Mouse Platelets Research Articles

Platelet RNA sequencing reveals profile of caffeic acid affecting hemostasis in mice

BackgroundCaffeic acid (CA) is a naturally occurring phenolic compound with diverse pharmacologic properties. CA plays a crucial role in hemostasis by increasing platelet count. However, the mechanism by which CA regulates platelets to promote hemostasis remains unclear. ObjectivesWe aim to identify the potential target pathways and genes by which CA regulates platelets to promote hemostasis. MethodsWe performed RNA sequencing (RNA-seq) analysis of mouse platelet pools in both the CA-gavaged group and phosphate-buffered saline–gavaged group. ResultsThe 12,934 expressed transcripts had been annotated after platelet RNA-seq. Compared with the phosphate-buffered saline group, 987 differentially expressed genes (DEGs) were identified, of which 466 were downregulated and 521 were upregulated in CA group. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Reactome gene set enrichment analysis demonstrated that upregulated DEGs were enriched in the pathways of hemostasis, platelet activation, signaling, aggregation, and degranulation. Moreover, Kyoto Encyclopedia of Genes and Genomes and Reactome gene set enrichment analysis revealed that 5 of the 25 cosignificantly upregulated DEGs were essential in CA-mediated platelet regulation to promote hemostasis. ConclusionOur findings of platelet RNA-seq analysis demonstrate that CA regulates the gene expression of hemostasis and platelet activation–related pathways to increase platelet count and promote hemostasis. It will also provide reference molecular resources for future research on the function and mechanism by which CA regulates platelets to promote hemostasis.

Myosin light chain 6 (Myl6) interacts with kindlin-3 and is required to support integrin αIIbβ3 activation in platelets in mice

BackgroundKindlin-3 in platelets plays an essential role in supporting integrin αIIbβ3 activation, platelet spreading, aggregation, and clot retraction by binding to the integrin β3 cytoplasmic tail. However, the mechanism by which kindlin-3 mediates the crosstalk between integrin αIIbβ3 and myosin in platelets remains unknown. ObjectivesTo examine the role of myosin light chain 6 (Myl6) in supporting integrin αIIbβ3 activation in platelets. MethodsMyl6fl/flPF4-Cre mice with a deficiency of Myl6 in the megakaryocyte lineage were generated, and integrin αIIbβ3 activation in Myl6-deficient platelets was analyzed. ResultsWe identified a novel kindlin-3 binding protein, Myl6, an essential light chain of myosin in platelets. Myl6fl/flPF4-Cre mice exhibited significant macrothrombocytopenia resulting from defective proplatelet formation. In the absence of Myl6, integrin αIIbβ3 activation in platelets was significantly suppressed, and platelet aggregation was substantially impaired. Interestingly, the deficiency of Myl6 in platelets preferentially affected the binding of a multivalent ligand compared to a monovalent ligand to integrin αIIbβ3 upon activation, indicating that Myl6 may contribute to the avidity modulation of integrin αIIbβ3 by binding to kindlin-3. Furthermore, blood coagulation ability was impaired in Myl6fl/flPF4-Cre mice, and consistently, these mice exhibited defects in both hemostatic and thrombotic functions. ConclusionIn summary, these results suggest that Myl6, as a novel kindlin-3 binding partner, is required to support integrin αIIbβ3 activation in platelets, which plays an important role in both hemostasis and thrombosis.

EcoHIV-Infected Mice Show No Signs of Platelet Activation.

Platelets express several surface receptors that could interact with different viruses. To understand the mechanisms of HIV-1's interaction with platelets, we chose the EcoHIV model. While EcoHIV is an established model for neuroAIDS, its effects on platelets are ill-defined. Our results indicate that EcoHIV behaves differently from HIV-1 and is cleared from circulation after 48 h post-infection. The EcoHIV course of infection resembles an HIV-1 infection under the effects of combined antiretroviral therapy (cART) since infected mice stayed immunocompetent and the virus was readily detected in the spleen. EcoHIV-infected mice failed to become thrombocytopenic and showed no signs of platelet activation. One explanation is that mouse platelets lack the EcoHIV receptor, murine Cationic Amino acid Transporter-1 (mCAT-1). No mCAT-1 was detected on their surface, nor was any mCAT-1 mRNA detected. Thus, mouse platelets would not bind or become activated by EcoHIV. However, impure virus preparations, generated by Polyethylene Glycol (PEG) precipitation, do activate platelets, suggesting that nonspecific PEG-precipitates may contain other platelet activators (e.g., histones and cell debris). Our data do not support the concept that platelets, through general surface proteins such as DC-SIGN or CLEC-2, have a wide recognition for different viruses and suggest that direct platelet/pathogen interactions are receptor/ligand specific.

