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Scientific Reports Jun 2024Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to...
Developing a reliable method to predict thrombocytopenia is imperative in drug discovery. Here, we establish an assay using a microphysiological system (MPS) to recapitulate the in-vivo mechanisms of platelet aggregation and adhesion. This assay highlights the role of shear stress on platelet aggregation and their interactions with vascular endothelial cells. Platelet aggregation induced by soluble collagen was detected under agitated, but not static, conditions using a plate shaker and gravity-driven flow using MPS. Notably, aggregates adhered on vascular endothelial cells under gravity-driven flow in the MPS, and this incident increased in a concentration-dependent manner. Upon comparing the soluble collagen-induced aggregation activity in platelet-rich plasma (PRP) and whole blood, remarkable platelet aggregate formation was observed at concentrations of 30 µg/mL and 3 µg/mL in PRP and whole blood, respectively. Moreover, ODN2395, an oligonucleotide, induced platelet aggregation and adhesion to vascular endothelial cells. SYK inhibition, which mediated thrombogenic activity via glycoprotein VI on platelets, ameliorated platelet aggregation in the system, demonstrating that the mechanism of platelet aggregation was induced by soluble collagen and oligonucleotide. Our evaluation system partially recapitulated the aggregation mechanisms in blood vessels and can contribute to the discovery of safe drugs to mitigate the risk of thrombocytopenia.
Topics: Platelet Aggregation; Humans; Thrombocytopenia; Blood Platelets; Collagen; Endothelial Cells; Platelet Adhesiveness; Syk Kinase; Platelet-Rich Plasma; Human Umbilical Vein Endothelial Cells; Microphysiological Systems
PubMed: 38898080
DOI: 10.1038/s41598-024-64063-y -
International Journal of Molecular... May 2024This study focuses on understanding the transcriptional heterogeneity of activated platelets and its impact on diseases such as sepsis, COVID-19, and systemic lupus...
This study focuses on understanding the transcriptional heterogeneity of activated platelets and its impact on diseases such as sepsis, COVID-19, and systemic lupus erythematosus (SLE). Recognizing the limited knowledge in this area, our research aims to dissect the complex transcriptional profiles of activated platelets to aid in developing targeted therapies for abnormal and pathogenic platelet subtypes. We analyzed single-cell transcriptional profiles from 47,977 platelets derived from 413 samples of patients with these diseases, utilizing Deep Neural Network (DNN) and eXtreme Gradient Boosting (XGB) to distinguish transcriptomic signatures predictive of fatal or survival outcomes. Our approach included source data annotations and platelet markers, along with SingleR and Seurat for comprehensive profiling. Additionally, we employed Uniform Manifold Approximation and Projection (UMAP) for effective dimensionality reduction and visualization, aiding in the identification of various platelet subtypes and their relation to disease severity and patient outcomes. Our results highlighted distinct platelet subpopulations that correlate with disease severity, revealing that changes in platelet transcription patterns can intensify endotheliopathy, increasing the risk of coagulation in fatal cases. Moreover, these changes may impact lymphocyte function, indicating a more extensive role for platelets in inflammatory and immune responses. This study identifies crucial biomarkers of platelet heterogeneity in serious health conditions, paving the way for innovative therapeutic approaches targeting platelet activation, which could improve patient outcomes in diseases characterized by altered platelet function.
Topics: Humans; COVID-19; Lupus Erythematosus, Systemic; Blood Platelets; Machine Learning; Single-Cell Analysis; Transcriptome; Sepsis; SARS-CoV-2; Gene Expression Profiling; Platelet Activation
PubMed: 38892129
DOI: 10.3390/ijms25115941 -
Scientific Reports Jun 2024Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is...
Familial platelet disorder with associated myeloid malignancies (FPDMM) is an autosomal dominant disease caused by heterozygous germline mutations in RUNX1. It is characterized by thrombocytopenia, platelet dysfunction, and a predisposition to hematological malignancies. Although FPDMM is a precursor for diseases involving abnormal DNA methylation, the DNA methylation status in FPDMM remains unknown, largely due to a lack of animal models and challenges in obtaining patient-derived samples. Here, using genome editing techniques, we established two lines of human induced pluripotent stem cells (iPSCs) with different FPDMM-mimicking heterozygous RUNX1 mutations. These iPSCs showed defective differentiation of hematopoietic progenitor cells (HPCs) and megakaryocytes (Mks), consistent with FPDMM. The FPDMM-mimicking HPCs showed DNA methylation patterns distinct from those of wild-type HPCs, with hypermethylated regions showing the enrichment of ETS transcription factor (TF) motifs. We found that the expression of FLI1, an ETS family member, was significantly downregulated in FPDMM-mimicking HPCs with a RUNX1 transactivation domain (TAD) mutation. We demonstrated that FLI1 promoted binding-site-directed DNA demethylation, and that overexpression of FLI1 restored their megakaryocytic differentiation efficiency and hypermethylation status. These findings suggest that FLI1 plays a crucial role in regulating DNA methylation and correcting defective megakaryocytic differentiation in FPDMM-mimicking HPCs with a RUNX1 TAD mutation.
