Platelet Coagulation Research Articles

Sialylated glycoproteins bind to Siglec-9 in a cis manner on platelets to suppress platelet activation.

Sialylated glycoproteins bind to Siglec-9 in a cis manner on platelets to suppress platelet activation.

Delphi consensus recommendations for neuraxial anesthesia in adults with platelet disorders and coagulation defects: communication from the ISTH SSC Subcommittee on von Willebrand Factor.

Delphi consensus recommendations for neuraxial anesthesia in adults with platelet disorders and coagulation defects: communication from the ISTH SSC Subcommittee on von Willebrand Factor.

Development of Iron-Modified Cotton Material: Surface Characterization, Biochemical Activity, and Cytotoxicity Assessment

Cotton, commonly used in wound care, has limitations such as quick saturation and wound adhesion, prompting surface modifications. In our studies, iron, which promotes platelet aggregation and coagulation, was deposited onto cotton via direct current (DC) magnetron sputtering. Thus, the biochemical properties of cotton fabrics were enhanced. Microscopic analyses revealed uniform iron coating on the fibers, and biochemical tests, such as activated partial thromboplastin time (aPTT) and prothrombin time (PT), showed that the modification did not affect the material’s coagulation activity. Measurements with the thiobarbituric acid (TBA) method (TBARS) showed that iron-modified cotton had antioxidant activity by lowering lipid peroxidation, which can be beneficial for better wound healing and lower infection risk. Moreover, our analysis showed the absence of cyto- and genotoxic properties against normal peripheral blood mononuclear cells (PBM cells). It was found that tested fabrics did not directly interact with DNA.

Velefibrinase: A Marine-Derived Fibrinolytic Enzyme with Multi-Target Antithrombotic Effects Across Diverse In Vivo Models

Background/Objectives: Thrombotic diseases (TDs), currently the number one killer worldwide, account for the highest mortality rate globally. In this study, we evaluated the antithrombotic efficacy of Velefibrinase, a marine bacteria-derived fibrinolytic enzyme, across multiple animal models. Results: The results demonstrated that Velefibrinase prolonged bleeding time (BT) and clotting time (CT), reduced mortality and thrombosis, relieved pulmonary alveolar structure degeneration in an acute pulmonary thromboembolism model, and inhibited carotid artery thrombosis and endothelial tissue damage in a rat model of FeCl3-induced carotid arterial thrombosis. Moreover, Velefibrinase reduced cerebral ischemia volume and ameliorated neurological deficits in a cerebral ischemia/reperfusion (I/R) injury model in rats. The putative underlying mechanisms were found to involve the inhibition of platelet aggregation and coagulation, along with the modulation of oxidative stress and inflammation levels. Conclusions: These results revealed that Velefibrinase exerts a notable thrombosis-preventive effect by interacting with multiple targets, thereby breaking the vicious cycle involving inflammation, oxidative stress, and thrombosis.

A Pump-Free, Hydraulic-Amplification Oscillatory Microfluidic Device for Continuous Particle and Cell Manipulation.

Microfluidics can achieve the spatiotemporal manipulation of particles and cells in the microscale fluids, but highly relies on the accuracy of the pumping systems. To overcome this issue, a pump-free, hydraulic-amplification oscillatory microfluidic (PHOMF) device is presented, which can be actuated by fingers to handle particles and cells within the microchannel. The PHOMF device has a hydraulic-amplification module for pressure transfer and a soft microchannel module for the generation of oscillatory flows. This is made possible by the periodic transfer of finger-driven liquid pressure to the soft microchannel. This pressure causes the soft microchannel to deform and then drives the reciprocating flow of fluid volumes within the microchannel. In the oscillatory flow, particles and cells achieve single-line focusing driven by the spatially accumulated inertial and elastic lift forces. The particle elasto-inertial focusing theory in the PHOMF microchannel has been revealed. To demonstrate the system's practicality, the PHOMF device is utilized to achieve the early observation of platelet clots (3min) and the rapid staining of cancer cells (8min). The PHOMF device provides a miniaturized, inexpensive, and efficient detection tool for lab-on-a-chip, and has the potential to become a mass-produced, widely available, and convenient disease detection product.