The Function of ASK1 in Sepsis and Stress-Induced Disorders.

Apoptosis signal-regulating kinase 1 (ASK1) is a serine-threonine kinase that is ubiquitously expressed in nucleated cells and is responsible for the activation of multiple mitogen-activated protein kinases (MAPK) to regulate cell stress. Activation of ASK1 via cellular stress leads to activation of downstream signaling components, activation of transcription factors, and proinflammatory cytokine production. ASK1 is also expressed in anucleate platelets and is a key player in platelet activation as it is important for signaling. Interestingly, the mechanism of ASK1 activation is cell type-dependent. In this review we will explore how ASK1 regulates a variety of cellular processes from innate immune function to thrombosis and hemostasis. We will discuss how ASK1 influences FcγRIIA-mediated platelet reactivity and how that reactivity drives platelet clearance. Furthermore, we will explore the role of ASK1 in thromboxane (TxA2) generation, which highlights differences in the way ASK1 functions in mouse and human platelets.

Viral uptake and pathophysiology of the lung endothelial cells in age-associated severe SARS-CoV-2 infection models.

Thrombosis is the major cause of death in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and the pathology of vascular endothelial cells (ECs) has received much attention. Although there is evidence of the infection of ECs in human autopsy tissues, their detailed pathophysiology remains unclear due to the lack of animal model to study it. We used a mouse-adapted SARS-CoV-2 virus strain in young and mid-aged mice. Only mid-aged mice developed fatal pneumonia with thrombosis. Pulmonary ECs were isolated from these infected mice and RNA-Seq was performed. The pulmonary EC transcriptome revealed that significantly higher levels of viral genes were detected in ECs from mid-aged mice with upregulation of viral response genes such as DDX58 and IRF7. In addition, the thrombogenesis-related genes encoding PLAT, PF4, F3 PAI-1, and P-selectin were upregulated. In addition, the inflammation-related molecules such as CXCL2 and CXCL10 were upregulated in the mid-aged ECs upon viral infection. Our mouse model demonstrated that SARS-CoV-2 virus entry into aged vascular ECs upregulated thrombogenesis and inflammation-related genes and led to fatal pneumonia with thrombosis. Current results of EC transcriptome showed that EC uptake virus and become thrombogenic by activating neutrophils and platelets in the aged mice, suggesting age-associated EC response as a novel finding in human severe COVID-19.

Radiolabeling of Platelets with 99mTc-HYNIC-Duramycin for In Vivo Imaging Studies

Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.

Clearance of Trehalose Lyophilized Platelets in Mice

Abstract The utilization of stored platelet transfusion has emerged as an effective approach in the management of thrombocytopenia. However, the limited availability of fresh platelets in veterinary medicine due to the challenging storage requirements poses a significant constraint. Lyophilized platelets offer extended shelf-life and convenient storage options, enhancing their accessibility for thrombocytopenic patients. However, only a limited number of studies have examined the clearance and survival rate of transfused lyophilized platelets, particularly regarding the lyophilization technique utilizing trehalose as a platelet stabilization agent. The objective of this study was to assess the recovery and survival rate of trehalose lyophilized platelets within the circulatory system. To investigate these parameters, CMFDA-labeled lyophilized platelets were administered to mice, and their recovery and survival rates were analyzed. Flow cytometric analysis revealed the rapid clearance of lyophilized platelets from the systemic circulation. The immediate post-infusion percent recovery of labeled platelet particles was 42.7 ± 8.15 %. The average survival rates at post-infusion time points at 15, 30, 45, and 60 minutes were 28.2 ± 4.31, 14.5 ± 3.56, 5.1 ± 2.02, and 0.82 ± 0.57, respectively. The calculated mean half-life was 8.39 ± 0.44 minutes. The most pronounced decrease in labeled lyophilized platelet count occurred during the 30-minute timeframe immediately following infusion. Subsequently, over 99% of lyophilized platelets were eliminated after 60 minutes post-infusion. These findings indicate that higher dosages and more frequent administration of trehalose lyophilized platelets might be necessary to achieve a therapeutic effect comparable to that of fresh platelets.

