Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent mitochondrial deacetylase that regulates protein acetylation and maintains mitochondrial homeostasis in nucleated cells. By deacetylating cyclophilin D (CypD), SIRT3 limits mitochondrial permeability transition pore (mPTP) opening and protects against mitochondrial dysfunction. Although SIRT3 is present in murine and human platelets, its contribution to platelet mitochondrial regulation and procoagulant platelet formation remains unknown. This study investigated whether platelet SIRT3 modulates CypD-dependent procoagulant platelet formation. Platelets obtained from platelet-specific Sirt3 knockout mice (Sirt3[plt-/-]) and littermate controls (Sirt3[plt+/+]) were analyzed under resting conditions and after activation with CRP-XL and thrombin. Flow cytometry was used to analyze platelet (activation) markers and procoagulant platelet formation, and mitochondrial respiration was assessed using a Seahorse extracellular flux analyzer. Platelets lacking SIRT3 showed no alterations in basal or agonist-stimulated mitochondrial respiration. Likewise, platelet Sirt3 deletion did not affect the generation of procoagulant platelets in response to strong dual agonist stimulation. These findings indicate that, despite its role in regulating mPTP opening in nucleated cells, platelet SIRT3 is not required for procoagulant platelet formation, suggesting that platelets rely on distinct mechanisms for mPTP regulation.

Metabolic regulation is increasingly recognized as an important determinant of megakaryocyte development and platelet production, yet the metabolite-level characteristics of primary megakaryocytes from distinct developmental stages and tissue origins remain incompletely defined. Here, using microscale targeted metabolomics, we profiled primary megakaryocytes isolated from mouse liver and bone marrow. This analysis revealed distinct metabolomic profiles between the two megakaryocyte populations and identified spermidine and lysine as metabolites enriched in liver-derived megakaryocytes. Functional analyses showed that spermidine, but not lysine, promoted fetal liver-derived megakaryocyte generation and platelet-like particle formation in vitro, whereas these effects were not observed in adult bone marrow-derived cultures under the tested conditions. Maternal dietary spermidine supplementation increased fetal liver megakaryocyte generation and fetal platelet production in vivo, with limited detectable effects on megakaryopoiesis in the maternal bone marrow. Single-cell transcriptomic analysis and pharmacological perturbation further linked endogenous spermidine biosynthesis to fetal liver megakaryopoiesis. Inhibition of Odc1 or Amd1 reduced intracellular spermidine levels, impaired fetal liver-derived megakaryocyte differentiation and platelet-like particle formation, and exogenous spermidine partially rescued DFMO-induced defects. Together, these findings define distinct metabolic features of mouse liver- and bone marrow-derived megakaryocytes and support a role for endogenous spermidine biosynthesis in fetal liver megakaryopoiesis and platelet production.

Considerable attention has been paid steadily for efficacy and safety of herb extracts with medicinal benefits, recently. Tranexamic acid (TXA) is a well-known hemostatic agent with strong efficacy but potential thrombus risk. Here, we identified oleanolic acid (OA) as a strong enhancer in promoting adhesion, spreading or aggregation of platelets on immobilized collagen, which exceeds TXA among six herb extracts. In addition, the study demonstrated that TXA could facilitate morphological changes of platelets on collagen, accelerate and enhance intracellular calcium bursting with and without mechanical stimulus, which might cause an excessive hemostatic response and lead to thrombus. While OA exerted a comparable effect specifically under low-shear flow regimes, providing a flow-assisted chemical activation that bypasses the need for high-magnitude mechanical tension but ensures localized efficacy. In summary, this study identified OA as a herbal coagulant that showed hemostatic efficacy similar to TXA, but with a better safety profile. It provided a new insight into the flow-assisted mechanism underlying hemostatic efficiency and should be useful in finding novel and safe hemostatic drugs and in developing a practical method for functional identification of hemostatic agents.

