascularization of ischemic and fabricated tissues is essential for successful tissue repair and replacement therapies. Endothelial cells (ECs) and mesenchymal stem/stromal cells (MSCs) in close proximity spontaneously organize into vessels after coimplantation in semisolid matrices. Thus, local injection of EC mixed with MSC may facilitate tissue (re)vascularization. The organization of these cells into vessels is accompanied by induction of a key regulator of vasculogenesis, activin A, in MSC through juxtacrine pathway. Mechanisms regulating activin A expression are poorly understood; therefore, the contributions of notch signaling pathways were evaluated in EC-adipose mesenchymal stromal cells (ASC) cocultures. Disruption of notch signaling in EC + ASC cocultures with a γ-secretase inhibitor, DAPT, completely abrogated both activin A induction and production, depending on the stage of vasculogenesis. While DAPT stimulated EC proliferation concurrent with increased secretion of vasculogenic factors, it also prevented the crucial transition of ASC from progenitor to smooth muscle cell phenotype, collectively resulting in ineffective tubulogenesis. Silencing Notch2 in ASC abolished activin A production in cocultures, but resulted in normal ASC maturation. In contrast, silencing Notch3 in ASC led to autonomous upregulation of mural cell markers, and intercellular contact with EC further enhanced upregulation of these markers, concurrent with amplified activin A secretion. Strong induction of activin A expression was achieved by exposing ASC to immobilized notch ligand jagged1, whereas jagged1 IgG, added to EC + ASC incubation media, prevented activin A expression. Overall, this study revealed that EC control activin A expression in ASC through trans juxtacrine notch signaling pathways, and uninterrupted notch signaling is required for activin A production, although signaling through Notch2 and Notch3 produce opposing effects.

Notch1 signalling controls the differentiation and function of myeloid-derived suppressor cells in systemic lupus erythematosus.

Emerging studies have reported the expansion of myeloid-derived suppressor cells (MDSCs) in some autoimmune disorders, such as systemic lupus erythematosus (SLE), but the detailed molecular mechanisms of the aberrant expansion in SLE are still unclear. In the present study, we confirmed that the increased MDSCs positively correlated with disease activity in SLE patients. The suppressive capacity of MDSCs from patients with high activity was lower than that of MDSCs from patients with low activity. Moreover, the potential precursors for MDSCs, common myeloid progenitors (CMPs) and granulocyte-monocyte progenitors (GMPs), were markedly increased in the bone marrow (BM) aspirates of SLE patients. As an important regulator of cell fate decisions, aberrant activation of Notch signalling was reported to participate in the pathogenesis of SLE. We found that the expression of Notch1 and its downstream target gene hairy and enhancer of split 1 (Hes-1) increased markedly in GMPs from SLE patients. Moreover, the Notch1 signalling inhibitor DAPT profoundly relieved disease progression and decreased the proportion of MDSCs in pristane-induced lupus mice. The frequency of GMPs was also decreased significantly in lupus mice after DAPT treatment. Furthermore, the inhibition of Notch1 signalling could limit the differentiation of MDSCs in vitro. The therapeutic effect of DAPT was also verified in Toll-like receptor 7 (TLR7) agonist-induced lupus mice. Taken together, our results demonstrated that Notch1 signalling played a crucial role in MDSC differentiation in SLE. These findings will provide a promising therapy for the treatment of SLE.

Augmentation by resveratrol of the inhibitory effect of ethanol on platelet aggregation.

Ethanol and resveratrol have been shown to inhibit platelet aggregation. The aim of this study was to determine whether resveratrol has an additional effect on ethanol-induced inhibition of platelet aggregation. Ca2+ entry and subsequent aggregation of human platelets were measured by the fluorescence method and light transmittance method, respectively. Thromboxane B2 concentrations in media containing platelets were measured by using the enzyme-linked immunosorbent assay. Platelet aggregation induced by thrombin (0.025 U/ml) was significantly inhibited by preincubation of platelets with ethanol (0.5%). Preincubation with resveratrol (3.125 µM), which did not affect thrombin-induced platelet aggregation, significantly augmented the inhibitory effect of ethanol on platelet aggregation. Similar synergic effects of ethanol and resveratrol were found on aggregatory responses to collagen (2 µg/ml) and arachidonic acid (0.25 mM). On the other hand, the thrombin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was not affected by ethanol alone, resveratrol alone or both ethanol and resveratrol together. In nominally Ca2+-free medium, arachidonic acid (0.75 mM) caused a potent platelet aggregation, which was not affected by the presence of ethanol alone, resveratrol alone, or both of them together. Thromboxane B2 formation induced by thrombin was significantly inhibited by ethanol (0.5%) alone and resveratrol (3.125 µM) alone, and these inhibitory effects were significantly augmented in the presence of both ethanol and resveratrol together. Resveratrol shows an additive effect on ethanol-induced inhibition of platelet aggregation. This effect by resveratrol is partly explained by its inhibitory action on thromboxane A2 production in platelets. In addition, both ethanol and resveratrol attenuate platelet aggregation through acting on the Ca2+-dependent intra-platelet pathway after an increase in [Ca2+]i induced by thrombin.

B Lymphocytes Balance Th1/Th2 to Participate in Fibrotic Hypersensitivity Pneumonia Induced by Pigeon Shedding.