Platelets from Ehlers-Danlos Syndrome Patients Exhibit Reduced GPVI Levels and Impaired Integrin αIIbβ3 inside-out Signaling

Background: Patients with Ehlers-Danlos syndrome (EDS) have a high susceptibility to bleeding. Easy bruising is included as a criterion for the classification of multiple types of EDS. Although the association between EDS and the tendency to bleed has been acknowledged for a long time, a definitive understanding of the underlying mechanisms contributing to bleeding diathesis in EDS patients is still lacking. Objective: To characterize platelet function in a cohort of EDS patients and a murine EDS model. Methods: A cohort of 53 patients with hypermobile (49%), classical (26%), classical-like (21%), or vascular (4%) types of EDS and a group of 53 healthy control subjects matched by age and gender were included in the study in accordance with the University of Iowa Institutional Review Board-approved protocol. The International Society of Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) was used to assess the bleeding tendency in study participants. Because a single mutation in hypermobile type is currently unknown, we used Col5a 1 +/− mice as a murine model of the classical type of EDS. The function of human and mouse platelets was evaluated using standardized agonist-induced in vitro assays. Results: The mean ISTH-BAT score was 0.1 in healthy controls and 9.2 in EDS patients ( P < 0.001). An abnormal ISTH-BAT score was observed in 33 out of 53 (62%) EDS patients and 0 of 53 healthy controls ( P < 0.001). Platelets from EDS patients exhibited reduced aggregation and αIIbβ3 activation (with normal αIIbβ3 surface exposure) in response to stimulation with collagen, collagen-related peptide, or thrombin receptor activator peptide-6, compared to healthy controls. Platelet secretion of dense and α-granules and surface phosphatidylserine exposure were comparable between EDS and healthy subjects. Evaluation of collagen receptors on the surface of resting platelets showed a mild deficiency of GPVI in EDS patients compared to healthy controls ( P < 0.001), while the integrin α2 levels were similar in both groups ( P = 0.152). Analysis of GPVI signaling revealed downregulation of Syk (Y525/526), PLCγ2 (Y1217), and talin (S425) phosphorylation in EDS patients ( P < 0.05 vs. healthy controls), suggesting a defect in collagen-induced integrin αIIbβ3 inside-out signaling. Consistent with our observations in EDS patients, Col5a1 +/− mice demonstrated a bleeding tendency characterized by prolonged tail-bleeding time ( P < 0.05 vs. wild-type control). Platelet function testing showed that Col5a 1 +/− mouse platelets recapitulated the abnormalities detected in human EDS platelets. Conclusions: Our data corroborate the observation that EDS patients exhibit a high risk of hemorrhagic complications.EDS platelets are characterized by reduced baseline GPVI expression that was associated with decreased agonist-induced platelet aggregation, αIIbβ3 activation, and impaired collagen-induced integrin αIIbβ3 inside-out signaling.

Upregulation of Platelet Transcripts As Biomarkers of Fibrotic Progression in Myeloproliferative Neoplasms

