Hemostasis in humans has traditionally been considered to be a function of platelets, coagulation, and the subendothelial matrix, but the role of red blood cells (RBCs) has been increasingly recognized. RBCs regulate hemostasis through biophysical and biochemical means. For the former, faster-moving RBCs in the center of vessels marginalize platelets and plasma to the vessel walls, where the platelets constantly probe the endothelial surface for injury. RBC counts also determine blood viscosity, which regulates the shear stress of laminar blood flow. For the latter, RBCs are the largest pool of adenosine triphosphate (ATP), which, upon release, is rapidly hydrolyzed to adenosine diphosphate (ADP). Both ATP and ADP activate platelets. Quantitative and qualitative abnormalities in RBCs have also been consistently identified as significant risk factors for arterial and venous thrombosis. Thrombosis is a major complication associated with diseases such as polycythemia vera, secondary erythrocytosis, and sickle cell anemia, all of which present with changes in numbers and physical properties of RBCs. Thrombosis is also common in conditions with significant hemolysis, such as paroxysmal nocturnal hemoglobinuria, severe infections, and when patients are on mechanical support. In this review, we discuss findings from clinical observations and mechanistic studies of how RBCs regulate hemostasis and contribute to thrombosis.
The development of blood-wetted artificial organs is limited by thrombosis on synthetic biomaterial surfaces. In contrast, the endothelium of the vasculature creates a natural barrier to both thrombosis and pannus growth. Consequently, efforts have been made to endothelialize synthetic biomaterials used in blood-wetted devices. Therefore, this study was undertaken to provide a numerical model to simulate the inhibitory effects of EC-derived nitric oxide (NO) on platelet deposition. An existing multi-constituent continuum model of thrombosis was amended to incorporate shear-dependent generation of NO as an anticoagulant. A simulation was performed of blood flow through a bipartite parallel plate channel having an endothelialized upstream layer followed by a pro-coagulant collagen surface downstream. The simulation showed that endothelial-derived NO inhibited downstream platelet deposition, reducing thrombus growth and creating a thrombus-free zone immediately downstream. This enhanced simulation model of thrombosis provides insights into factors that may guide future endothelialization of artificial organs and other blood-wetted devices.
Inflammation-induced injury of venous endothelium is the first trigger of deep vein thrombosis (DVT). We previously showed NLRP3 regulates platelet function and arterial thrombosis. However, whether platelet NLRP3 involves in venous thrombosis remains unclear. In this study, we intended to investigate the role of NLRP3 in venous thrombosis by using NLRP3 knockout mice and platelet-specific NLRP3 knockout mice. DVT model was established through ligation of the inferior vena cava. After 48 hours of ligation, inferior vena cava sample was excised to measure thrombi length and weight, the recruitment of platelets, neutrophils, monocytes, and neutrophil extracellular trap (NET) formation by immunofluorescence staining. Deficiency of NLRP3 reduced the incidence and severity of venous thrombosis, inhibited the recruitment and accumulation of platelets, neutrophils, and monocytes in venous thrombi, and reduced NET formation as well as interleukin (IL)-1β and IL-18 release. Additionally, platelet NLRP3 is the major source of elevated IL-1β in the venous thrombi, and adoptive transfer of platelets to NLRP3-/- mice increased IL-1β level in thrombi and promoted venous thrombosis and NET formation. Moreover, platelet NLRP3 deficiency inhibited NET formation induced by activated platelets in vitro. Our study demonstrated that deficiency of NLRP3 reduces the incidence and the severity of venous thrombosis with platelet NLRP3 playing a predominant role, indicating that NLRP3 might be a therapeutic target for the treatment of venous thrombosis.
