Aging involves morphological and functional changes across different organs, but how these changes are linked among the different organs remains to be elucidated. Here, we uncover a central role of platelets in systemic aging. In aged mice, the levels of platelet-secreted pro-inflammatory factors (PSPF) increased greatly in the serum and platelets, leading to a diffuse increase of platelet infiltration in the brain, liver, lung, kidney, and aortic root. The RNA-binding protein HuR/ELAVL1, a major regulator of RNA metabolism, promoted the production of PSPF in platelets. Platelet-specific deletion of HuR reduced the expression of PSPF in platelets, alleviated platelet infiltration in the brain, liver, lung, kidney, and aortic root, and delayed systemic aging. By using single-nucleus sequencing, platelet-specific HuR ablation was found to alleviate p53 and pro-inflammatory signaling pathways in liver, lung, and brain tissues in aged mice. Our findings highlight a role of platelets in coordinating aging traits across organs.

Rationale:Acrosomal developmental defects are associated with severe sperm morphogenetic abnormalities and male infertility; however, the specific molecular determinants underlying these defects remain poorly defined. Endosome/lysosome-associated apoptosis and autophagy regulator 1 (ELAPOR1) is important for cellular membrane dynamics and organelle functions, indicating that it plays an essential role in spermatogenesis, which needs further investigation. This study aimed to explore the specific role of ELAPOR1 in spermatogenesis and male fertility. Methods:Single-cell RNA-sequencing datasets from human testes were obtained to investigate the expression of ELAPOR1 in specific cell types. An Elapor1 germ cell-specific knockout mouse line (Elapor1cKO ) was established, and the morphology and function of the testis and sperm were assessed through breeding capacity, fertilization capacity, testicular histology, sperm staining, concentration and motility, immunofluorescence, quantitative PCR, immunoblotting, and transmission electron microscopy analyses. Moreover, mass spectrometry and enrichment analyses were employed to identify the proteins that interact with ELAPOR1. The interactions were verified through proximity labeling, coimmunoprecipitation, and immunofluorescence staining. Results:ELAPOR1 is highly expressed and its protein colocalized with the acrosome during the early stage of acrosome formation. Elapor1cKO mice produced deformed sperm with decreased concentration, impaired motility, and defective fertilization capacity. Moreover, ELAPOR1 deficiency led to impaired fusion of proacrosomal vesicles. Mechanistically, ELAPOR1 functioned in regulating the transport of the Golgi and early endosome-related vesicles. It interacted with VPS54 and affected VPS54-associated assembly of the GARP complex in the testis. Conclusions:Our findings reveal the essential role of ELAPOR1 in acrosome formation during spermatogenesis and male fertility. ELAPOR1 potentially influences the trafficking, integration, and fusion of proacrosomal vesicles through VPS54-mediated GARP complex assembly. These findings provide novel insights into the interaction of the ELAPOR1-GARP complex in acrosome-related reproductive failure, suggesting that Elapor1 deficiency or mutation could be considered a potential genetic risk factor for human infertility.

Pulmonary thrombosis is a fatal complication observed in patients following severe trauma or pulmonary infections. Patients with existing heart failure (HF) are more susceptible to infection-induced lung thrombosis; however, the underlying mechanisms remain poorly understood. This study introduces a severe mouse pulmonary thrombosis model utilizing CD40 knockout (KO) mice following transverse aortic constriction (TAC)-induced HF. While CD40 KO was found to have no detectable effect on left ventricular (LV) structure or function in mice either after TAC or under control conditions, the CD40 KO mice developed profound pulmonary micro-thrombosis after TAC. CD40 deficiency also significantly exacerbated TAC-induced pulmonary leukocyte infiltration (such as total CD45+ cells, Mac2+ cells and CD3+ cells), ∼1.7-fold more pulmonary fibrosis, and pulmonary vessel remodeling, as well as the consequent right ventricular hypertrophy. Mechanistically, our findings indicate that CD40 KO significantly enhanced TAC-induced pulmonary oxidative stress and pulmonary vascular endothelial cell activation, as indicated by upregulated vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 expression (ICAM-1). Moreover, CD40 KO and TAC synergistically augmented thrombin (49.1±3.0 in CD40 KO TAC group vs 27.2±4.8 in WT TAC group), collagen (51.4±2.5 in CD40 KO TAC group vs 37.8±2.4 in WT TAC group), and ADP-induced platelet aggregation (40.1±2.1 in CD40 KO TAC group vs 31.0±1.8 in WT TAC group) and blood clot contraction(0.92±0.01 in CD40 KO TAC group vs 0.72±0.03 in WT TAC group) in mice. Furthermore, mild but significant pulmonary micro-thrombosis and increased blood clot retraction (0.80±0.02 in CD40 KO sham group vs 0.66±0.02 in WT sham group) were also observed in CD40 KO mice under baseline (control) conditions. Collectively, these results demonstrate that the profound pulmonary micro-thrombosis observed in CD40 KO mice is the outcome of a synergistic effect involving an inherent platelet defect in CD40 KO mice, combined with HF-induced pulmonary endothelial oxidative stress, endothelial activation, and systemic platelet activation. This unique lung micro-thrombosis model may serve as a useful tool for investigating the mechanisms and therapeutic strategies for pulmonary micro-thrombosis, particularly under conditions of existing HF.