Mitogen-activated protein kinase 1 regulates platelet function and thrombus formation.

Mitogen-activated protein kinase 1 regulates platelet function and thrombus formation.

BAD-Glucokinase Axis Regulates Platelet Activation and Thrombosis.

BAD (Bcl2-associated death promoter), a member of the Bcl2 proapoptotic family, promotes cell apoptosis by sequestering the prosurvival proteins Bcl-XL and Bcl2 from the proapoptotic proteins BAK (Bcl2 homologous antagonist/killer) and BAX (Bcl2-associated X protein) in nucleated cells. BAD is also expressed in platelets, playing a role in regulating platelet lifespan, apoptosis, and clearance. However, whether BAD regulates platelet activation and arterial thrombosis remains unclear. The role of BAD in platelet activation and arterial thrombosis was investigated using BAD-deficient mice (Bad-/-), in vitro functional studies, and arterial thrombosis models. The regulatory effect of BAD on platelet energy metabolism was detected using a Seahorse Extracellular Flux Analyzer. The regulatory effect of BAD on glucokinase was investigated by coimmunoprecipitation and activity measurement. The glucokinase heterozygous knockout mice (Gck+/-) and activator were used to study its role in platelet activation. BAD-deficient mice (Bad-/-) and wild-type mice transfused with Bad-/- platelets displayed prolonged tail bleeding and arterial occlusion times. Bad-/- platelets exhibited decreased aggregation in response to stimulations by proteinase-activated receptor 4-activating peptide, thrombin, and U46619. Furthermore, BAD ablation suppressed platelet integrin αIIbβ3 activation, granule secretion, and clot retraction induced by these agonists. Mechanistically, BAD interacted with glucokinase, and BAD deficiency resulted in decreased platelet glucokinase activity, mitochondrial oxidative phosphorylation, and mitochondrial ATP production. The partial loss of glucokinase (Gck+/-) phenocopied platelet function defects caused by BAD deficiency, and a glucokinase activator rescued the impaired mitochondrial ATP production and function of Bad-/- platelets. Additionally, the glucokinase activator enhanced human platelet activation. Our findings demonstrate the critical role of the BAD-glucokinase axis in platelet activation and thrombosis, suggesting a potential target for antithrombotic therapy.

Dendrobium nobile lindl extract modulates integrin αIIbβ3-mediated signaling pathways to inhibit platelet activation and thrombosis.

Dendrobium nobile lindl extract modulates integrin αIIbβ3-mediated signaling pathways to inhibit platelet activation and thrombosis.

Soluble FMS-Like Tyrosine Kinase-1 (sFLT-1) and Soluble Endoglin (sEng) Induce Platelet Hyperfunction in a Rat Model of Severe Preeclampsia

