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JCI Insight May 2024Identifying immune correlates of protection is a major challenge in AIDS vaccine development. Anti-Envelope antibodies have been considered critical for protection...
Identifying immune correlates of protection is a major challenge in AIDS vaccine development. Anti-Envelope antibodies have been considered critical for protection against SIV/HIV (SHIV) acquisition. Here, we evaluated the efficacy of an SHIV vaccine against SIVmac251 challenge, where the role of antibody was excluded, as there was no cross-reactivity between SIV and SHIV envelope antibodies. After 8 low-dose intrarectal challenges with SIVmac251, 12 SHIV-vaccinated animals demonstrated efficacy, compared with 6 naive controls, suggesting protection was achieved in the absence of anti-envelope antibodies. Interestingly, CD8+ T cells (and some NK cells) were not essential for preventing viral acquisition, as none of the CD8-depleted macaques were infected by SIVmac251 challenges. Initial investigation of protective innate immunity revealed that protected animals had elevated pathways related to platelet aggregation/activation and reduced pathways related to interferon and responses to virus. Moreover, higher expression of platelet factor 4 on circulating platelet-leukocyte aggregates was associated with reduced viral acquisition. Our data highlighted the importance of innate immunity, identified mechanisms, and may provide opportunities for novel HIV vaccines or therapeutic strategy development.
Topics: Animals; Simian Acquired Immunodeficiency Syndrome; Simian Immunodeficiency Virus; SAIDS Vaccines; Macaca mulatta; Immunity, Innate; CD8-Positive T-Lymphocytes; Antibodies, Viral; Male; Vaccines, Attenuated
PubMed: 38912579
DOI: 10.1172/jci.insight.175800 -
Heliyon Jun 2024Inferior vena cava filters have been shown to be effective in preventing deep vein thrombosis and its secondary complication, pulmonary embolism, thereby reducing the...
BACKGROUND AND OBJECTIVE
Inferior vena cava filters have been shown to be effective in preventing deep vein thrombosis and its secondary complication, pulmonary embolism, thereby reducing the high mortality rate. Although inferior vena cava filters have evolved, specific complications like inferior vena cava thrombosis-induced deep vein thrombosis worsening and recurrent pulmonary embolism continue to pose challenges. This study analyzes the effects of geometric parameter variations of inferior vena cava filters, which have a significant impact on the thrombus formation inside the filter, the capture, dissolution, and hemodynamic flow of thrombus, as well as the shear stress on the filter and vascular wall.
METHODS
This study used computational fluid dynamic simulations with the carreau model to investigate the impact of varying inferior vena cava filter design parameters (number of struts, strut arm length, and tilt angle) on hemodynamics.
RESULTS
Recirculation and stagnation areas due to flow velocity and pressure, along with wall shear stress values, were identified as key factors. It is important to find a balance between wall shear stress high enough to aid thrombolysis and low enough to prevent platelet activation. The results of this paper show that the risk of platelet activation and thrombus filtration may be lowest when the wall shear stress of the filter ranges from 0 to 4 [Pa], minimizing stress concentration within the filter.
CONCLUSION
16 arm struts with a length of 20 mm and a tilt angle of 0° provide the best balance between thrombus capture and minimization of hemodynamic disturbance. This configuration minimizes the size of the stagnation and recirculation zones while maintaining sufficient wall shear stress for thrombus dissolution.
PubMed: 38912484
DOI: 10.1016/j.heliyon.2024.e32667 -
TH Open : Companion Journal To... Apr 2024Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under...
Agonist-induced platelet activation, with the integrin αIIbβ3 conformational change, is required for fibrinogen binding. This is considered reversible under specific conditions, allowing a second phase of platelet aggregation. The signaling pathways that differentiate between a permanent or transient activation state of platelets are poorly elucidated. To explore platelet signaling mechanisms induced by the collagen receptor glycoprotein VI (GPVI) or by protease-activated receptors (PAR) for thrombin that regulate time-dependent αIIbβ3 activation. Platelets were activated with collagen-related peptide (CRP, stimulating GPVI), thrombin receptor-activating peptides, or thrombin (stimulating PAR1 and/or 4). Integrin αIIbβ3 activation and P-selectin expression was assessed by two-color flow cytometry. Signaling pathway inhibitors were applied before or after agonist addition. Reversibility of platelet spreading was studied by microscopy. Platelet pretreatment with pharmacological inhibitors decreased GPVI- and PAR-induced integrin αIIbβ3 activation and P-selectin expression in the target order of protein kinase C (PKC) > glycogen synthase kinase 3 > β-arrestin > phosphatidylinositol-3-kinase. Posttreatment revealed secondary αIIbβ3 inactivation (not P-selectin expression), in the same order, but this reversibility was confined to CRP and PAR1 agonist. Combined inhibition of conventional and novel PKC isoforms was most effective for integrin closure. Pre- and posttreatment with ticagrelor, blocking the P2Y adenosine diphosphate (ADP) receptor, enhanced αIIbβ3 inactivation. Spreading assays showed that PKC or P2Y inhibition provoked a partial conversion from filopodia to a more discoid platelet shape. PKC and autocrine ADP signaling contribute to persistent integrin αIIbβ3 activation in the order of PAR1/GPVI > PAR4 stimulation and hence to stabilized platelet aggregation. These findings are relevant for optimization of effective antiplatelet treatment.