Platelet-erythrocyte (PE) complexes, composed of single erythrocytes bound to platelets, are a physiological component of the bloodstream and contribute to erythrocyte turnover. Conditions in which PE complex production is increased or their clearance from the circulation is impeded are associated with hypercoagulable states and thrombocytopenia. Sepsis is a serious acute inflammatory condition that is often complicated by major hemostatic derangements, including thrombosis, anemia and thrombocytopenia, and an important driver of sepsis pathology are extracellular histones, which are released into the circulation from activated neutrophils. Histone-neutralizing small polyanions (SPAs) have been developed to block the cytotoxic effects of histones and are beneficial in treating sepsis. This study investigated whether histones may stimulate increased PE complex production as a potential cause of the hemostatic complications in disease, and whether SPAs can prevent complex generation. Treatment with histones in co-incubated human erythrocytes and platelets, or murine blood, increased PE complex production approximately 10-fold compared with control conditions. Mice administered histones by intravenous injection displayed reduced platelet counts but sustained complex levels, suggesting complex production was enhanced within the diminished platelet pool. The platelets in the histone-stimulated complexes were also more activated than cell-free platelets, according to increased surface expression of P-selectin and the fibrin-binding form of integrin αIIbβ3, determined using flow cytometry, and their altered shape and presence of filopodia observed by scanning electron microscopy. Inclusion of SPAs in the histone-treated cells and blood prevented histone-stimulated production of complexes, indicating the importance of the histone-mediated cell injury in their generation and identifying PE complexes as a treatment target.

Efgartigimod, a neonatal Fc receptor (FcRn) antagonist, represents a novel therapeutic approach for refractory immune thrombocytopenia (ITP). However, its clinical positioning and durability of response in real-world settings remain unclear. We describe a case of refractory ITP in which efgartigimod induced a partial response that was enhanced by the subsequent addition of a thrombopoietin receptor agonist (TPO-RA), resulting in durable remission after efgartigimod discontinuation. Subsequent hematologic workup revealed that platelet recovery was accompanied by a reciprocal shift in immature platelet indices, with immature platelet fraction percentage decreasing and absolute immature platelet fraction increasing. Although there is a paucity of literature on immature platelet indices during ITP treatment, these parameters may serve as useful biomarkers for monitoring therapeutic response. Notably, efgartigimod was discontinued after 22 weeks, with sustained response maintained for an additional 24 weeks on TPO-RA monotherapy. This treatment course highlights the potential of FcRn inhibition to induce durable immunomodulatory effects and demonstrates synergistic benefits with TPO-RAs-insights that extend beyond the scope of conventional clinical trial protocols.

Platelet activation is energy-dependent and associated with a hyperglycolytic phenotype, yet the role of key glycolytic enzymes remains unclear. We investigated the role of platelet hexokinases (HK1 and HK2) in regulating thrombin- and convulxin-stimulated bioenergetics, functions, and hemostasis using pharmacological inhibitors 2-deoxyglucose (2DG; pan-HK) and 3-bromopyruvate (3BP; HK2-preferential). Platelet activation increased HK activity, with convulxin producing a stronger response than thrombin. Using 2DG and 3BP, we found that activation-induced glucose uptake, glycolytic ATP production, and glycolysis were dependent on both HK isoforms. Platelet aggregation was reduced but not abolished by HK inhibition, indicating that platelet function is underpinned by metabolic flexibility. Integrin activation and pro-coagulant platelet formation were suppressed by both inhibitors. In contrast, α-granule secretion markers (CD62P, CD40L), platelet spreading, and convulxin-induced reactive oxygen species (ROS) generation were more sensitive to 3BP, suggesting a prominent role for HK2 in secretion, outside-in signaling, and ROS regulation. In vitro thrombus formation over collagen was significantly reduced by both inhibitors. In addition, HKs play a pivotal role in the regulation of in vivo hemostasis with increased bleeding time in mice treated with HK inhibitor. These findings identify HK as a central regulator of platelet metabolism and function, with distinct contributions of HK isoforms to specific platelet responses.