Background: Placental ischemia is a key initiating event in the pathophysiology of preeclampsia (PE) that triggers the release of anti-angiogenic factors into the maternal circulation, including sFLT-1 and sEng. These placental factors cause systemic vascular dysfunction and ultimately hypertension and end-organ damage in PE. Furthermore, PE has been associated with placental thrombosis and increased risk of thrombotic events during and after pregnancy, being recognized as a hypercoagulable state. However, the mechanisms of hypercoagulability in PE remain to be fully elucidated. Previous studies have shown that, similar to patients with severe PE, chronic sFLT-1 and sEng infusion into pregnant rats promotes placental pathology, hypertension, and organ damage failure. Our study aimed to determine the contributions of platelets versus fibrin to the coagulation process in this model of severe PE. Methods: 12-14-week-old Sprague-Dawley rats were either implanted intraperitoneally with an osmotic mini-pump containing sFLT-1 and another one loaded with sEng (4.7 and 7.0 µg/kg/day, respectively) or underwent a sham surgery on gestational day (GD)12 (n=4-8/group, depending on the assay). On GD19, arterial blood was drawn into collection tubes with anticoagulant to run viscoelastic assays to measure all phases of hemostasis using the thromboelastography (TEG) 6s Hemostasis Analyzer. Citrated blood was used in global hemostasis with lysis assay to assess the relative contribution of platelets and fibrinogen to overall clot strength, whereas heparinized blood was utilized in platelet mapping assay to assess adenosine diphosphate (ADP)- as well as arachidonic acid (AA)-stimulated platelet clot strength. EDTA plasma was used in qRT-PCR to quantify cell-free mitochondrial (mt) DNA, a damage-associated molecular pattern released from hypoxic and injured tissue with platelet-activation effects. Results: sFLT-1/sEng-infused pregnant rats had decreased platelet cell count compared to sham pregnant rats (5.85±0.28 vs. 6.9 ±0.34 x 10^8 cells/mL; P=0.0274). TEG-based citrated-kaolin (CK) and heparinized-kaolin heparinase (HKH) assays, which identify the hemostatic characteristics of blood in response to thrombin and kaolin showed that maximal amplitude (MA) are similar in sFLT-1/sEng-infused and sham pregnant rats (74.30±0.66 vs. 74.50±1.55 and 75.61±0.76 vs. 76.42±1.02 mm, respectively; both p>0.05). In the platelet-mapping TEG assay, reptilase and Factor XIII (activator F; ActF) are utilized to initiate fibrin-only clot formation, allowing the relative contribution of fibrin polymerization and platelet aggregation to total MA to be distinguished. ActF MA was lower in sFLT-1/sEng-infused pregnant rats than in sham counterparts (25.42±1.53 vs. 34.27±3.85 mm; P= 0.0426). Consequently, platelet contribution to HKH MA was higher in sFLT-1/sEng-infused pregnant rats than in sham counterparts (66.17±1.98 vs. 55.49±4.50 %; P=0.0405). Heparinized-ADP and -AA assays showed similar MA values between sFLT-1/sEng-infused and sham pregnant rats (40.13±4.89 vs. 46.76±5.06 and 73.45±0.77 vs. 73.52±0.62 mm, respectively; both p>0.05). Finally, plasma mtDNA levels were increased in sFLT-1/sEng-infused pregnant rats compared to sham pregnant rats on GD19 (0.067±0.029 vs. 0.003±0.001 ng/mL; P= 0.0121). Conclusion: These findings indicate that platelets have a larger contribution to coagulation than fibrin in sFLT-1/sEng-infused pregnant rats, despite exhibiting reduced number of circulating platelets. In addition, sFLT-1/sEng induced increased circulating mtDNA levels, which could serve as one of the mechanisms leading to platelet hyperfunction in our model of severe PE. This research was supported by the National Institutes of Health (K01HL159032, K08GM138812, R01HL148191, and U54GM115428). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Assessing direct transfusion feasibility with syringe-based collection of whole blood from universal donors.

The anticipated multidomain operations of future battlefields necessitates minimalistic medical strategies for treatment of combat wounded. Direct transfusion, involving the administration of blood directly from a donor to a recipient, may be a solution for treating hemorrhagic shock patients in an austere environment where the tools to execute clinical practice guidelines are unavailable. Six participants were enrolled in this observational pilot study. Blood was drawn through medical tubing using a 60 mL syringe and pushed through a second medical tubing set with output to a waste container. Three participants were collected with untreated lines, and three collections were performed with heparin flushed lines. Visible clotting was assessed, and blood samples were collected from the output at baseline, after 240 mL of blood collected, and at study endpoint, which was 400 mL collection or termination due excessive clotting or draw difficulties. Coagulation, hematology, and chemistry testing were performed on the samples. Visible clotting was observed at endpoint in the four procedures with the longest draw times. There were statistically significant differences in platelet count and fibrinogen concentration between baseline and endpoint samples of the non-heparin group. Clot strength decreased over time in the non-heparin group. The association between longer procedure times and visible clotting indicates direct transfusions should be completed based on time constraints rather than target volumes. Maintenance of platelet count, fibrinogen concentration, and clot strength in the heparin group suggest that heparin coated lines may enhance the safety of direct transfusions.