Nanoplastics (NPs) are emerging environmental pollutants with potential health risks, yet their role in cancer progression remains poorly understood. Here, this is demonstrated that oral exposure to 100 nm polystyrene nanoplastics (PS-NPs) accelerates tumor initiation and metastasis in a murine triple-negative breast cancer (TNBC) model, without affecting primary tumor growth. PS-NPs do not directly alter TNBC cell behavior in vitro but induced gut microbiota dysbiosis, characterized by Alloprevotella enrichment and elevated systemic glutamate levels, both identified as key mediators of PS-NPs-driven tumor promotion. Moreover, PS-NPs enhanced platelet activation, evidenced by increased aggregation, microthrombus formation at metastatic sites, and upregulation of CD36 and Serpine1. Collectively, these findings uncover a synergistic mechanism whereby oral PS-NPs promote TNBC progression via a gut microbiota-derived metabolite-platelet axis, establishing an unrecognized link between environmental nanoplastic exposure and cancer progression, and highlighting potential therapeutic targets for intervention.

Certain secretory proteins are known to be critical for maintaining the stemness of stem cells through autocrine signaling. However, the processes underlying the biogenesis, maturation, and secretion of these proteins remain largely unknown. Here we demonstrate that many secretory proteins produced by hematopoietic stem cells (HSCs) undergo exosomal maturation and release that is controlled by vacuolar protein sorting protein 33b (VPS33B). Deletion of VPS33B in either mouse or human HSCs resulted in impaired exosome maturation and secretion as well as loss of stemness. Additionally, VPS33B deficiency led to a dramatic delay in leukemogenesis. Exosomes purified from either conditioned medium or human plasma could partially rescue the defects of HSCs and leukemia-initiating cells (LICs). VPS33B co-existed in exosomes with GDI2, VPS16B, FLOT1, and other known exosome markers. Mechanistically, VPS33B interacted with the GDI2/RAB11A/RAB27A pathway to regulate the trafficking of secretory proteins as exosomes. These findings reveal an essential role for VPS33B in exosome pathways in HSCs and LICs. Moreover, they shed light on the understanding of vesicle trafficking in other stem cells and on the development of improved strategies for cancer treatment.