Myeloproliferative neoplasms (MPN) are a group of clonal hemopoietic disorders that have a proliferative phase (including essential thrombocythemia (ET) and polycythemia vera (PV)), and which can progress to advanced disease such as myelofibrosis (MF) or acute leukemia. This is thought to be due in part to progressively dysfunctional megakaryocytes driving reticulin deposition in the bone marrow. In previous work, we demonstrated that the platelet transcriptome in MPN differs from normal, and the difference is most marked with increased reticulin (Guo et al., 2020). A putative 3-gene signature ( CCND1, H2AFX and CEP55) could discriminate between patients with and without increased reticulin fibrosis. To progress this, we have profiled the transcriptome of platelets from a further 173 MPN patients were analyzed using next generation sequencing. CCND1, H2AFX and CEP55 were upregulated in platelets from patients with MF compared to ET (4.6-fold, padj<0.001, 9.0-fold, padj<0.001 and 10.5-fold, padj<0.001, respectively) and to PV (3.9-fold, padj<0.001, 9.2-fold, padj<0.001, 12.0-fold, padj<0.001). Application of the established 3-gene signature had a positive predictive value of 78%, a negative prediction value of 90% and accuracy of 88% for MF. To assess the temporal changes in these gene transcripts in platelets and the potential utility to predict fibrotic progression, we then performed longitudinal analyses in two MPN mouse models. Jak2V617F knock-in mice ( n=25) with stable serially transplantable disease, which resembles PV (Mullally et al., 2010), were sacrificed at 4, 8, 12, 16 and 20 weeks post-transplant, and controls ( n=5) at 20 weeks. Blood counts were performed, and spleen and sternum harvested and stained with hematoxylin and eosin, and for reticulin. By 20-weeks, the platelet and leucocyte counts and hemoglobin had increased as expected. There was splenomegaly with extramedullary hematopoiesis and a mild increase in marrow reticulin. There was no significant up-regulation of Ccnd1, H2afx or Cep55 in platelet transcripts at any timepoint compared to the control mice. This data showed stability of the mouse PV phenotype and platelet transcripts, in keeping with the chronic phase of MPN. Mice with MPLW515L ( n=8) (Pikman et al., 2006) were sacrificed at 3 and 6 weeks post-transplant, and control mice ( n=4) at 6 weeks. At 3-weeks post-transplant Cep55 (131.6-fold, padj<0.001) and H2afx (1.7-fold, padj<0.01) had increased in the MPLW515L mice and there was no increase in reticulin in the bone marrow. At 6-weeks platelet and leucocyte counts had increased, there was splenomegaly with extramedullary hematopoiesis including marked megakaryocytosis and marrow reticulin. On platelet transcript analysis Cep55 (154.6-fold, padj<0.001) and H2afx (2.4-fold, padj<0.001) were significantly increased; there was no significant change for Ccnd1. Upregulation of Cep55 and H2afx in the MPLW515L mouse occurred prior to the accumulation of bone marrow reticulin and development of MF phenotype. This suggests that up-regulation of Cep55 and H2afx in platelets may be a predictor of disease progression from ET and PV to MF. For MPN patients, a deviation from steady-state platelet transcript levels, specifically for H2AFX and CEP55, may infer a biological transition towards the fibrotic phenotype. This novel approach utilizing platelet transcript analysis may therefore have potential for serially monitoring MPN patients and the early identification of genomic changes that may herald disease transformation to MF.

Adipose-Derived Mesenchymal Stem Cell Facilitate Hematopoietic Stem Cell Proliferation via the Jagged-1/Notch-1/Hes Signaling Pathway.