Storage of whole blood in the plasma bag of an ordinary blood component collection set as an emergency preparedness measure.

Demand for whole blood (WB) transfusions can increase dramatically during a disaster, but it is difficult to predict the need for blood products in the early phase of an event. Generally, blood services have large stocks of blood component collection sets but lack bags for the collection of WB for transfusion. While the collection bag in conventional blood component sets often lacks transfusion ports and cannot be used for WB transfusion, the plasma bags in these same sets have transfusion ports and are without filters or additives. This study aimed to evaluate the feasibility of transfer to and storage of WB in plasma bags from blood component collection sets. Blood was collected in 10 Reveos (REV) and 10 Macopharma (MP) blood bag systems. Samples were taken from the units on days 1, 14, 21, 28, and 35 after donation. At each sample point, hematological, metabolic, and coagulation parameters were analyzed. All results from both groups were in accordance with European regulatory requirements until Day 28, where three and one bags from the REV and MP groups, respectively, exhibited hemolysis at or above the 0.8% threshold. By day 35, this rose to seven and three bags, respectively. Platelet count, coagulation function, and factor VIII levels decreased but remained detectable throughout storage. We conclude that storage of WB in the plasma bag of an ordinary blood component collection set as an emergency preparedness measure can maintain quality for a minimum of 21 days.

Biosynthesis of a N-Acetylated Tricyclic Carbazole with Antithrombotic Activity.

Tricyclic carbazoles are significant pharmacophores. Herein, heterologous expression of the carbazole-3,4-quinone (1) biosynthetic pathway in the chassis host Streptomyces albus J1074 yielded a previously chemosynthesized orthoquinone carbazole (2) and three new N-acetylated carbazoles (3-5). Their structures were established by a combination of HR-ESI-MS, NMR, and X-ray crystallographic analysis. Compound 2, the deaminated precursor of 4 and 5, was enzymatically synthesized, indicating the substrate tolerance of the key enzymes in the bacterial tricyclic-carbazole biosynthetic pathway. Mutagenetic analysis revealed an arylamine N-acetyltransferase homologous gene required for the production of compounds 3-5. Bioactivity analysis using the zebrafish model demonstrated that compound 5 has significant antithrombotic activity, potentially by downregulating the genes involved in the platelet activation and coagulation cascade. These findings expand the natural strategies for structural diversification of the tricyclic carbazole alkaloids.

Abstract 6313: Context-aware pathway enrichment clustering and interpretation using large language models