BACKGROUND:Mounting evidence indicates that nuclear receptors play a critical regulatory role in platelet pathophysiology and thrombotic disorders. Although NR4A (the nuclear receptor subfamily 4 group A) plays an important role in cardiovascular pathophysiology, the expression profile and biological function of NR4A member 1 (NR4A1) in platelets have never been reported. METHODS:We evaluated the functions and the underlying mechanisms of NR4A1 in platelet activation and thrombus formation using platelet-specific NR4A1-deficient mice and NR4A1-specific agonists. Using a hyperlipidemic mouse model and platelets from patients with hypercholesterolemia, we explored the influence of hypercholesterolemia on NR4A1 expression and the effects of NR4A1-specific agonists on platelet hyperreactivity induced by hypercholesterolemia. RESULTS:NR4A1 was expressed in both human and mouse platelets. Platelet-specific NR4A1 deletion accelerated FeCl3-induced carotid arterial occlusive thrombus formation, enhanced collagen/epinephrine-induced pulmonary thromboembolism, and exacerbated microvascular microthrombi obstruction and infarct expansion in an acute myocardial infarction model. NR4A1-deficient platelets exhibited enhanced agonist-induced aggregation responses, integrin αIIbβ3 activation, dense granule release, α-granule release, platelet spreading, and clot retraction. Consistently, pharmacological activation of NR4A1 by specific agonists decreased platelet activation in both mouse and human platelets. Mechanistically, CAP1 (adenylyl cyclase-associated protein 1) was identified as the direct downstream interacting protein of NR4A1. NR4A1 deletion decreased cAMP levels and phosphorylation of VASP (vasodilator-stimulated phosphoprotein), while NR4A1-specific agonists increased cAMP levels and phosphorylation of VASP in platelets. Importantly, NR4A1 expression in platelets was upregulated in the setting of hypercholesterolemia, which was derived from its upregulation in megakaryocytes in a reactive oxygen species-dependent manner. Platelets from hypercholesterolemic patients and mice exhibited hyperreactivity. However, NR4A1-specific agonists significantly inhibited the activation of hypercholesterolemic platelets to the levels of healthy control platelets. CONCLUSIONS:We provide the first evidence that nuclear receptor NR4A1 negatively regulates platelet activation and thrombus formation. NR4A1 may serve as a novel therapeutic target for managing thrombosis-based cardiovascular diseases, especially with hypercholesterolemia.

A comprehensive understanding of the evolution of the immune landscape in humans across the entire lifespan at single-cell transcriptional and protein levels, during development, maturation and senescence is currently lacking. We recruited a total of 220 healthy volunteers from the Shanghai Pudong Cohort (NCT05206643), spanning 13 age groups from 0 to over 90 years, and profiled their peripheral immune cells through single-cell RNA-sequencing coupled with single T cell and B cell receptor sequencing, high-throughput mass cytometry, bulk RNA-sequencing and flow cytometry validation experiments. We revealed that T cells were the most strongly affected by age and experienced the most intensive rewiring in cell–cell interactions during specific age. Different T cell subsets displayed different aging patterns in both transcriptomes and immune repertoires; examples included GNLY+CD8+ effector memory T cells, which exhibited the highest clonal expansion among all T cell subsets and displayed distinct functional signatures in children and the elderly; and CD8+ MAIT cells, which reached their peaks of relative abundance, clonal diversity and antibacterial capability in adolescents and then gradually tapered off. Interestingly, we identified and experimentally verified a previously unrecognized ‘cytotoxic’ B cell subset that was enriched in children. Finally, an immune age prediction model was developed based on lifecycle-wide single-cell data that can evaluate the immune status of healthy individuals and identify those with disturbed immune functions. Our work provides both valuable insights and resources for further understanding the aging of the immune system across the whole human lifespan. In this Resource, authors profile peripheral immune cells from a total of 220 healthy volunteers from birth to over 90 years. This revealed that T cells were most affected by aging with divergent aging patterns in different subsets and identified a population of cytotoxic B cells that were enriched in children.