Poor graft function (PGF) is a life-threatening complication following hematopoietic stem cell transplantation (HSCT). Current therapies, such as CD34+ cell infusion, have shown limited effectiveness. Conversely, mesenchymal stem cells (MSCs) show potential in addressing PGF. Adipose-derived mesenchymal stem cells (ADSCs) effectively support long-term hematopoietic stem cell proliferation. Therefore, this study aimed to investigate the mechanisms underlying the long-term hematopoietic support provided by ADSCs. ADSCs were isolated from mice and subsequently identified. In vitro experiments involved coculturing ADSCs as feeders with Lin-Sca-1+c-kit+ (LSK) cells from mice for 2 and 5 weeks. The number of LSK cells was quantified after coculture. Scanning electron microscopy was utilized to observe the interaction between ADSCs and LSK cells. Hes-1 expression was assessed using western blot and real-time quantitative PCR. An γ-secretase inhibitor (GSI) was used to confirm the involvement of the Jagged-1/Notch-1/Hes-1 pathway in LSK cell expansion. Additionally, Jagged-1 was knocked down in ADSCs to demonstrate its significance in ADSC-mediated hematopoietic support. In vivo experiments were conducted to study the hematopoietic support provided by ADSCs through the infusion of LSK, LSK + fibroblasts, and LSK + ADSCs, respectively. Mouse survival, platelet count, leukocyte count, and hemoglobin levels were monitored. ADSCs showed high-Jagged-1 expression and promoted LSK cell proliferation. There was a direct interaction between ADSCs and LSK cells. After coculture, Hes-1 expression increased in LSK cells. Moreover, GSI-reduced LSK cell proliferation and Hes-1 expression. Knockdown of Jagged-1 attenuated ADSCs-mediated promotion of LSK cell proliferation. Furthermore, ADSCs facilitated hematopoietic recovery and promoted the survival of NOD/SCID mice. The hematopoietic support provided by ADSCs both in vivo and in vitro may be mediated, at least in part, through the Jagged-1/Notch-1 signaling pathway. These findings provide valuable insights into the mechanisms underlying ADSCs-mediated hematopoietic support and may have implications for improving the treatment of PGF following HSCT.

Superiority of CS585 as a selective prostacyclin receptor agonist in prevention of thrombosis

Abstract Background A major challenge with development of new targets for antithrombotic treatment is the sustained effect on the platelet and the target selectivity. While increasing cAMP in the platelet through activation of the prostacyclin (IP) receptor represents a viable antiplatelet target for prevention of thrombosis, current IP receptor agonists lack stability and selectivity. Development of a selective IP receptor agonist that is stable in the blood and selective for the IP receptor would represent a new approach for prevention of platelet activation and thrombosis in the blood and expand the utility of IP receptor agonists which are currently used for the treatment of pulmonary arterial hypertension (PAH). Purpose Develop an IV and oral IP receptor agonist, CS585, with sustained activity in vivo in the blood and eliminating the off-target effects observed with other IP agonists not having selective activation of the IP receptor. These characteristics would make CS585 an attractive new therapeutic approach for limiting platelet activation and thrombosis. Methods We assessed platelet activation and thrombosis in human blood ex vivo and mouse models in vivo. For mouse models, platelets and fibrin were labelled and accumulation at the site of injury was measured using intravital microscopy. Thrombosis in the vessel was assessed in the laser-induced cremaster thrombosis assay. Selectivity was assessed in human blood pharmacologically using prostaglandin receptor inhibitors and in mouse with an IP receptor knockout mouse. Flow cytometry, aggregometry, and western blot techniques were used to assess selectivity. Results IV and oral administration of CS585 resulted in sustained inhibited clot formation and fibrin formation at the site of injury in the laser-induced cremaster arteriole thrombosis assay in wild-type mice but was unable to alter platelet function in IP knockout mice. CS585 inhibition of platelet activation was fully prevented in platelets from IP knockout mice as assessed by flow cytometry (integrin activation and granule secretion) and by VASP phosphorylation. Inhibition of human platelet activation by CS585 was inhibited by pharmacological inhibition of the IP receptor, but not by inhibition of the DP1, EP2, or EP4 receptors as assessed by aggregation, flow cytometry, and VASP phosphorylation. Conclusions We have shown for the first time that CS585, a novel IP receptor agonist, not only inhibits platelet activation and clot formation, but gives sustained inhibition through multiple routes of administration in an in vivo mouse model of thrombosis. Importantly, the challenge in selectivity observed with other IP receptor agonists is not observed with CS585 supporting its development for a number of diseases in the cardiovascular system including thrombosis, VTE, secondary prevention after MI and stroke, and PAH.CS585 inhibition of thrombosis in vivoCS585 selectively signals through IP

Platelets Sequester Extracellular DNA, Capturing Tumour-Derived and Free Fetal DNA