The chemiluminescence resonance energy transfer (CRET)-based method was applied to the detect of CD61 and CD62P (P-selectin) on the surface of platelet in patients with acute myocardial infarction (AMI) and healthy controls. Patients admitted to the Department of Emergency in The First Affiliated Hospital of Soochow University from May 2025 to December 2025 were recruited. We studied 22 AMI patients who were matched with 22 controls. The coefficient of variations and standard curves of indexes were calculated. Expressions of CD61 and CD62P in plasma were evaluated by CRET assay. Standard curve property for CD61 and CD62P: R2 .995 and 0.9999. The coefficient of variation in CRET system was 3.39%, 1.76% (QC high), 6.25% and 7.16% (QC low) for CD61 and CD62P, respectively. CRET data showed the agonist (arachidonic acid and ADP) concentration dependency on platelet activation. AMI patients had higher basal CD62P levels when compared to controls. The platelet aggregation rate of AMI patients had a strong positive correlation with the expression of CD62P (r = 0.8354, p < .0001). In the healthy controls, the platelet CD61 and CD62P expression detected by flow cytometry showed a positive correlation with that evaluated by CRET (r = 0.4296, p = .0460; r = 0.5662, p = .0060, respectively). After platelet activation with AA or ADP, the expression levels of CD62P detected by flow cytometry were positively correlated with those measured by the CRET method (r = 0.6195, p = .0021; r = 0.7663, p < .0001, respectively). Expressions of CD62P elevated after agonist stimulation in AMI patients and controls in vitro. With the biomarker of CD62P as the model, the Area Under Curve (AUC) was 0.9236 (p < .0001). The cutoff value of CD62P to evaluate platelet reactivity by CRET was 3.985%. This one-step CERT assay provided a new approach to detect platelet activity of acute myocardial infarction.

Platelet spreading assays are widely used to assess platelet adhesion, cytoskeletal remodeling and receptor‑specific signaling, but current GPVI ligands such as Collagen Related Peptide (CRP‑XL) suffer from non‑defined structure, batch variability, and high cost. Nanobodies targeting GPVI offer an attractive alternative due to their defined stoichiometry and recombinant production. Here, we evaluated whether GPVI‑binding nanobodies can support GPVI‑dependent platelet spreading. Using automated image analysis, we compared spreading on monovalent Nb2, trivalent Nb2‑3 and CRP‑XL. Monovalent Nb2 supported full spreading in only a subset of platelets, with reduced adhesion compared to CRP‑XL. In contrast, trivalent Nb2‑3 induced platelet adhesion and spreading indistinguishable from CRP‑XL, with comparable morphology, actin organization and spread area at concentrations ≥0.1 µg mL-1. Spreading on Nb2‑3 was inhibited by soluble Nb2 and by Src kinase blockade, confirming GPVI‑dependent signaling, while the non‑blocking nanobody Nb28 had no inhibitory effect. Notably, Nb28 alone supported spreading, demonstrating that ligand‑induced GPVI clustering, rather than binding site location, drives activation. These findings show that Nb2‑3 is a potent and reproducible alternative to CRP‑XL, offering a well‑defined and standardizable reagent for GPVI‑dependent static platelet spreading assays.

Platelets are small, anucleate cells with a primary physiological role in vascular damage repair (hemostasis) and initiation of thrombus formation in response to vascular injury. Platelets circulate approximately 7-10 days, slowly undergoing age-related changes in molecular composition, morphology, activation capacity, function, and surface receptor density. As older platelets are associated with poor clinical outcome, no in vitro tests are available to predict platelet age, or to determine the fitness of platelet transfusion products. In this study, we developed a convolutional neural network model that could determine platelets' chronological age from confocal microscopic images. The model was trained using platelets stored in platelet-rich plasma up to 8 hours and using routine platelet concentrates up to 10 days. The model predicted chronological age of stored platelets with >97% accuracy. To test our model in vivo, we analyzed a cohort of patients with acute myeloid leukemia, experiencing thrombocytopenia due to chemotherapy. Our model could reliably distinguish in vivo between samples with younger and older platelets during the course of treatment. This study demonstrates the ability to predict platelets' chronological age both in vitro during storage and in vivo, which may impact clinical transfusion medicine and the diagnosis and treatment of patients with platelet disorders.

Studies investigating the role of differential platelet function across various ancestry groups have been ongoing since at least the middle twentieth century. Consistently identified trends include differential frequency of CD36 deficiencies, variability in the efficacy of the antiplatelet functions of clopidogrel, increased PAR4 reactivity observed among African ancestry individuals, and reduced ristocetin-induced platelet aggregation also observed in African ancestry. Nonetheless, consensus is lacking in many areas. Replication in many of these studies is poor, utilized models may lack the necessary covariates to effectively capture environmental effects, and the represented cohorts disproportionately lean North American. Further comparative research of platelet phenotypes may yield important clinical insights and benefits; though to accomplish this a collaborative, international effort between institutes would be required. In this review, we discuss what is currently understood regarding differential platelet reactivity between ancestry groups, what areas are inadequately characterized, and the technical and organizational aspects which should be considered in future research.