Prevention of thromboembolism (TE) remains a priority in children with a high thrombotic risk. While recent trials using direct oral anticoagulants (DOACs) for TE prevention have been completed, these trials had significant limitations. This study reviewed the methodology of pediatric DOAC trials for TE prevention to identify design and execution challenges and opportunities for improvement. We performed a systematic review of MEDLINE, EMBASE, the Cochrane Library, and clinicaltrials.gov, from January 2002 to April 2025, to identify pediatric trials evaluating DOACs for TE prevention. We compared registered information in clinicaltrials.gov, trial design manuscripts, and final result manuscripts of pivotal TE prevention DOAC trials to highlight changes in methods, study duration, and outcomes. Eight publications from 4 trials (1 design and 1 final manuscript each) were included. Each trial targeted a pediatric subpopulation: 3 in cardiac disease and 1 in oncology. Together, 984 children enrolled in the 4 trials. All trials had some modification to methdology from original design, including changes in eligibility criteria, sample size, timing of end point ascertainment, outcomes, and analysis. All but 1 required a longer duration to complete than planned. Only 1 trial had definitive statistical power calculations. The pediatric DOAC trials for TE prevention provide an opportunity to understand the unique successes and limitations in studying TE prevention in children. Given lack of power, inconsistent definitions, and limited follow-up duration, questions remain regarding safety, dose intensity, end points, and appropriate selection of high-risk populations. Future research should use innovative methods to overcome these limitations.
In patients with iliofemoral deep vein thrombosis (DVT), early thrombus removal reduces the risk of post-thrombotic syndrome (PTS). It remains uncertain if mechanical thrombectomy (MT) using the ClotTriever system may offer advantages as compared with rheolytic thrombectomy (RT) using the AngioJet ZelanteDVT. In our multicenter, retrospective, observational study, we included 122 patients (mean age, 48 years; 57% women) with iliofemoral (78%) or iliocaval DVT (22%). All underwent early thrombus removal with either MT (n = 40) or RT (n = 82) and had a minimum of 3 months of follow-up. Periprocedural outcomes included periprocedural thrombolytic use, access complications, and stent placement rate. Clinical outcomes included stent patency rate and freedom from PTS. The median follow-up was 25 months (interquartile range, 11-52 months). Compared with RT, MT was associated with lower periprocedural thrombolytic use (38% vs 95%; P < .01) and a lower rate of stent placement (70% vs 98%; P < .01). Postprocedural access site thrombosis of the popliteal vein occurred in 5 MT patients (13%) and in none of the RT patients. At 1 year, primary and secondary patency rates were 80% (95% confidence interval [CI], 67%-95%) and 97% (95% CI, 93%-100%) in the MT group, and 88% (95% CI, 81%-96%) and 97% (95% CI, 94%-100%) in the RT group, respectively. Freedom from PTS at latest follow-up was observed in 98% of the MT group (95% CI, 93%-100%) and 94% of RT patients (95% CI, 87%-100%). Both MT and RT were associated with high patency rates and freedom from PTS. MT may decrease the need for thrombolysis and venous stent placement. Popliteal vein thrombosis from large-bore access in MT patients requires further investigation.
Antiplatelet drugs have represented a milestone in treating patients at high risk of thrombosis. However, their clinical use remains limited by bleeding-associated risk and limited efficacy. Excessive reactive oxygen species (ROS) produced by damaged vascular endothelial cells have been shown to stimulate thrombosis. Here we propose that a ROS-chemotactic nanoscavenger (MDCP), formed by crosslinking melanin and catalase, prevents acute thrombosis by protecting vascular endothelial cells from oxidative stress. We demonstrate that treatment with MDCP inhibits ROS-induced apoptosis of endothelial cells, thereby maintaining endothelial integrity and preventing collagen exposure, which consequently prevents platelet activation and thrombosis. By avoiding direct interference with platelet function, this modulation of vascular redox homeostasis via MDCP provides a promising alternative antithrombotic strategy that addresses the bleeding risk of current clinical antithrombotic drugs.