Abstract Pathway enrichment analysis is pivotal for elucidating key oncogenic processes from high-throughput cancer profiling. However, the inherent redundancy and interdependence in biological pathway annotations complicate the meaningful interpretation of enrichment results. This challenge necessitates the effective clustering of enriched pathways. Traditional clustering methods predominantly rely on structural matrices, such as gene overlap within hierarchical pathway graphs. However, these methods do not account for the biological context of the experiment, potentially overlooking critical, context-specific insights tied to cancer types, stages, and other key factors. To address these limitations, we present a context-aware approach for clustering and interpretating pathway enrichment results using emerging large language models (LLMs). Our method involves: 1) generating contextually enriched pathway summaries based on original definitions, using LLMs with prompt engineering techniques; 2) embedding these summaries into high-dimensional quantitative representations, to capture pathway-level contextual semantics; and 3) clustering pathways into biologically coherent themes by computing pairwise similarities between embeddings. We applied this approach to a case study involving 144 up-regulated Reactome pathways from pediatric acute myeloid leukemia (AML) samples compared with normal controls in the TARGET dataset. A widely used overlap-based method, EnrichmentMap, yielded clusters with uneven sizes and fragmented groupings. For instance, the largest cluster contained 79 pathways spanning diverse processes, while immune-related pathways formed several separate clusters. In contrast, our LLM-based approach produced more cohesive and interpretable results, with 11 clusters ranging from 5 to 25 pathways. These clusters successfully delineated distinct biological processes, such as platelet activation and coagulation, and chromatin remodeling. Notably, our approach demonstrated the value of contextualizing pathways. For example, the “pentose phosphate pathway” was grouped with iron metabolism and autophagy pathways, emphasizing its role in providing NADPH for iron metabolism, which is relevant to AML. This insight was missed by EnrichmentMap, as this pathway was left unclustered. Additionally, “nuclear events stimulated by ALK signaling in cancer” was grouped with oncogenic signaling, reflecting the role of ALK in nuclear processes that drive survival, transformation, and apoptosis escape. Overall, this pilot study underscores the potential of LLM-generated contextual pathway summaries and embeddings to produce biologically coherent pathway clusters that highlight their collective roles. Our approach represents a novel strategy for enhancing the interpretability of pathway analysis results, particularly in complex disease contexts such as cancer. Citation Format: Yibing Guo, Yanhao Tan, Li-Ju Wang, Chien-Hung Shih, Yu-Chiao Chiu. Context-aware pathway enrichment clustering and interpretation using large language models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6313.

Effect of Tofacitinib on Hemostasis in Patients with Ulcerative Colitis: A Comparative Ex Vivo Study.

Background: Tofacitinib is effective for refractory ulcerative colitis (UC), a chronic inflammatory disease of the colonic mucosa. However, its use has been associated with an increased risk of thromboembolic events, prompting regulatory restrictions. Understanding the pathophysiological mechanisms contributing to these potential risks is critical for patient safety. We aim to evaluate and compare ex vivo the effects of tofacitinib and anti-TNF on coagulation parameters and platelet function. Methods: Whole blood and platelet-rich plasma from 10 active UC (aUC) and 10 quiescent UC (qUC) patients and 10 healthy controls (HC) were spiked ex vivo with tofacitinib, anti-TNF (as comparator), or a sterile solution. Coagulation kinetics were measured by rotational thromboelastometry (ROTEM), platelet aggregation by aggregometry, and platelet activation by flow cytometry. The study was conducted at Hospital Universitario de La Princesa. Results: Flow cytometry showed increased expression of activation markers CD62P and CD63 and higher PAC-1 binding in platelets from both aUC and qUC patients incubated with either tofacitinib or anti-TNF versus no drug. No differences were found between the drugs. CD63 expression also increased in HC after drug exposure, with no differences between anti-TNF or tofacitinib. Platelet aggregation and coagulation parameters did not differ between tofacitinib, anti-TNF, and no drug in aUC, qUC, and HC. Conclusions: Tofacitinib does not alter platelet function or coagulation in UC patients under ex vivo conditions compared to anti-TNF. The increased thromboembolic risk observed in some populations treated with tofacitinib cannot be attributed to these factors in UC patients.

Okra polysaccharide mitigates carrageenan-induced thrombosis in mice by regulating inflammation and oxidative stress.