Bone maintains a dynamic and stable state through the orchestration of osteoclasts and osteoblasts. Osteoblasts are derived mainly from mesenchymal stem cell (MSC) and are responsible for bone formation. The inhibition of osteoblast proliferation and differentiation is involved in many diseases, including osteoporosis, osteoarthritis, infected bone defects, and inflammatory aseptic loosening of implants. Given the currently limited treatment options, exploring new methods to promote bone formation is an important focus significant for orthopedists. Platelet-rich plasma (PRP), an autologous substance that is rich in various growth factors, is widely used in regenerative medicine. However, the effect of PRP on inflammatory bone destruction remains unclear. We investigated the effects of PRP on the viability of MSC, and the impact of different concentrations of PRP (1% and 3%, respectively) on cell death, proliferation, and differentiation. Furthermore, we tested the therapeutic effect of different concentrations of PRP (1% and 3%) on LPS-induced inflammatory bone destruction in vivo. PRP enhanced the cellular activity of MSC and promoted osteogenesis. A higher concentration of PRP (3%) primarily reduced the death and increased the proliferation of in LPS-treated MSC via the PI3K/AKT pathway, while a lower concentration of PRP (1%) promoted MSC differentiation into osteoblasts through the MAPK pathway. Consistent with in vitro experiments, we validated the protective effect of PRP against LPS-induced bone loss by increasing bone formation in vivo. These results suggest that different concentrations of PRP can ameliorate LPS-induced inflammatory bone loss through distinct mechanisms.

Thrombopoietin receptor agonists (TPO-RAs) represent a cornerstone in immune thrombocytopenia (ITP) management, yet their molecular mechanisms remain incompletely elucidated. This study systematically deciphered the key targets and signaling networks of four TPO-RAs (romiplostim, eltrombopag, avatrombopag, hetrombopag) in ITP pathogenesis. Network pharmacology was integrated with single-cell high-dimensional weighted gene co-expression network analysis (hdWGCNA) using bone marrow scRNA-seq data from ITP patients and healthy controls. Metacell-based co-expression modules to hematopoietic bone marrow cells were identified. Drug targets were curated from multiple databases, and candidate genes were screened by intersecting differentially expressed genes (DEGs), cell specific modules, and TPO-RA targets. Molecular docking, pseudotime trajectory analysis, and in silico gene knockdown were employed for functional validation. Intersection analysis revealed five key genes (CACNA1A, CSF1R, PKN1, CD9, DSTYK). Molecular docking demonstrated strong binding affinities between TPO-RAs and key targets. The ITP bone marrow niche exhibited rewired cell-cell communication, with enhanced T cell-initiated signaling and aberrant megakaryocyte-T cell interactions. Pseudotime analysis uncovered disrupted megakaryocyte maturation dynamics. In silico knockdown revealed CACNA1A, CSF1R, and PKN1 dysregulation exacerbated neutrophil hyperactivity, while CD9 and DSTYK knockdown impaired mitotic regulation. This study delineated mechanisms of TPO-RAs, highlighting five key genes that orchestrate dysregulated thrombopoiesis and immune dysfunction in ITP. The integration of in silico strategies identified novel targets for optimizing ITP therapy.

Peripheral helper T (Tph) cells, a distinct T-cell subset defined as CD4[+]PD-1[hi]CXCR5[-] phenotypically, are increasingly recognized as key drivers of antibody-mediated autoimmunity; however, their role in primary immune thrombocytopenia (ITP) remains poorly defined. In this study, we evaluated circulating Tph cells in newly diagnosed ITP and characterized their dynamic changes following dexamethasone therapy. Circulating Tph cells were significantly expanded in patients with ITP and were positively correlated with bleeding severity, suggesting involvement of Tph cells in ITP pathophysiology. High-dose dexamethasone markedly reduced circulating Tph frequencies both in vitro and in vivo. Notably, persistently elevated Tph levels were observed in patients who were non-responsive to dexamethasone. Furthermore, plasma interleukin-21 levels declined in parallel with Tph reduction following dexamethasone treatment, and were positively correlated with Tph frequencies in untreated, newly diagnosed ITP patients, consistent with Tph-associated B-cell helper activity. Collectively, these findings identify Tph cells as a potential indicator of extrafollicular B-cell activation in ITP and highlight their relevance to dexamethasone therapeutic responsiveness.