Patients with decompensated cirrhosis commonly have indications for anticoagulation, including stroke prevention in atrial fibrillation and the prevention and treatment of venous thromboembolism, particularly portal vein thrombosis. While the rates of anticoagulation in those with indications for therapy have improved over time, the concern for bleeding due to altered hepatic elimination of anticoagulants and exclusion of patients with cirrhosis from pivotal randomized trials of direct oral anticoagulants and warfarin continue to create challenges in defining best practices for anticoagulation in patients with cirrhosis, especially in decompensated disease. In this review, we present 3 commonly encountered clinical scenarios in patients with decompensated cirrhosis requiring consideration of anticoagulation: a patient with atrial fibrillation and elevated baseline international normalized ratio, a patient with atrial fibrillation and recurrent bleeding while on anticoagulation, and a patient with portal vein thrombosis and thrombocytopenia. For each scenario, we discuss the available literature and propose a management approach.
Sepsis is characterized by a systemic inflammation and microvascular thrombosis induced by infection. The nucleotide-oligomerization domain-like receptor family pyrin domain containing 6 protein (NLRP6) possesses both proinflammatory and anti-inflammatory abilities with cell type-specific or tissue-specific functions. However, the role of cell type-specific NLRP6 in sepsis remains poorly understood. In this study, we detected NLRP6 expression in platelets. By using platelet-specific NLRP6 knockout mice and the cecal ligation and puncture model of sepsis, we demonstrated that deletion of platelet NLRP6 increased the mortality; enhanced microvascular thrombosis in the lung and liver; and promoted platelet activation, platelet-neutrophil interactions, as well as the neutrophil extracellular trap (NET) formation after sepsis. Platelet function analysis in vitro showed that deletion of NLRP6 enhanced platelet aggregation, activation, and granules release. In addition, NLRP6 deletion promoted platelet NF-κB signaling via sustaining transforming growth factor-β activated kinase 1-binding protein 1 (TAB1) expression independent of the inflammasome. Moreover, inhibition of NF-κB signaling abolished the aggravated effects of the absence of platelet NLRP6 on the intravascular microthrombosis and NET formation in sepsis and increased the overall survival. Mechanistically, NLRP6 facilitated the interaction between tripartite motif-containing protein 21 (TRIM21) and TAB1 in activated platelets, resulting in K48-linked polyubiquitination of TAB1 and subsequent degradation. Finally, sepsis plasma triggered TAB1 degradation mediated by NLRP6/TRIM21 in normal healthy platelets through toll-like receptor 4/myeloid differentiation primary response 88. Our study identifies a novel protective role of platelet NLRP6 in microvascular thrombosis during sepsis, implying it as a novel target for the treatment of sepsis.
Small-diameter vascular grafts are indispensable in coronary and peripheral artery bypass surgeries to reestablish the blood flow when blood vessels block. Despite advances in biomaterials' design, current clinical practice still relies heavily on autologous vessels, as synthetic grafts for small-diameter applications present high failure rates due to thrombosis and neointimal hyperplasia. Hence, there is a need for antithrombogenic synthetic small-diameter vascular grafts. Antithrombogenicity can be ensured by passive strategies, which aim to minimize nonspecific protein adsorption and platelet adhesion/activation onto the biomaterial's surface, hence preventing the onset steps of thrombus formation. This review aims to shed light on passive strategies reported in literature to improve antithrombogenicity and performance of vascular grafts. Such strategies include either surface modifications of pre-existing materials composing the backbone of vascular grafts (such as expanded polytetrafluorethylene or polyethylene terephthalate), or the creation of novel intrinsically antithrombogenic bulk materials for fabrication of such conduits. Endowing vascular grafts with antithrombogenic properties has been attempted using a variety of macromolecules, from hydrophilic polymers such as the classically-explored poly(ethylene glycol), to more recently proposed hydrophobic lubricant-infused surfaces. Here, the physicochemical phenomena underlying each surface type is carefully analyzed, reporting both in vitro and in vivo results. By critically examining both theoretical and translational aspects of each strategy, this review provides key insights to guide future innovations in vascular graft design and development. STATEMENT OF SIGNIFICANCE: Small-diameter vascular grafts, used in bypass surgeries when blood vessels block, still fail due to thrombosis. This review highlights passive strategies - i.e. strategies which aim to minimize nonspecific protein adsorption and platelet adhesion - to prevent thrombosis in vascular grafts. By looking into the types of polymers explored, evaluating their molecular mechanisms, reporting in vitro and in vivo results, and critically analyzing translational aspects, it provides a comprehensive guide and future perspective of passive strategies. As such, it appeals to authors interested in the development of vascular grafts, as well as other blood-contacting devices.