Lymphatic vessels (LVs) interdigitated with blood vessels, travel and form an extensive transport network in the musculoskeletal system. Blood vessels in bone regulate osteogenesis and hematopoiesis, however, whether LVs in bone affect fracture healing is unclear. Here, by near infrared indocyanine green lymphatic imaging (NIR-ICG), we examined lymphatic draining function at the tibial fracture sites and found lymphatic drainage insufficiency (LDI) occurred as early as two weeks after fracture. Sufficient lymphatic drainage facilitates fracture healing. In addition, we identified that lymphatic platelet thrombosis (LPT) blocks the draining lymphoid sinus and LVs, caused LDI and then inhibited fracture healing, which can be rescued by a pharmacological approach. Moreover, unblocked lymphatic drainage decreased neutrophils and increased M2-like macrophages of hematoma niche to support osteoblast (OB) survival and bone marrow-derived mesenchymal stem cell (BMSC) proliferation via transporting damage-associated molecular patterns (DAMPs). These findings demonstrate that LPT limits bone regeneration by blocking lymphatic drainage from transporting DAMPs. Together, these findings represent a novel way forward in the treatment of bone repair.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease without effective pharmacological approaches. The nuclear hormone receptor LXRα (liver X receptor α), encoded by the NR1H3 gene, serves as a critical transcriptional mediator linked to several vascular pathologies, but its role in AAA remains elusive. METHODS: Through integrated analyses of human and murine AAA gene expression microarray data sets, we identified NR1H3 as a candidate gene regulating AAA formation. To investigate the role of LXRα in AAA formation, we used global Nr1h3 -knockout and vascular smooth muscle cell–specific Nr1h3 -knockout mice in 2 AAA mouse models induced with angiotensin II (1000 ng·kg·min; 28 days) or calcium chloride (CaCl 2 ; 0.5 mol/L; 42 days). RESULTS: Upregulated LXRα was observed in the aortas of patients with AAA and in angiotensin II– or CaCl 2 -treated mice. Global or vascular smooth muscle cell–specific Nr1h3 knockout inhibited AAA formation in 2 mouse models. Loss of LXRα function prevented extracellular matrix degeneration, inflammation, and vascular smooth muscle cell phenotypic switching. Uhrf1 , an epigenetic master regulator, was identified as a direct target gene of LXRα by integrated analysis of transcriptome sequencing and chromatin immunoprecipitation sequencing. Susceptibility to AAA development was consistently enhanced by UHRF1 (ubiquitin-like containing PHD and RING finger domains 1) in both angiotensin II– and CaCl 2 -induced mouse models. We then determined the CpG methylation status and promoter accessibility of UHRF1-mediated genes using CUT&Tag (cleavage under targets and tagmentation), RRBS (reduced representation bisulfite sequencing), and ATAC-seq (assay for transposase-accessible chromatin with sequencing) in vascular smooth muscle cells, which revealed that the recruitment of UHRF1 to the promoter of miR-26b led to DNA hypermethylation accompanied by relatively closed chromatin states, and caused downregulation of miR-26b expression in AAA. Regarding clinical significance, we found that underexpression of miR-26b-3p correlated with high risk in patients with AAA. Maintaining miR-26b-3p expression prevented AAA progression and alleviated the overall pathological process. CONCLUSIONS: Our study reveals a pivotal role of the LXRα/UHRF1/miR-26b-3p axis in AAA and provides potential biomarkers and therapeutic targets for AAA.

CD44 is associated with a high risk of metastasis, recurrence, and drug resistance in various cancers. Here we report that platelet endothelial aggregation receptor 1 (PEAR1) is a CD44 chaperone protein that protected CD44 from endocytosis-mediated degradation and enhances cleavage of the CD44 intracellular domain (CD44-ICD). Furthermore, we found that lysyl oxidase-like protein 2 (LOXL2), an endogenous ligand of PEAR1, bound to the PEAR1-EMI domain and facilitated the interaction between PEAR1 and CD44 by inducing PEAR1 Ser891 phosphorylation in a manner that was independent of its enzyme activity. Levels of PEAR1 protein and PEAR1 phosphorylation at Ser891 were increased in patients with triple-negative breast cancer (TNBC), were positively correlated with expression of LOXL2 and CD44, and were negatively correlated with overall survival. The level of PEAR1 Ser891 phosphorylation was identified as the best independent prognostic factor in TNBC patients. The prognostic efficacy of the combination of PEAR1 phosphorylation at Ser891 and CD44 expression was superior to that of PEAR1 phosphorylation at Ser891 alone. Blocking the interaction between LOXL2 and PEAR1 with monoclonal antibodies significantly inhibited TNBC metastasis, representing a promising therapeutic strategy for TNBC.