PubMed: 38911141
DOI: 10.1055/s-0044-1786987 -
Platelets Dec 2024Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass...
Receptor-induced tyrosine phosphorylation of spleen tyrosine kinase (Syk) has been studied extensively in hematopoietic cells. Metabolic mapping and high-resolution mass spectrometry, however, indicate that one of the most frequently detected phosphorylation sites encompassed S297 (S291 in mice) located within the linker B region of Syk. It has been reported that Protein kinase C (PKC) phosphorylates Syk S297, thus influencing Syk activity. However, conflicting studies suggest that this phosphorylation enhances as well as reduces Syk activity. To clarify the function of this site, we generated Syk S291A knock-in mice. We used platelets as a model system as they possess Glycoprotein VI (GPVI), a receptor containing an immunoreceptor tyrosine-based activation motif (ITAM) which transduces signals through Syk. Our analysis of the homozygous mice indicated that the knock-in platelets express only one isoform of Syk, while the wild-type expresses two isoforms at 69 and 66 kDa. When the GPVI receptor was activated with collagen-related peptide (CRP), we observed an increase in functional responses and phosphorylations in Syk S291A platelets. This potentiation did not occur with AYPGKF or 2-MeSADP, although they also activate PKC isoforms. Although there was potentiation of platelet functional responses, there was no difference in tail bleeding times. However, the time to occlusion in the FeCl injury model was enhanced. These data indicate that the effects of Syk S291 phosphorylation represent a significant outcome on platelet activation and signaling but also reveals its multifaceted nature demonstrated by the differential effects on physiological responses .
Topics: Animals; Syk Kinase; Blood Platelets; Mice; Signal Transduction; Phosphorylation; Immunoreceptor Tyrosine-Based Activation Motif; Gene Knock-In Techniques; Humans; Platelet Membrane Glycoproteins; Platelet Activation
PubMed: 38904212
DOI: 10.1080/09537104.2024.2369766 -
BMC Medical Genomics Jun 2024Mediators, genomic and epigenomic characteristics involving in metabolism of arachidonic acid by cyclooxygenase (COX) and lipoxygenase (ALOX) and hepatic activation of...
BACKGROUND
Mediators, genomic and epigenomic characteristics involving in metabolism of arachidonic acid by cyclooxygenase (COX) and lipoxygenase (ALOX) and hepatic activation of clopidogrel have been individually suggested as factors associated with resistance against aspirin and clopidogrel. The present multi-center prospective cohort study evaluated whether the mediators, genomic and epigenomic characteristics participating in arachidonic acid metabolism and clopidogrel activation could be factors that improve the prediction of the aspirin and clopidogrel resistance in addition to cardiovascular risks.
METHODS
We enrolled 988 patients with transient ischemic attack and ischemic stroke who were evaluated for a recurrence of ischemic stroke to confirm clinical resistance, and measured aspirin (ARU) and P2Y12 reaction units (PRU) using VerifyNow to assess laboratory resistance 12 weeks after aspirin and clopidogrel administration. We investigated whether mediators, genotypes, and promoter methylation of genes involved in COX and ALOX metabolisms and clopidogrel activation could synergistically improve the prediction of ischemic stroke recurrence and the ARU and PRU levels by integrating to the established cardiovascular risk factors.
RESULTS
The logistic model to predict the recurrence used thromboxane A synthase 1 (TXAS1, rs41708) A/A genotype and ALOX12 promoter methylation as independent variables, and, improved sensitivity of recurrence prediction from 3.4% before to 13.8% after adding the mediators, genomic and epigenomic variables to the cardiovascular risks. The linear model we used to predict the ARU level included leukotriene B4, COX2 (rs20417) C/G and thromboxane A2 receptor (rs1131882) A/A genotypes with the addition of COX1 and ALOX15 promoter methylations as variables. The linear PRU prediction model included G/A and prostaglandin I receptor (rs4987262) G/A genotypes, COX2 and TXAS1 promoter methylation, as well as cytochrome P450 2C19*2 (rs4244285) A/A, G/A, and *3 (rs4986893) A/A genotypes as variables. The linear models for predicting ARU (r = 0.291, R = 0.033, p < 0.01) and PRU (r = 0.503, R = 0.210, p < 0.001) levels had improved prediction performance after adding the genomic and epigenomic variables to the cardiovascular risks.