Hemostasis prevents bleeding by forming clots, although disruptions can cause insufficient or excessive clotting. Shear stress is a critical factor influencing the normal function of the blood and can stimulate the formation of unnecessary clots. Stenosed vessels significantly contribute to this phenomenon by increasing shear stress on the vessel walls. This study aims to quantify the effects of shear stress and stenosis severity on thrombus formation in patient-specific left coronary bifurcations. Three-dimensional patient-specific geometries were reconstructed from angiographic data. Blood flow was modeled using the Brinkman equation, while transport and interactions of coagulation factors were simulated through the convection-diffusion-reaction equation. Model predictions were validated using literature data. The findings showed that all patients experienced significant shear stress at the stenosed regions, which are highly susceptible to clot formation. Shear stress was found to be inversely related to vessel diameter and directly related to the stenosis degree. Furthermore, in bifurcated vessels, blood flow may reach zero before complete occlusion by the clot, emphasizing the role of hydrodynamic resistance in redirecting blood flow. Among these factors, the stenosis degree emerges as the most significant predictor of blood flow cessation time. A relationship was developed allowing physicians to accurately and rapidly monitor a patient's condition using angiographic images, providing new insights into the hemodynamic mechanisms of coronary thrombosis and supporting improved diagnosis and treatment of cardiovascular diseases.
Background and Objectives: Access-site complications (ASCs) remain clinically relevant after peripheral endovascular procedures, particularly with large femoral sheaths and complex anatomy. While randomized coronary trials show non-inferiority of vascular closure devices (VCDs) versus manual compression (MC), real-world data in peripheral interventions performed under systematic ultrasound-guided access are limited. Materials and Methods: This retrospective single-center cohort included consecutive peripheral arterial revascularizations (2010-2023) performed via common femoral access under real-time ultrasound guidance. Hemostasis was achieved using MC or VCDs, categorized as collagen plug-based, suture-mediated, or clip-based systems. The primary endpoint was 30-day ASCs, defined as hematoma requiring management, pseudoaneurysm, bleeding requiring transfusion, access-site thrombosis/occlusion, arteriovenous fistula, or infection. The secondary endpoint was VCD failure, defined as unsuccessful hemostasis requiring adjunctive measures. Multivariable logistic regression adjusted for prespecified anatomical and procedural covariates, including sheath size > 6 Fr and puncture-site calcification. Results: Among 231 procedures, VCDs were used in 139 (60.2%) and MC in 92 (39.8%). ASC occurred in 28 cases (12.1%), with higher rates in the MC group compared with VCDs (18.5% vs. 9-14% across device types; p = 0.044). In adjusted analyses, MC (vs any VCD) (odds ratio [OR] 2.41, 95% confidence interval [CI] 1.06-5.47; p = 0.035), sheath size > 6 Fr, and puncture-site calcification were independently associated with ASCs. VCD failure occurred in 5 cases (3.6%) and was not observed with collagen plug-based devices. Conclusions: In this ultrasound-guided real-world peripheral cohort, VCD use was associated with lower 30-day ASC rates and low device failure rates compared with MC. Given the retrospective and non-randomized design, these findings should be considered hypothesis-generating and support individualized, imaging-guided strategies for femoral closure in peripheral interventions.
