Platelet hyperactivation is a major factor in thrombotic complications such as myocardial infarction and ischemic stroke. Ginsenoside Rg5 is a minor ginsenoside, and among its various beneficial pharmacological effects, its antithrombotic potential has not been extensively studied. Human platelets were isolated and treated with Rg5 (35-100 μM) before stimulation with agonists such as collagen, thrombin, and U46619. Platelet aggregation, granule secretion, calcium mobilization, thromboxane A₂ production, fibrinogen binding, and clot retraction were evaluated. The effects of Rg5 on signaling pathways were determined via Western blot analysis of key proteins. In vivo, the antithrombotic efficacy was assessed using ferric chloride (FeCl₃)-induced thrombosis in mice. Rg5 dose-dependently inhibited collagen-induced platelet aggregation (IC₅₀ = 42.5 μM) and selectively inhibited GPVI-mediated signaling compared to thrombin and U46619. Rg5 suppressed intracellular calcium mobilization, granule secretion, and thromboxane A₂ production, with no cytotoxicity observed. Rg5 downregulated key signaling proteins (p-PI3K, p-AKT, p-cPLA2, and p-p38) while upregulating p-VASP (S157 and S239), suggesting its role in elevating cyclic nucleotide signaling. Additionally, Rg5 inhibited CD162 expression that was induced in the presence of collagen and oxidized low-density lipoprotein. It also prevented fibrinogen and fibronectin binding and significantly reduced clot retraction. In vivo, Rg5 (20 mg/kg) significantly prolonged the carotid artery occlusion time and prevented thrombus formation, outperforming aspirin (100 mg/kg). Ginsenoside Rg5 exhibits potent antiplatelet activity by selectively targeting GPVI-mediated platelet activation and modulating key intracellular signaling pathways. These results suggest that Rg5 could be utilized to develop safer and natural antiplatelet therapies.

Cardiovascular diseases (CVDs) have been the major cause of mortality all around the globe. Lespedeza cuneata abbreviated as L. cuneata with the authority name of Dumont de Courset (G. Don) is a perennial flowering plant commonly grown in Asian countries such as Korea, Japan, China, and Taiwan. We aimed to investigate the L. cuneata extracts antiplatelet and antithrombotic properties as GC-MS analysis indicated that the extract contained short-chain fatty acids, which have been reported to possess beneficial cardiovascular effects. L. cuneata was extracted using water, 50 EtOH, 70 EtOH, and 100 EtOH. For in vitro antiplatelet analysis, washed platelets were prepared and incubated with L. cuneata with 200μg/mL of 50 EtOH in the presence of 1mM of CaCl₂ for 1minute followed by agonist (collagen 2.5μg/mL or ADP 10μM or thrombin 0.1U/mL) stimulation for 5minutes over light transmission aggregometer. Scanning electron microscopy was performed to assess platelet shape change. ATP release and intracellular calcium mobilization were quantified to assess the granular content. Fibrinogen-binding assay and clot retraction assay assessed integrin αIIbβ3-mediated inside-out and outside-in signaling. Protein phosphorylation expression was investigated by western blot analysis. Finally, the in vivo antithrombotic efficacy was investigated by oral dosage of L. cuneata 200 and 400mg/kg and aspirin 100mg/kg for 7days, and tail bleeding and FeCl₃-induced murine thrombus model were performed. In vitro platelet aggregation and platelet shape change were dose-dependently suppressed by L. cuneata. Calcium mobilization, dense granules secretion, integrin αIIbβ3-mediated inside-out and outside-in signaling, and protein phosphorylation of MAPK and PI3K/Akt pathways were significantly inhibited. In vivo assays revealed that L. cuneata prevents side effects of synthetic drugs via nonsignificantly increasing bleeding time and improving coronary artery blood flow and animal survival. Our results demonstrate that L. cuneata exhibited potent antiplatelet and antithrombotic effects and can be considered a potential herbal medicine with cardioprotective effects.

Cardiovascular disease remains a leading cause of morbidity and mortality worldwide, frequently arising from platelet hyperactivation and subsequent thrombus formation. Although conventional antiplatelet therapies are available, challenges, such as drug resistance and bleeding complications, require the development of novel agents. In this study, dihydrogeodin (DHG) was isolated from Fennellia flavipes and evaluated using platelets derived from Sprague–Dawley rats. Platelet aggregation induced by collagen, adenosine diphosphate, or thrombin was assessed by light transmission aggregometry; DHG significantly reduced aggregation in a dose-dependent manner. Further assays demonstrated that DHG suppressed intracellular calcium mobilization, adenosine triphosphate release, and integrin αIIbβ₃-dependent fibrinogen binding, thereby impairing clot retraction. Western blot analysis revealed that DHG reduced the phosphorylation of mitogen-activated protein kinases (ERK, JNK, p38) and PI3K/Akt, indicating inhibition across multiple platelet-signaling pathways. Additionally, SwissADME-assisted pharmacokinetics predicted favorable properties without violations of the Lipinski (Pfizer) filter, Muegge (Bayer) filter, Ghose filter, Veber filter, and Egan filter, and network pharmacology revealed inhibition of calcium and MAPK pathways. These results highlight the potential of DHG as a novel antiplatelet agent with broad-spectrum activity and promising drug-like characteristics. Further studies are warranted to assess its therapeutic window, safety profile, and potential for synergistic use with existing antiplatelet drugs.