Thrombosis is a serious health hazard, which has been paid more and more attention.Okra polysaccharide (OP) is a biologically active substance extracted from okra which exhibits anti-inflammation and anti-oxidative properties. Nevertheless, the effect of OP on thrombosis is still unknown. In this study, we determined whether OP can suppress carrageenan-induced mice thrombosis and its involved mechanism. Twenty-four BALB/c mice were assigned to four groups randomly (6 mice/group): Ctrl, Model, OP low lose (OP-L, 200 mg/kg body weight), and OP high lose (OP-H,400 mg/kg body weight) were administered via intragastric administration for 9 days. Tails were photographed before collecting for H&E and Masson staining. Liver and lung tissues were collected for H&E staining, RT-qPCR, Western blot and GSH content detection. Injury or dysfunction of endothelial cells (ECs) was assessed using RT-qPCR, Western blot and cell adhesion assays. OP can effectively improve carrageenan-induced thrombosis in tissues of mice (tail, liver, and lung) in vivo. In addition, OP inhibited inflammation by suppressing the toll-like receptor 4 (TLR4)/nuclear factor κB (NF-κB) pathway and reduced oxidative damage by elevating the level of GSH and antioxidant enzyme in liver and lung tissues. In vitro, OP inhibited thrombin-induced human platelet clots retraction, and decreased lipopolysaccharide (LPS)-activated adhesion of THP-1 monocytes to human umbilical vein endothelial cells(HUVECs) by suppressing intercellular adhesion molecule-1 (ICAM-1) level. In conclusion, OP can inhibit thrombosis in mouse model by regulating inflammation and oxidative stress, which suggest that OP could act as a potential functional food for prevention of thrombus.

Tumor Microenvironment Triggered In Situ Coagulation of Supramolecularly Engineered Platelets for Precise Tumor Embolization.

Although embolization therapy has demonstrated success in impeding tumor growth, concerns persist regarding potential tumor recurrence and inadvertent embolization of non-target tissues. In this study, drawing inspiration from the natural targeting and coagulation process of platelets in injured blood vessels, platelets are engineered by integrating acid-sensitive, morphology-transformable nanoparticles onto their surface through supramolecular conjugation (PLT-NP). The nanoparticles are constructed through the self-assembly of a β-amyloid derived peptide (FFVLK) terminally functionalized with Fmoc, hexahistidine (His6), and a polyethylene glycol (PEG)-functionalized cyclodextrin (CD). The supramolecularly engineered platelets actively accumulate in the tumor tissue upon inducing a tumor blood vessel injury through tumor resection. In response to the local acidic microenvironment, the nanoparticles undergo a morphological transformation into nanofibers via spontaneous assembly of FFLVK into fibril structures through hydrogen bonding and β-sheet interactions, to artificially enhance the coagulation and aggregation of platelets, causing occlusion of tumor blood vessels. The supramolecularly engineered platelets efficiently embolize tumor blood vessels in a specific manner, effectively suppressing tumor growth, metastasis, and recurrence, thus offering a promising paradigm for combating cancer.

Variability in Coagulation Profiles in Patients with Chronic Kidney Disease and Peripheral Artery Disease.

Variability in Coagulation Profiles in Patients with Chronic Kidney Disease and Peripheral Artery Disease.

The Possible Role of Metformin and Fibroblast Growth Factor-21 in Multiple Sclerosis Neuropathology: Birds of a Feather Flock Together.

Multiple sclerosis (MS) is a progressive demyelinating disease of the CNS, characterized by inflammation, the formation of CNS plaques, and damage to the neuronal myelin sheath (Graphical abstract). Fibroblast growth factor 21 (FGF21) is involved in various metabolic disorders and neurodegenerative diseases. FGF21 and its co-receptor β-Kloth are essential in the remyelination process of MS. Metformin, an insulin-sensitizing drug that is the first-line treatment for type 2 diabetes mellitus (T2DM), may have a potential neuroprotective impact by up-regulating the production of FGF21, which may prevent the onset of neurodegenerative diseases including MS. The purpose of this review is to clarify how metformin affects MS neuropathology mechanistically via modifying FGF21. Metformin increases the expression of FGF21. Metformin also increases the expression of β-Klotho, modulates oxidative stress, reduces glutamate-induced excitotoxicity, and regulates platelet function and coagulation cascades. In conclusion, metformin can enhance the functional activity of FGF21 in counteracting the development and progression of MS. Preclinical and clinical studies are warranted in this regard.