Portal vein tumor thrombosis (PVTT) and portal hypertension are major contributors to gastrointestinal bleeding in hepatocellular carcinoma (HCC), yet the molecular programs linking vascular pathology, immune dysregulation, and bleeding risk remain incompletely defined. Bulk transcriptomic datasets related to PVTT (GSE77509 and GSE69164) and noncirrhotic portal hypertension (GSE77627) were analyzed using differential expression and weighted gene coexpression network analysis to identify robust disease-associated genes. A six-gene core signature was derived by intersecting differentially expressed genes with intramodular hub genes across datasets. Bleeding risk-associated biological programs, including coagulation, complement activation, angiogenesis, hypoxia, and inflammatory signaling, were quantified using ssGSEA. Immune infiltration was estimated using CIBERSORTx. Single-cell RNA-sequencing data from PVTT (GSE149614) were analyzed to resolve cell type-specific expression patterns and intercellular communication. Associations with survival, DNA methylation, immune infiltration, drug sensitivity, and molecular interactions were evaluated using public cancer genomics resources. Functional validation was performed using siRNA-mediated knockdown and drug treatment assays in HepG2 and Huh7 cells, followed by proliferation, colony formation, and wound healing assays. Bleeding risk-related biological programs exhibited dataset-specific activation patterns and correlated with expression of the six-gene signature. Single-cell analysis revealed heterogeneous, cell type-specific expression across malignant, stromal, endothelial, and immune populations. OGFRL1 and WDR62 were significantly associated with overall survival and showed methylation-linked transcriptional regulation. Genetic silencing or pharmacological targeting of these genes significantly suppressed HCC cell proliferation, clonogenicity, and migration in vitro. Drug signature analysis and molecular docking supported potential interactions of nocodazole with OGFRL1 and testosterone with WDR62, which phenocopied knockdown effects. These findings identify immune-coagulation dysregulation as a molecular link between PVTT, portal hypertension, and gastrointestinal bleeding risk in HCC and functionally validate OGFRL1 and WDR62 as biologically and therapeutically relevant targets.
Factor VIII (FVIII), a critical cofactor protein traditionally acknowledged for its deficiency in haemophilia A (HA), has been attracting interest for its substantial role in vascular disease. Recent data highlights its essential role beyond haemostasis, in the development of venous thrombosis (VT) and endothelial dysfunction influenced by genetic and acquired factors. This review summarizes the biology, synthesis, and activation of FVIII, emphasizing its role in thrombin generation and endothelial dysfunction. FVIII is implicated in inflammatory and thrombotic disorders, such as COVID-19, sepsis, and cancer-associated thrombosis. Although anticoagulant medication indirectly reduces elevated FVIII levels, direct intervention is constrained by the associated bleeding risks. Novel approaches like RNA interference, gene editing, and endothelial-specific modulation might offer compelling opportunities for the regulation of FVIII. This study highlights the potential of FVIII as a diagnostic and therapeutic target in thrombosis by integrating molecular insights with clinical data, hence promoting future precision-based therapies.