Megakaryocytes (MKs) are large cells derived from hematopoietic stem cells (HSCs) through a process of differentiation and maturation. Mature MKs extend long processes called proplatelets, which are released into bone marrow sinusoids under the shear forces of blood flow. These proplatelets also form platelets in the lungs. Platelets are small cell fragments that play crucial roles in hemostasis, thrombosis, and immunity. The process of platelet generation from megakaryocytes is intricately orchestrated by non-coding RNAs, such as miRNAs. Another non-coding RNA, circRNA, is highly abundant in platelets, but its role in platelet production remains unclear. We performed RNA-seq and bioinformatics analysis to identify circRNAs during the differentiation of megakaryocytes from umbilical cord blood hematopoietic stem cells. We found that circFUT8, a novel circRNA, increases with megakaryocyte differentiation and is enriched in mature megakaryocytes. Knockdown and overexpression studies showed that circFUT8 promotes human megakaryocyte proplatelet formation (PPF) in vitro. We also found that circFUT8 is highly conserved between humans and mice. In vitro, knockdown of circFUT8 decreases mouse megakaryocyte PPF. In vivo, it reduces murine platelet counts, prolongs tail bleeding time, and reduces the number of MKs in contact with sinusoids. Furthermore, knockdown of circFUT8 reduces F-actin polymerization and impairs spreading on collagen. Additionally, we revealed that circFUT8 interacts with insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) and stabilizes tensin 1 (TNS1) mRNA. We observed that knockdown of TNS1 disrupts the actin cytoskeleton and decreases PPF. Our study highlights the crucial functions of circRNAs in platelet production and the rearrangement of the actin cytoskeleton.

Impaired differentiation of megakaryocytes constitutes the principal etiology of thrombocytopenia. The signal transducer and activator of transcription 3 (STAT3) is a crucial transcription factor in regulating megakaryocyte differentiation, however the precise mechanism of its activation remains unclear. PALLD, an actin-associated protein, has been increasingly recognized for its essential functions in multiple biological processes. This study revealed that megakaryocyte/platelet-specific knockout of Palld in mice exhibited thrombocytopenia due to diminished platelet biogenesis. In megakaryocytes, PALLD deficiency led to impaired proplatelet formation and polyploidization, ultimately weakening their differentiation for platelet production. Mechanistic studies demonstrated that PALLD bound to STAT3 and interacted with its DNA-binding domain and Src homology 2 domain via immunoglobulin domain 3. Moreover, the absence of PALLD attenuated STAT3 Y705 phosphorylation and impeded STAT3 nuclear translocation. Based on the PALLD-STAT3 binding sequence, we designed a peptide C-P3, which can facilitate megakaryocyte differentiation and accelerate platelet production in vivo. In conclusion, this study highlights the pivotal role of PALLD in megakaryocyte differentiation and proposes a novel approach for treating thrombocytopenia by targeting the PALLD-STAT3 interaction.

Breakthrough treatment for refractory and relapsed immune thrombocytopenia (ITP) patients is urgently needed. Autoantibody- mediated platelet clearance and megakaryocyte dysfunction are important pathogenic mediators of ITP. Glycoprotein (GP) Ibα is a significant autoantigen found in ITP patients and is associated with poor response to standard immunosuppressive treatments. Here, we engineered human T cells to express a chimeric autoantibody receptor (CAAR) with GPIbα constructed into the ligand-binding domain fused to the CD8 transmembrane domain and CD3ζ-4-1BB signaling domains. We performed cytotoxicity assays to assess GPIbα CAAR T-cell selective cytolysis of cells expressing anti-GPIbα B-cell receptors in vitro. Furthermore, we demonstrated the potential of GPIbα CAAR T cells to persist and precisely eliminate GPIbα-specific B cells in vivo. In summary, we present a proof of concept for CAAR T-cell therapy to eradicate autoimmune B cells while sparing healthy B cells with GPIbα CAAR T cells that function like a Trojan horse. GPIbα CAAR T-cell therapy is a promising treatment for refractory and relapsed ITP patients.

Abstract Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbβ, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.

BACKGROUND:Snake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. OBJECTIVES:To explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. METHODS:The effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic, ITGA2B and ITGB3-deficient mice, and Bernard-Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbβ3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. RESULTS:Botrocetin could induce aggregation of platelets from a Bernard-Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbβ3 and facilitated αIIbβ3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbβ3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbβ3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbβ3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. CONCLUSION:Our study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbβ3 and GPIbα.