CONCLUSIONS
This study demonstrates that different mediators, genomic and epigenomic characteristics of arachidonic acid metabolism and clopidogrel activation synergistically improved the prediction of the aspirin and clopidogrel resistance together with the cardiovascular risk factors.
TRIAL REGISTRATION
URL: https://www.
CLINICALTRIALS
gov ; Unique identifier: NCT03823274.
Topics: Humans; Clopidogrel; Male; Female; Aspirin; Drug Resistance; Middle Aged; Aged; Epigenomics; Genomics; Prospective Studies; Platelet Aggregation Inhibitors; DNA Methylation
PubMed: 38902747
DOI: 10.1186/s12920-024-01936-1 -
PloS One 2024The underlying causes of breast cancer are diverse, however, there is a striking association between type 2 diabetes and poor patient outcomes. Platelet activation is a...
Platelet-derived microvesicles isolated from type-2 diabetes mellitus patients harbour an altered miRNA signature and drive MDA-MB-231 triple-negative breast cancer cell invasion.
The underlying causes of breast cancer are diverse, however, there is a striking association between type 2 diabetes and poor patient outcomes. Platelet activation is a common feature of both type 2 diabetes and breast cancer and has been implicated in tumourigenesis through a multitude of pathways. Here transcriptomic analysis of type 2 diabetes patient-derived platelet microvesicles revealed an altered miRNA signature compared with normoglycaemic control patients. Interestingly, interrogation of these data identifies a shift towards an oncogenic signature in type 2 diabetes-derived platelet microvesicles, with increased levels of miRNAs implicated in breast cancer progression and poor prognosis. Functional studies demonstrate that platelet microvesicles isolated from type 2 diabetes patient blood are internalised by triple-negative breast cancer cells in vitro, and that co-incubation with type 2 diabetes patient-derived platelet microvesicles led to significantly increased expression of epithelial to mesenchymal transition markers and triple-negative breast cancer cell invasion compared with platelet microvesicles from healthy volunteers. Together, these data suggest that circulating PMVs in type 2 diabetes patients may contribute to the progression of triple-negative breast cancer.
Topics: Humans; Triple Negative Breast Neoplasms; Diabetes Mellitus, Type 2; Female; Blood Platelets; MicroRNAs; Cell-Derived Microparticles; Cell Line, Tumor; Neoplasm Invasiveness; Middle Aged; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic
PubMed: 38900754
DOI: 10.1371/journal.pone.0304870 -
Circulation Jun 2024There is significant variability in the efficacy and safety of oral P2Y12 inhibitors, which are used to prevent ischemic outcomes in common diseases such as coronary and... (Review)
Review
There is significant variability in the efficacy and safety of oral P2Y12 inhibitors, which are used to prevent ischemic outcomes in common diseases such as coronary and peripheral arterial disease and stroke. Clopidogrel, a prodrug, is the most used oral P2Y12 inhibitor and is activated primarily after being metabolized by a highly polymorphic hepatic cytochrome CYP2C219 enzyme. Loss-of-function genetic variants in are common, can result in decreased active metabolite levels and increased on-treatment platelet aggregation, and are associated with increased ischemic events on clopidogrel therapy. Such patients can be identified by genetic testing and can be treated with alternative therapy. Conversely, universal use of potent oral P2Y12 inhibitors such as ticagrelor or prasugrel, which are not dependent on CYP2C19 for activation, has been recommended but can result in increased bleeding. Recent clinical trials and meta-analyses have demonstrated that a precision medicine approach in which loss-of-function carriers are prescribed ticagrelor or prasugrel and noncarriers are prescribed clopidogrel results in reducing ischemic events without increasing bleeding risk. The evidence to date supports genetic testing before oral P2Y12 inhibitors are prescribed in patients with acute coronary syndromes or percutaneous coronary intervention. Clinical implementation of such genetic testing will depend on among multiple factors: rapid availability of results or adoption of the concept of performing preemptive genetic testing, provision of easy-to-understand results with therapeutic recommendations, and seamless integration in the electronic health record.
PubMed: 38899464
DOI: 10.1161/CIR.0000000000001257 -
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