Introduction & Hypothesis Platelets are small, multi-functional cells that lack a nucleus but contain RNA and translational machinery for protein synthesis. Platelet RNA mostly derives from parent megakaryocytes, but they also sense and sequester endogenous and pathogen-derived nucleic acids during circulation (D'Ambrosi, 2021; Koupenova, 2019). Nucleated cells release DNA after cell death or aberrant mitosis, resulting in ‘cell-free’ DNA in plasma (cfDNA). Excess cfDNA is deleterious. cfDNA isolated from platelet-poor plasma is emerging as a major liquid biopsy tool in cancer and antenatal screening, but a major limitation is its low abundance, especially in early-stage disease. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets may clear cfDNA from plasma, and that clinically-relevant insights may be derived from the analysis of DNA fragments contained in platelets. Methods Live/fixed imaging using specific DNA probes and fluorescence in-situ hybridization (FISH), droplet digital PCR (ddPCR), NGS and flow cytometry were applied to platelets isolated from Streck, EDTA tubes or apheresis bags from healthy donors, pregnant women, patients with cancer and murine platelets from cancer models. DNA was extracted from platelet pellets or platelet-depleted plasma (cfDNA) using the Qiagen QIAamp Circulating Nucleic Acid Kit. Results Using live cell imaging and flow cytometry, significant DNA content (Draq5+) was detected in ~8% of platelets from healthy donors, including in platelets with low RNA content (SYTO-13-negative). Overall Draq5+ platelets were larger and primed for agonist activation. FISH and ddPCR of platelets from pregnant women carrying male fetuses detected Y-chromosome fragments (n=10), confirming that platelet DNA is not solely derived from parent megakaryocytes but also sequestered during circulation (Fig. 1a). In contrast, Y-chromosome genes were not detected in DNA isolated from mononuclear cells or red blood cells pellets. Acute platelet depletion in an ITP mouse model (n=20) led to a >2-fold increase in cfDNA extracted from platelet-poor plasma, suggesting a role for platelets in plasma DNA clearance. Platelet uptake in vitro was rapid, visible using live cell microscopy within 2 minutes of co-culture with cancer cells labelled with a probe that irreversibly intercalates to nuclear DNA. ddPCR of platelet DNA following co-culture of healthy donor platelets with cancer cell lines detected a range of canonical cancer driver mutations, including in PI3K, BRAF and JAK2. DNase treatment of co-cultured platelets did not reduce mutant allele content, indicating that platelets encapsulate DNA and protect it from degradation. We explored the mechanism of platelet DNA uptake. Platelets internalized DNA contained in extracellular vesicles (EVs). Platelet uptake of “free” DNA was also confirmed, with uptake of synthetic DNA fragments of varying lengths (120 bp to 650 bp). Importantly, platelet DNA detection was enhanced following exocytosis inhibition, confirming that platelet DNA uptake and release is an active process. Whole genome sequencing revealed that platelets contain a repertoire of DNA fragments that map across the nuclear genome, similar to cfDNA, and that the majority of platelet DNA is of nuclear not mitochondrial origin. We also found tumor-derived DNA showing multiple copy number aberrations in a patient with pancreatic carcinoma. In a transgenic mouse model of colorectal cancer, mutant KRASG12D alleles were readily detectable in platelet DNA and, notably, in higher abundance in platelets than platelet-poor plasma (cfDNA) in 11/16 mice (Fig. 1b). Similarly, KRASG12D was detected in platelets of mice with orthotopic pancreatic adenocarcinoma (n=4). Finally, to explore utility in cancer screening, we analysed patients with high-risk, pre-malignant colonic lesions (serrated polyps). Remarkably, the driver mutation BRAFV600E was detected in platelets in 16% patients despite the small-size lesions (5/30, Fig.1b). Conclusions This study establishes a role for platelets in sequestration of cfDNA, an aspect of platelet biology that has not previously been highlighted and is of substantial clinical relevance. Their abundance, ease of isolation, and continuous tissue perfusion make platelets ideal ‘sentinels’ for genetic perturbations including in early stage/pre-malignant disease.

Inhibition of cancer cell‑platelet adhesion as a promising therapeutic target for preventing peritoneal dissemination of gastric cancer.