Aims MicroRNA-126 (miR-126), one of the most abundant microRNAs in platelets, is involved in the regulation of platelet activity and the circulating miR-126 is reduced during antiplatelet therapy. However, whether intraplatelet miR-126 plays a role in thrombosis and platelet inhibition remains unclear. Methods and results Here, using tissue-specific knockout mice, we reported that the deficiency of miR-126 in platelets and vascular endothelial cells significantly prevented thrombosis and prolonged bleeding time. Using chimeric mice, we identified that the lack of intraplatelet miR-126 significantly prevented thrombosis. Ex vivo experiments further demonstrated that miR-126-deficient platelets displayed impaired platelet aggregation, spreading, and secretory functions. Next, miR-126 was confirmed to target phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2) in platelet, which encodes a negative regulator of the phosphoinositide 3-kinase/protein kinase B pathway, enhancing platelet activation through activating the integrin αIIbβ3-mediated outside-in signalling. After undergoing myocardial infarction (MI), chimeric mice lacking intraplatelet miR-126 displayed reduced microvascular obstruction and prevented MI expansion in vivo. In contrast, overexpression of miR-126 by the administration of miR-126 agonist (agomiR-126) in wild-type mice aggravated microvascular obstruction and promoted MI expansion, which can be almost abolished by aspirin administration. In patients with cardiovascular diseases, antiplatelet therapies, either aspirin alone or combined with clopidogrel, decreased the level of intraplatelet miR-126. The reduction of intraplatelet miR-126 level was associated with the decrease in platelet activity. Conclusion Our murine and human data reveal that (i) intraplatelet miR-126 contributes to platelet activity and promotes thrombus formation, and (ii) the reduction of intraplatelet miR-126 contributes to platelet inhibition during antiplatelet therapy.

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets, bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody, which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification, Elisa assay for antigen binding, cytotoxicity assays, T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially, BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover, PHE1 domain was further shortened with D14G and R41S mutations, named PHE-S, and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo, bringing more possibilities for the development and optimization of different bsAbs. To sum up, PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy.

Portal vein tumor thrombosis (PVTT), a severe complication of hepatocellular carcinoma (HCC), markedly influences patient prognosis by fostering a hypercoagulable state. However, its molecular underpinnings remain largely unexplored. This study sheds light on the critical role of the KIT ligand (KITLG) in modulating expression of the collagen gene COL4A1 via the STAT3-SMAD2 signaling pathway, thereby influencing platelet activation and PVTT development. Extensive analysis of PVTT tissue samples, alongside in vitro and in vivo experiments including cell-platelet interaction assays and PVTT animal models, revealed the mechanism through which KITLG regulates COL4A1 expression, as well as its downstream effects on platelet behavior and the coagulation cascade. Our findings revealed that marked upregulation of COL4A1 expression, mediated by KITLG through the STAT3-SMAD2 pathway, led to increased platelet activation and PVTT formation. KITLG and COL4A1 expression was markedly higher in PVTT tissues than primary HCC tissues, thus highlighting their critical role in the pathophysiological trajectory leading to thrombosis. The finding that the KITLG-COL4A1 signaling axis is a crucial mediator in PVTT development may offer promising new directions for developing targeted diagnostic and therapeutic strategies. This study underscores the importance of the KITLG-COL4A1 axis in PVTT formation and its potential as a therapeutic target in HCC treatment protocols.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has a global prevalence of about 25% and no approved therapy. Using metabolomic and proteomic analyses, we identified high expression of hepatic transketolase (TKT), a metabolic enzyme of the pentose phosphate pathway, in human and mouse MAFLD. Hyperinsulinemia promoted TKT expression through the insulin receptor-CCAAT/enhancer-binding protein alpha axis. Utilizing liver-specific TKT overexpression and knockout mouse models, we demonstrated that TKT was sufficient and required for MAFLD progression. Further metabolic flux analysis revealed that Tkt deletion increased hepatic inosine levels to activate the protein kinase A-cAMP response element binding protein cascade, promote phosphatidylcholine synthesis, and improve mitochondrial function. Moreover, insulin induced hepatic TKT to limit inosine-dependent mitochondrial activity. Importantly, N-acetylgalactosamine (GalNAc)-siRNA conjugates targeting hepatic TKT showed promising therapeutic effects on mouse MAFLD. Our study uncovers how hyperinsulinemia regulates TKT-orchestrated inosine metabolism and mitochondrial function and provides a novel therapeutic strategy for MAFLD prevention and treatment.