Topics: Core Binding Factor Alpha 2 Subunit; Humans; DNA Methylation; Megakaryocytes; Proto-Oncogene Protein c-fli-1; Cell Differentiation; Induced Pluripotent Stem Cells; Mutation; Blood Platelet Disorders; Transcriptional Activation; Hematopoietic Stem Cells; Leukemia, Myeloid, Acute; Blood Coagulation Disorders, Inherited
PubMed: 38890442
DOI: 10.1038/s41598-024-64829-4 -
The Journal of Extra-corporeal... Jun 2024Cardiopulmonary bypass is an essential component of cardiothoracic surgeries. However, significant complications such as systemic inflammatory response syndrome (SIRS)...
INTRODUCTION
Cardiopulmonary bypass is an essential component of cardiothoracic surgeries. However, significant complications such as systemic inflammatory response syndrome (SIRS) resulting from cardiopulmonary bypass (CPB) are a common occurrence due to contact between circulating blood and foreign surfaces that leads to platelet activation. It is suggested that different available CPB circuit coatings can potentially reduce platelet activation. However, there have been no published evidence-based reports confirming these claims. In addition, there is no well-established protocol for studying platelet activation biomarkers during CPB in vitro in a laboratory setting.
METHODS
CPB was simulated in the laboratory using bovine blood in two different types of coated CPB circuits: Trillium Biosurface by Medtronic, and Xcoating Surface by Terumo. Fresh bovine blood samples were collected and circulated through the CPB circuit following the standard protocol used in the operation rooms. Blood samples were then collected at 5 min, 30 min, and 55 min during the circulation. Blood plasmas were separated and subjected to enzyme-linked immunosorbent assay to measure most established platelet activation markers P-selectin, Platelet Factor 4 (PF4), Glycoprotein IIb/IIIa (GPIIb/IIIa), and β-thromboglobulin (β-TG) at different time points.
RESULTS
The biomarker values at 30 min and 55 min were compared to the base values at 5 min for each type of CPB circuit. The results of the means from all measured biomarkers showed data measurements that indicated no significant variability within each coating. All collected data points fell within ±2 SD of the means, which was considered acceptable variations across technical replicates. Conclusion: In this study, we were able to establish an in vitro protocol in the laboratory setting that is precise and reliable with minimum intra-variability. This established protocol will allow for future studies in which different coated CPB circuits can be compared for their effectiveness in blocking platelet activation during the CPB.
Topics: Cardiopulmonary Bypass; Platelet Activation; Animals; Biomarkers; Cattle; Coated Materials, Biocompatible; Materials Testing
PubMed: 38888546
DOI: 10.1051/ject/2024003 -
Cell Communication and Signaling : CCS Jun 2024HRAS/NRAS double knockout mice exhibit exceedingly high rates of perinatal lethality due to respiratory failure caused by a significant lung maturation delay. The few...
BACKGROUND
HRAS/NRAS double knockout mice exhibit exceedingly high rates of perinatal lethality due to respiratory failure caused by a significant lung maturation delay. The few animals that reach adulthood have a normal lifespan, but present areas of atelectasis mixed with patches of emphysema and normal tissue in the lung.
METHODS
Eight double knockout and eight control mice were analyzed using micro-X-ray computerized tomography and a Small Animal Physiological Monitoring system. Tissues and samples from these mice were analyzed using standard histological and Molecular Biology methods and the significance of the results analyzed using a Student´s T-test.
RESULTS
The very few double knockout mice surviving up to adulthood display clear craniofacial abnormalities reminiscent of those seen in RASopathy mouse models, as well as thrombocytopenia, bleeding anomalies, and reduced platelet activation induced by thrombin. These surviving mice also present heart and spleen hyperplasia, and elevated numbers of myeloid-derived suppressor cells in the spleen. Mechanistically, we observed that these phenotypic alterations are accompanied by increased KRAS-GTP levels in heart, platelets and primary mouse embryonic fibroblasts from these animals.
CONCLUSIONS
Our data uncovers a new, previously unidentified mechanism capable of triggering a RASopathy phenotype in mice as a result of the combined removal of HRAS and NRAS.
Topics: Animals; Proto-Oncogene Proteins p21(ras); Phenotype; Mice; Mice, Knockout; GTP Phosphohydrolases; Membrane Proteins; Platelet Activation; Spleen; Monomeric GTP-Binding Proteins
PubMed: 38886790
DOI: 10.1186/s12964-024-01717-4 -
Life Sciences Jun 2024This research aimed to study the changes in platelet function and their underlying mechanisms in iron deficiency anemia.
AIMS
This research aimed to study the changes in platelet function and their underlying mechanisms in iron deficiency anemia.