Platelets are key elements of hemostasis which play an important role in pathological processes, especially in the context of cancer. They are small, disc-shaped structures equipped with a rich network of organelles and a cytoskeleton. In healthy individuals, the range of the platelet count is 150-350*103/uL. They contain numerous receptors that are responsible for diverse cellular responses triggered by physiological and pathological signals. The process of their production, known as thrombopoiesis, is regulated by many factors, which allows stem cells to transform into megakaryocytes and facilitates further platelet production. In the context of cancer, platelets play a special role in thrombosis, which is a common complication in cancer patients. Moreover, through the cytokines and adhesion molecules they secrete, cancers affect platelets, increasing their prothrombotic potential. Chemotherapy and radiotherapy can also damage blood vessels, promoting thrombosis. Platelets also support the process of cancer metastasis, affecting different stages of its evolution. By forming aggregates with cancer cells and leukocytes, they protect cancer cells from mechanical forces in the bloodstream and from recognition by the immune system. In addition, platelets secrete growth factors such as PDGF and TGF-β, which support cancer cell proliferation and angiogenesis, which in turn stimulates further tumor development. Leukocytes, enter into cooperation with platelets, supporting the adhesion of cancer cells to the vascular endothelium and facilitating their migration to distant tissues. A high platelet count is an unfavorable prognostic factor in many cancers, associated with poorer survival rates, a greater tendency to form metastases, and lower efficacy of anticancer treatment. Thrombocytosis is also associated with an increased risk of thrombotic complications in cancer patients, which requires intensive monitoring and antithrombotic prophylaxis. Platelets can also increase the resistance of cancer cells to chemotherapy and radiotherapy by supporting DNA repair in damaged cancer cells and protecting them from cytotoxic drugs. Surface receptors such as P-selectin and GP IIb/IIIa, facilitate platelets in forming aggregates with CTCs, which enhances their survival and promotes the formation of metastases. Intensive research is underway concerning the possibility of using antiplatelet therapy, such as the use of aspirin or P2Y12 receptor inhibitors, as potential methods for treating cancer patients. Some studies have shown that aspirin can prevent the formation of pre-metastatic niches, and P2Y12 inhibitors, such as clopidogrel, reduce the incidence of metastases in animal models. In addition, platelet apheresis is gaining importance as a therapeutic method in cancer, particularly in the case of thrombocytosis. In summary, platelets play an important role not only in hemostasis, but also in the development and progression of cancer, which makes them an important target in the context of both antithrombotic and anticancer therapy. Their activity and number affect the course of the disease, the response to treatment and the risk of thrombotic complications, which emphasizes the need for further research on their role in oncology.
Combined oral contraceptives (COCs) remain one of the most popular reversible contraceptive methods worldwide. Still, regardless of the drug composition and duration of therapy, almost all COCs are associated with the risk of venous thrombosis. This review highlights the main pathogenetic mechanisms of thrombosis development during oral contraceptive use. Increase the production of certain clotting factors; a decrease in antithrombin and protein S levels; acquired resistance to activated protein C; a reduction in tissue factor pathway inhibitor (TFPI); indirect endothelial activation; inhibition of endogenous fibrinolysis; regulation of tissue factor by estradiol-sensitive microRNA; homocysteine imbalance caused by decreased intestinal reabsorption of folates and vitamin B-12; reduced bioavailability of nitric oxide (NO) due to high homocysteine levels; higher blood pressure, water retention, insulin resistance, increased levels of pro-inflammatory C-reactive protein (CRP) and uric acid, and antifibrinolytic (plasminogen activator inhibitor 1 type, PAI-1) biomarkers as consequences of NO deficiency; increased platelet adhesiveness and ADP-induced aggregation, which promote fibrinogen binding; and increased expression of pro-inflammatory cytokines are the main thrombotic effects of COCs use. Clinicians should carefully evaluate each patient's individual risk factors when prescribing COCs and conduct regular monitoring to reduce the risk of complications.
In situ pulmonary arterial thrombosis (iPAT), occurring without concurrent deep vein thrombosis, is a life-threatening complication associated with various pathological conditions. The etiological mechanisms underlying iPAT remain poorly understood. Several studies suggest that circulating tissue factor (cTF) may contribute to the development of thrombotic complications; however, there is no direct in vivo evidence supporting the role of cTF in the pathogenesis of iPAT. Furthermore, although in vitro studies suggest that platelet anionic phospholipids enable cTF-initiated coagulation, how platelets contribute to cTF-dependent iPAT in vivo remains unknown. In the current study, we used quantitative fluorescence intravital lung microscopy to investigate the development of cTF-induced iPAT in live mice following intravascular administration of thromboplastin. To dissect the interplay between coagulation and platelet procoagulant activity, we assessed the effects of coagulation and platelet inhibition on iPAT development in vivo. Additionally, we conducted an in vitro clotting time assay using mouse plasma samples. Thromboplastin triggered iPAT in mice in a dose-dependent manner. IPAT involved the formation of platelet-rich thrombi at the bottle-neck junctions of pulmonary arterioles and capillaries, which was prevented by heparin. Notably, pretreatment of mice with annexin A5 or eptifibatide also completely abrogated thromboplastin-induced iPAT. These intravital microscopy findings were further corroborated by the in vitro clotting time assay. Our study provides in vivo evidence that cTF contributes to the development of iPAT. We demonstrate that the prothrombotic effect of cTF is dependent on platelet-αIIbβ3 signaling, which enhances platelet procoagulant activity, leading to accelerated coagulation and development of iPAT.