INTRODUCTION: We investigated the molecular mechanism by which B lymphocytes regulate Th1/Th2 imbalance to participate in the pulmonary fibrosis in hypersensitivity pneumonia induced by pigeon shedding in rats. METHODS: CD19+ rats and CD19- rats were used to construct animal models of fibrotic hypersensitivity pneumonia. DAPT was used to inhibit the Notch signaling pathway. The pathological changes were assessed with HE and Masson staining. Protein level was detected with Western blot. Th1/Th2 ratio was analyzed with flow cytometry. Cytokine levels were measured with ELISA. RESULTS: The pathological changes of pulmonary fibrosis were not obvious in the CD19- rats and after DAPT treatment. Notch signaling pathway proteins, including Notch1, Notch2, Jag1, Jag2, DLL1, and DLL4, in lung tissues of model rats were all significantly upregulated than those in control rats. However, these proteins in CD19- rats were lower in CD19+ rats, suggesting that B cells play a key role in inducing pneumonia. Besides, the Th1/Th2 ratio in the BALF of model rats decreased, which was further reversed by DAPT. However, we found that in CD19- rats, the regulation of the Th1/Th2 ratio by the Notch signaling pathway was lost. CONCLUSION: Deleting B lymphocytes or blocking the Notch pathway both reversed the Th1/Th2 imbalance in fibrotic hypersensitivity pneumonia and inhibited pulmonary fibrosis.

Pharmacological Actions of 5-Hydroxyindolin-2 on Modulation of Platelet Functions and Thrombus Formation via Thromboxane A2 Inhibition and cAMP Production.

Platelets play a very significant role in hemostasis while simultaneously posing a risk for the development of various cardiovascular diseases. Platelet-mediated issues can occur in blood vessels and trigger various medical problems. Therefore, controlling platelet function is important in the prevention of thrombosis. In this regard, we need to find compounds that provide potent antiplatelet activity with minimum side effects. Therefore, we examined the effect of 5-hydroxyindolin-2-one isolated from Protaetia brevitarsis larvae having antiplatelet properties and investigated different pathways that mediate the antiplatelet activity. We examined the effect of 5-hydroxyindolin-2-one (5-HI) on the regulation of phosphoproteins, thromboxane A2 generation, and integrin αIIbß3 action. Our data showed that human platelet aggregation was inhibited by 5-HI (75, 100, 150, 200 µM) without cytotoxicity, and it suppressed intracellular Ca2+ concentration through the regulation of inositol 1, 4, 5-triphosphate receptor I (Ser1756) and extracellular signal-regulated kinase (ERK). Moreover, collagen-elevated thromboxane A2 production and αIIbß3 action were inhibited by 5-HI through the regulation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinase p38 (p38MAPK), vasodilator-stimulated phosphoprotein (VASP), phosphoinositide 3-kinase (PI3K), and Akt (protein kinase B). Therefore, we suggested that 5-HI could be a potential substance for the prevention of thrombosis-mediated thrombosis.

A Comprehensive Literature Review on Cardioprotective Effects of Bioactive Compounds Present in Fruits of Aristotelia chilensis Stuntz (Maqui).

Some fruits and vegetables, rich in bioactive compounds such as polyphenols, flavonoids, and anthocyanins, may inhibit platelet activation pathways and therefore reduce the risk of suffering from CVD when consumed regularly. Aristotelia chilensis Stuntz (Maqui) is a shrub or tree native to Chile with outstanding antioxidant activity, associated with its high content in anthocyanins, polyphenols, and flavonoids. Previous studies reveal different pharmacological properties for this berry, but its cardioprotective potential has been little studied. Despite having an abundant composition, and being rich in bioactive products with an antiplatelet role, there are few studies linking this berry with antiplatelet activity. This review summarizes and discusses relevant information on the cardioprotective potential of Maqui, based on its composition of bioactive compounds, mainly as a nutraceutical antiplatelet agent. Articles published between 2000 and 2022 in the following bibliographic databases were selected: PubMed, ScienceDirect, and Google Scholar. Our search revealed that Maqui is a promising cardiovascular target since extracts from this berry have direct effects on the reduction in cardiovascular risk factors (glucose index, obesity, diabetes, among others). Although studies on antiplatelet activity in this fruit are recent, its rich chemical composition clearly shows that the presence of chemical compounds (anthocyanins, flavonoids, phenolic acids, among others) with high antiplatelet potential can provide this berry with antiplatelet properties. These bioactive compounds have antiplatelet effects with multiple targets in the platelet, particularly, they have been related to the inhibition of thromboxane, thrombin, ADP, and GPVI receptors, or through the pathways by which these receptors stimulate platelet aggregation. Detailed studies are needed to clarify this gap in the literature, as well as to specifically evaluate the mechanism of action of Maqui extracts, due to the presence of phenolic compounds.

Reversible Platelet Integrin αIIbß3 Activation and Thrombus Instability.

Integrin αIIbß3 activation is essential for platelet aggregation and, accordingly, for hemostasis and arterial thrombosis. The αIIbß3 integrin is highly expressed on platelets and requires an activation step for binding to fibrinogen, fibrin or von Willebrand factor (VWF). A current model assumes that the process of integrin activation relies on actomyosin force-dependent molecular changes from a bent-closed and extended-closed to an extended-open conformation. In this paper we review the pathways that point to a functional reversibility of platelet αIIbß3 activation and transient aggregation. Furthermore, we refer to mouse models indicating that genetic defects that lead to reversible platelet aggregation can also cause instable thrombus formation. We discuss the platelet agonists and signaling pathways that lead to a transient binding of ligands to integrin αIIbß3. Our analysis points to the (autocrine) ADP P2Y1 and P2Y12 receptor signaling via phosphoinositide 3-kinases and Akt as principal pathways linked to reversible integrin activation. Downstream signaling events by protein kinase C, CalDAG-GEFI and Rap1b have not been linked to transient integrin activation. Insight into the functional reversibility of integrin activation pathways will help to better understand the effects of antiplatelet agents.