MAIN METHODS
Initially, we evaluated platelet function in an IDA mice model. Due to the inability to accurately reduce intracellular Fe concentrations, we investigated the impact of Fe on platelet function by introducing varying concentrations of Fe. To probe the underlying mechanism, we simultaneously examined the dynamics of calcium in the cytosol, and integrin αIIbβ3 activation in Fe-treated platelets. Ferroptosis inhibitors Lip-1 and Fer-1 were applied to determine whether ferroptosis was involved in this process.
KEY FINDINGS
Our study revealed that platelet function was suppressed in IDA mice. Fe concentration-dependently facilitated platelet activation and function in vitro. Mechanistically, Fe promoted calcium mobilization, integrin αIIbβ3 activation, and its downstream outside-in signaling. Additionally, we also demonstrated that ferroptosis might play a role in this process.
SIGNIFICANCE
Our data suggest an association between iron and platelet activation, with iron deficiency resulting in impaired platelet function, while high concentrations of Fe contribute to platelet activation and function by promoting calcium mobilization, αIIbβ3 activation, and ferroptosis.
PubMed: 38885879
DOI: 10.1016/j.lfs.2024.122848 -
Clinical and Translational Science Jun 2024This cohort study aims to assess the connection between cytochrome P450 family 2 subfamily C member 19 (CYP2C19) genotyping, platelet aggregability following oral...
This cohort study aims to assess the connection between cytochrome P450 family 2 subfamily C member 19 (CYP2C19) genotyping, platelet aggregability following oral clopidogrel administration, and the occurrence of postoperative atrial fibrillation (POAF) after off-pump coronary artery bypass graft (CABG) surgery. From May 2017 to November 2022, a total of 258 patients undergoing elective first-time CABG surgery, receiving 100 mg/day oral aspirin and 75 mg/day oral clopidogrel postoperatively, was included for analysis. These patients were categorized based on CYP2C19 genotyping. Platelet aggregability was assessed serially using multiple-electrode aggregometry before CABG, 1 and 5 days after the procedure, and before discharge. The incidences of POAF were compared using the log-rank test for cumulative risk. CYP2C19 genotyping led to categorization into CYP2C19*1*1 (WT group, n = 123) and CYP2C19*2 or *3 (LOF group, n = 135). Baseline characteristics and operative data showed no significant differences between the two groups. The incidence of POAF after CABG was 42.2% in the LOF group, contrasting with 22.8% in the WT group (hazard risk [HR]: 2.061; 95% confidence interval [CI]: 1.347, 3.153; p = 0.0013). Adenosine diphosphate-stimulated platelet aggregation was notably higher in the LOF group compared to the WT group 5 days after CABG (30.4% ± 6.5% vs. 17.9% ± 4.1%, p < 0.001), remaining a similar higher level at hospital discharge (25.6% ± 6.1% vs. 12.2% ± 3.5%, p < 0.001). The presence of CYP2C19 LOF was linked to a higher incidence of POAF and relatively elevated platelet aggregation after CABG surgery under the same oral clopidogrel regimen.
Topics: Humans; Cytochrome P-450 CYP2C19; Atrial Fibrillation; Male; Female; Aged; Coronary Artery Bypass; Middle Aged; Clopidogrel; Postoperative Complications; Genotype; Platelet Aggregation Inhibitors; Platelet Aggregation; Incidence; Aspirin
PubMed: 38877696
DOI: 10.1111/cts.13862 -
Medicine Jun 2024A prospective cohort study investigated the effectiveness of platelet-rich plasma (PRP) infusion for refractory thin endometrium in 38 infertile patients. Patients...
Treating refractory thin endometrium through a novel way of activation and administration of Platelet-rich plasma in sexually active women: An interventional prospective cohort clinical study.
A prospective cohort study investigated the effectiveness of platelet-rich plasma (PRP) infusion for refractory thin endometrium in 38 infertile patients. Patients showed significant improvement in endometrial thickness post-PRP injection, leading to successful implantation and pregnancy. The study revealed a negative correlation between antimullerian hormone (AMH) levels and the need for PRP interventions, suggesting higher ovarian reserve may reduce the necessity for repeated treatments. This implies AMH levels could serve as a prognostic indicator for treatment outcomes, aiding clinicians in optimizing protocols and reducing patient burden. Further research is needed to confirm these findings in larger and more diverse populations, along with exploring long-term reproductive success rates post-PRP treatment.
Topics: Humans; Female; Platelet-Rich Plasma; Prospective Studies; Adult; Endometrium; Infertility, Female; Anti-Mullerian Hormone; Pregnancy; Ovarian Reserve; Treatment Outcome
PubMed: 38875415
DOI: 10.1097/MD.0000000000038554 -
Circulation Jun 2024Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate...
BACKGROUND
Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization.
METHODS
EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene (). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers.
RESULTS
In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic mice or after silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models.
CONCLUSIONS
Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.
PubMed: 38873770
DOI: 10.1161/CIRCULATIONAHA.124.068726 -
Frontiers in Cardiovascular Medicine 2024
PubMed: 38873263
DOI: 10.3389/fcvm.2024.1433858