Platelets form complexes with gastric cancer (GC) cells via direct contact, enhancing their malignant behavior. In the present study, the molecules responsible for GC cell-platelet interactions were examined and their therapeutic application in inhibiting the peritoneal dissemination of GC was investigated. First, the inhibitory effects of various candidate surface molecules were investigated on platelets and GC cells, such as C-type lectin-like receptor 2 (CLEC-2), glycoprotein VI (GPVI) and integrin αIIbβ3, in the platelet-induced enhancement of GC cell malignant potential. Second, the therapeutic effects of molecules responsible for the development and progression of GC were investigated in a mouse model of peritoneal dissemination. Platelet-induced enhancement of the migratory ability of GC cells was markedly inhibited by an anti-GPVI antibody and inhibitor of galectin-3, a GPVI ligand. However, neither the CLEC-2 inhibitor nor the integrin-blocking peptide significantly suppressed this enhanced migratory ability. In experiments using mouse GC cells and platelets, the migratory and invasive abilities enhanced by platelets were significantly suppressed by the anti-GPVI antibody and galectin-3 inhibitor. Furthermore, in vivo analyses demonstrated that the platelet-induced enhancement of peritoneal dissemination was significantly suppressed by the coadministration of anti-GPVI antibody and galectin-3 inhibitor, and was nearly eliminated by the combined treatment. The inhibition of adhesion resulting from GPVI-galectin-3 interaction may be a promising therapeutic strategy for preventing peritoneal dissemination in patients with GC.

Age-Dependent Surface Receptor Expression Patterns in Immature Versus Mature Platelets in Mouse Models of Regenerative Thrombocytopenia.

Aging is a multifaceted process that unfolds at both the individual and cellular levels, resulting in changes in platelet count and platelet reactivity. These alterations are influenced by shifts in platelet production, as well as by various environmental factors that affect circulating platelets. Aging also triggers functional changes in platelets, including a reduction in RNA content and protein production capacity. Older individuals and RNA-rich immature platelets often exhibit hyperactivity, contributing significantly to pathologic conditions such as cardiovascular diseases, sepsis, and thrombosis. However, the impact of aging on surface receptor expression of circulating platelets, particularly whether these effects vary between immature and mature platelets, remains largely unexplored. Thus, we investigated the expression of certain surface and activation receptors on platelets from young and old mice as well as on immature and mature platelets from mouse models of regenerative thrombocytopenia by flow cytometry. Our findings indicate that aged mice show an upregulated expression of the platelet endothelial cell adhesion molecule-1 (CD31), tetraspanin-29 (CD9), and Toll-like receptor 2 (TLR2) compared to their younger counterparts. Interestingly, when comparing immature and mature platelets in both young and old mice, no differences were observed in mature platelets. However, immature platelets from young mice displayed higher surface expression compared to immature platelets from old mice. Additionally, in mouse models of regenerative thrombocytopenia, the majority of receptors were upregulated in immature platelets. These results suggest that distinct surface receptor expressions are increased on platelets from old mice and immature platelets, which may partially explain their heightened activity and contribute to an increased thrombotic risk.

Molecular mechanocytometry using tension-activated cell tagging.

Flow cytometry is used routinely to measure single-cell gene expression by staining cells with fluorescent antibodies and nucleic acids. Here, we present tension-activated cell tagging (TaCT) to label cells fluorescently based on the magnitude of molecular force transmitted through cell adhesion receptors. As a proof-of-concept, we analyzed fibroblasts and mouse platelets after TaCT using conventional flow cytometry.

PB0397 Differential Requirement for Ligand Valency in the Activation of CLEC-2 in Human and Humanised Mouse Platelets

PB0397 Differential Requirement for Ligand Valency in the Activation of CLEC-2 in Human and Humanised Mouse Platelets

OC 22.1 Proteome and Phosphoproteome Analyses of SARS-CoV-2 Infected Humanized ACE2 Mice Platelets Revealed Reprogramming of Platelets from Early Hyperreactive to Late Antiviral Phenotypes

OC 22.1 Proteome and Phosphoproteome Analyses of SARS-CoV-2 Infected Humanized ACE2 Mice Platelets Revealed Reprogramming of Platelets from Early Hyperreactive to Late Antiviral Phenotypes

Essential role of CD38 in platelet aggregation through the PKC-mediated internalization and activation.