Lespedeza cuneata (LC) is a perennial plant used in herbal medicine to treat numerous diseases, including prostatic hyperplasia, diabetes, early atherosclerosis, and hematuria. Reference collections of bioactive compounds of LC are crucial for the determination of their pharmacological properties. However, little is known regarding its anti-oxidative and anti-inflammatory effects in alveolar macrophage (MH-S) cells. This study examined whether LC can inhibit reactive oxygen species and Coal fly ash (CFA) induced inflammation in MH-S cells. The anti-oxidative effects of LC were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays, anti-inflammatory effects were examined using nitric oxide (NO) assay, and cytotoxicity was analyzed using methyl thiazolyl tetrazolium assay. The expression of inflammatory cytokine genes was assessed through a reverse-transcription polymerase chain reaction. Our results revealed that LC exhibited high radical scavenging activity and a dose-dependent (7.8–1,000 μg/mL) inhibition of oxidation as compared to ascorbic acid and Trolox. It also inhibited CFA-induced NO production in MH-S cells. Moreover, it suppressed the CFA exposure-mediated expression of pro-inflammatory mediators and cytokines, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-1β, IL-6, and tumor necrosis factor-α. These results suggest that LC is a potent antioxidant and anti-inflammatory agent that can be useful as a nutraceutical product.

Sepsis is a life-threatening condition characterized by systemic inflammation and thrombo-inflammation, which trigger platelet activation and platelet–leukocyte aggregate (PLA) formation. While Rg3-enriched red ginseng extract (Rg3-RGE) has demonstrated anti-inflammatory and antithrombotic properties, its role in modulating PLAs during sepsis remains poorly understood. The in vivo effects of Rg3-RGE were analyzed in a lipopolysaccharide (LPS)-induced murine model of sepsis. Flow cytometry was used to evaluate platelet–leukocyte activation markers. Confocal and scanning electron microscopy (SEM) provided visual evidence of aggregate formation. ELISA was used to quantify plasma levels of tissue factor (TF) and platelet factor 4 (PF4). HPLC analysis revealed the presence of Rb1, Rg3 [20(S) and 20(R)], Rc, Rd, Rf, Rh1, Rb2, and Rg2 [20(S)] in Rg3-RGE. Network pharmacology was employed to identify target interactions and assess their involvement in the thrombo-inflammatory pathway. Rg3-RGE significantly reduced the activated platelets (CD41+CD62P+), platelet–neutrophil aggregates (PNAs; CD41+Ly6G+), and platelet–monocyte aggregates (PMAs; CD41+CD115+). Confocal imaging confirmed a reduction in PNAs. SEM revealed decreased fibrin-like structures and fewer platelet–leukocyte interactions. Rg3-RGE lowered plasma TF levels, indicating a potential effect on the coagulation cascade, though PF4 levels were not significantly altered. Network pharmacology analysis of ginsenosides in Rg3-RGE identified 235 overlapping targets associated with sepsis, inflammation, platelet activation, and PLA formation. Enrichment analysis revealed key thrombo-inflammatory pathways, including platelet activation, MAPK signaling, PI3K-Akt signaling, sphingosine-1-phosphate receptor signaling, and EGFR signaling. Rg3-RGE effectively inhibits platelet–leukocyte aggregate formation and mitigates sepsis-induced platelet–leukocyte interactions.

Platelet hyperactivation and thrombo-inflammatory processes are critical drivers of cardiovascular diseases. Although conventional antiplatelet therapies reduce thrombotic risk, their use is limited by bleeding complications and drug resistance. Ginsenoside Rs3 (G-Rs3) exhibits anti-inflammatory potential; however, the specific mechanisms underlying the antiplatelet and thrombo-inflammatory activity of ginsenoside Rs3 (G-Rs3) remain unclear. Human platelets were treated with G-Rs3 in vitro to evaluate its effects on aggregation, intracellular signaling, granule secretion, and thrombo-inflammatory signaling markers. Network pharmacological analyses were performed to predict pathway-level modulations. The in vivo antithrombotic efficacy of G-Rs3 was assessed using a murine FeCl₃-induced thrombosis model. G-Rs3 inhibited platelet aggregation in a dose-dependent manner following stimulation with collagen, thrombin, and U46619. It suppressed Ca²⁺ mobilization, dense granule secretion, and phosphorylation of key signaling molecules, including InsP₃R, ERK1/2, MAPKs, PI3K, Akt, GSK3, and Syk. Additionally, G-Rs3 inhibited integrin αIIbβ3 activation, reduced platelet adhesion, and enhanced cyclic nucleotide–mediated negative regulation, as evidenced by elevated VASP phosphorylation and increased cAMP/cGMP levels. Notably, G-Rs3 attenuated oxLDL-enhanced upregulation of the pro-inflammatory platelet markers CD62P and CD162, highlighting its potential to suppress platelet-driven thrombo-inflammatory responses under atherogenic conditions. Further, network pharmacology analysis revealed that G-Rs3 targets multiple interconnected thrombo-inflammatory pathways, including MAPK, PI3K-Akt, Ras, calcium, and cyclic nucleotide signaling. In vivo, G-Rs3 significantly delayed thrombus formation and moderately prolonged occlusion time. These findings highlight G-Rs3 as a novel natural antithrombotic agent with anti-thrombo-inflammatory properties. Further studies are required to determine its translational potential and establish its therapeutic viability and safety profile.