Exploring Mechanisms of Ephx2 in Treating Atherosclerosis Using Independent Cascade Model and Adverse Outcome Pathways.

Atherosclerosis (AS) is a leading cause of cardiovascular diseases, characterized by lipid accumulation in arterial walls. The enzyme Ephx2 (soluble epoxide hydrolase, sEH) is implicated in AS development, but its precise mechanisms and therapeutic potential are not fully understood. This study aimed to analyze gene expression data from low-density lipoprotein receptor knockout (LDLR⁸/⁸) and LDLR⁸/⁸sEH⁸/⁸ mice to identify significant genes associated with AS. A directed compound-protein interaction network was constructed based on these genes and related pathways from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In the end, through resistance distance (RD) between any two nodes in this network, the Independent Cascade (IC) model was applied to explore Ephx2 mechanisms in AS, such as important Adverse Outcome Pathways (AOPs). Several AOPs were identified as critical in AS treatment via Ephx2. The key AOPs included inflammatory response and cytokine release, cholesterol deposition and oxidation, disruption of plaque stability, smooth muscle cell proliferation and migration, and platelet activation and coagulation. Within the top AOPs of inflammatory response and cytokine release, potential target genes were identified, such as Mapk3, PiK3cd, Gnai2, Mapk10, Arnt, and RhoA. Critical paths from Ephx2 to these target genes were established, suggesting mechanisms by which Ephx2 may influence AS pathogenesis. By defining the AS network and corresponding RD, this study elucidated potential mechanisms by which Ephx2 affects AS through specific KEGG pathways, AOPs, and target genes. These findings enhanced the understanding of AS pathogenesis and highlighted potential targets like Mapk3 for developing therapeutic strategies in AS prevention and treatment.

Innovative Application of Medicinal Insects: Employing UHPLC-MS, Bioinformatics, In Silico Studies and In Vitro Experiments to Elucidate the Multi-Target Hemostatic Mechanism of Glenea cantor (Coleoptera: Cerambycidae) Charcoal-Based Medicine.

Background: Longhorn beetles, a widely recognized group of Chinese traditional medicinal insects, are characterized by their notable hemostatic properties. However, the comprehensive understanding of their medicinal potential has been hindered by the limitations of current research methodologies. Methods: This study focuses on the species Glenea cantor (Fabricius), which can produce several generations per year, and introduces a novel method using microwave carbonization techniques. By employing an in vitro coagulation test, UHPLC-MS, network pharmacology, molecular docking, and molecular dynamics simulation, the hemostatic efficacy and mechanism of action of Glenea cantor charcoal medicine (GC-CM) were thoroughly studied. Results: In vitro coagulation tests showed that GC-CM significantly reduced the activated partial thromboplastin time (APTT) and prothrombin time (PT), indicating its ability to enhance the coagulation cascade and preliminarily confirming its hemostatic efficacy (p < 0.01 vs. blank control group). The analysis revealed that GC-CM comprises 453 components, including 137 bioactive components with high human utilization. After predictions via databases such as SwissTargetPrediction and deduplication, 215 targets linked to hemostatic specificity were identified. These targets regulate signaling pathways such as platelet activation, complement and coagulation cascades, and cGMP-PKG. Molecular docking demonstrated strong affinities between key targets such as SRC and PIK3R1 and compounds such as 2',6'-dihydroxy 4'-methoxydihydrochalcone, and 1-monolinoleoyl-rac-glycerol (binding energy < -5 kcal/mol). Molecular dynamics simulations show good binding capacity between core components and targets Conclusions: The aim of this study was to elucidate the material basis and mechanism of the hemostatic efficacy of GC-CM, offering a model for exploring other insect-based medicinal resources.