CD38 is a multifunctional enzyme with a potent Ca2+ mobilizing effect, cyclic ADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP). Here, we aimed to demonstrate the role of CD38 in platelets via protein kinase C (PKC)-mediated internalization and activation. Mouse platelets were used in this study. Thrombin, an agonist of platelet function, provoked a prompt and long-lasting increase in intracellular Ca2+ concentration ([Ca2+]i), resulting from an interplay of multifold Ca2+ mobilizing messengers.The signaling pathway was delineated using different inhibitors and techniques such as platelet aggregation assay, intracellular calcium measurements, immunoprecipitation, immunoblotting, and flow cytometry. We observed a sequential formation of cADPR and NAADP through CD38 activation by PKC of non-muscle myosin heavy chain IIA (MHCIIA), resulting in phospholipase C (PLC) activation in the thrombin-stimulated platelets. These findings reveal that PKC is fundamental in activating CD38 and elicits a physiological response in the murine platelets. PKC is involved in many signaling pathways. Specifically, PKC is involved in the internalization of CD38 via MHCIIA in CD38+/+ wild-type (WT) and CD38-/- knockout mice (KO). CD38 generates calcium-mobilizing agents that act on specific receptors of the calcium stores. Calcium triggered platelet aggregation while serving as a secondary messenger.

Phosphoinositides take a central stage in regulating blood platelet production and function

Blood platelets are produced by megakaryocytes through a complex program of differentiation and play a critical role in hemostasis and thrombosis. These anucleate cells are the target of antithrombotic drugs that prevent them from clumping in cardiovascular disease conditions. Platelets also significantly contribute to various aspects of physiopathology, including interorgan communications, healing, inflammation, and thromboinflammation. Their production and activation are strictly regulated by highly elaborated mechanisms. Among them, those involving inositol lipids have drawn the attention of researchers. Phosphoinositides represent the seven combinatorially phosphorylated forms of the inositol head group of inositol lipids. They play a crucial role in regulating intracellular mechanisms, such as signal transduction, actin cytoskeleton rearrangements, and membrane trafficking, either by generating second messengers or by directly binding to specific domains of effector proteins. In this review, we will explore how phosphoinositides are implicated in controlling platelet production by megakaryocytes and in platelet activation processes. We will also discuss the diversity of phosphoinositides in platelets, their role in granule biogenesis and maintenance, as well as in integrin signaling. Finally, we will address the discovery of a novel pool of phosphatidylinositol 3-monophosphate in the outerleaflet of the plasma membrane of human and mouse platelets.

Immunological Role of Megakaryocytes and Platelets during Influenza A Virus Infection

Influenza viruses pose a serious threat to public health, with severe cases often characterized by lung damage and inflammation. However, the underlying mechanisms of these processes remain poorly understood. This study aimed to investigate the essential role of megakaryocytes (MKs) and platelets (PLTs) in influenza A virus (IAV) infections. Conducted at the Department of Rare Respiratory Diseases, Cystic Fibrosis, and Pulmonology, Nord University Hospital, Marseille, France, the study collected seventy blood samples between October 2018 and March 2019. Samples were obtained from healthy individuals and patients diagnosed with IAV. Messenger RNA was extracted from isolated PLTs and subjected to quantitative real-time-polymerase chain reaction using sets of primers targeting immune marker genes. Western blot analysis was also performed for confirmation, focusing on Fas Ligand (FasL). Results showed that PLTs from IAV-infected individuals expressed the FasL, tumor necrosis factor-related apoptosis-inducing ligand, and Granulysin (GNLY) receptors when activated. Furthermore, an in vitro assay revealed the presence of FasL receptors on infected CMK cell lines. In vivo investigations demonstrated that activated MKs and PLTs in mice also expressed FasL. Interestingly, none of the immune receptors under investigation were found in both MKs and PLTs in mouse model studies. In conclusion, MKs and PLTs play a significant role in influencing immune responses that may help prevent viral spread during infection. However, further examination of their mechanisms of action is warranted. Understanding the involvement of these cells in influenza pathogenesis could offer valuable insights for developing potential therapeutic strategies.