Cardiovascular and cerebrovascular diseases are the leading causes of death worldwide. Pathological thrombosis is a major underlying mechanism, where excessive platelet activation triggered by vascular injury constitutes a key event. Liquiritigenin, a key flavonoid constituent of Glycyrrhiza glabra l., has been reported to possess diverse pharmacological properties, including anti-inflammatory, anti-tumor, neuroprotective, and cardiovascular protective effects. However, its specific role in platelet activation remains unclear. This study aims to explore the mechanism through which liquiritigenin inhibits platelet activation to attenuate thrombosis. The inhibitory effect of Liquiritigenin on platelet activation was evaluated using thrombin, ADP, and collagen as agonists. A co-culture system of platelets with human umbilical vein endothelial cells (HUVECs) on a collagen-coated surface was established to simulate platelet adhesion to vascular endothelia and subendothelial matrix under flow conditions. Flow cytometry, transmission electron microscopy, fluorescence labeling, and Western blotting were employed to assess P-selectin (CD62P) expression, dense granule secretion, and platelet adhesion following activation. The effects of Liquiritigenin on the Src/PLCγ2 signaling pathway, cytoplasmic Ca²⁺ mobilization, and the interaction between P2Y12R and the guanine nucleotide-binding protein Gi subunit alpha-1 (Gαi1) were further investigated. Additionally, integrated analysis of multiple single-cell transcriptome datasets was performed to identify cell types interacting with activated platelets in heart and lung tissues. The anti-thrombotic efficacy of liquiritigenin was evaluated in two in vivo models: acute pulmonary thromboembolism (APE) and coronary microvascular dysfunction (CMD). Liquiritigenin demonstrated a more potent inhibitory effect on ADP-activated platelets than on those activated by thrombin or collagen. It significantly suppressed ADP-induced P-selectin expression, dense granule secretion, and platelet adhesion to HUVECs. Furthermore, Liquiritigenin inhibited the ADP-triggered Src/PLCγ2 signaling pathway, reduced cytoplasmic Ca²⁺ mobilization, and attenuated the interaction between P2Y12R and Gαi1. Molecular modeling indicated that Liquiritigenin binds to key P2Y12R residues-LYS179, CYS97, and ASN191-which are also involved in ADP binding. In animal models, Liquiritigenin administration significantly reduced thrombus formation in the lung tissue of APE and within the coronary micro vessels of CMD model animals. The findings indicate that Liquiritigenin inhibits platelet activation by targeting P2Y12R. This action disrupts the Src/PLCγ2 signaling pathway and subsequently reduces cytoplasmic calcium mobilization. These results suggest that Liquiritigenin may have therapeutic potential for the treatment of thrombotic diseases associated with platelet activation.
Large, randomized trials testing omega-3 polyunsaturated fatty acid (ω-3 PUFA) supplementation to reduce cardiovascular events have reported contradictory results. Interpretation of these trials is challenging, because different dosages and formulations of ω-3 PUFA were tested. Furthermore, the exact mechanisms for the reduction in cardiovascular events are unclear. In this study, we investigated the effects of ω-3 PUFA on platelet adhesion, degranulation, and aggregation in vitro and in patients with cardiovascular disease using different formulations of ω-3 PUFA. We also investigated the effects of ω-3 PUFA in rodent models of arterial thrombosis and in tail bleeding assays, including in cyclooxygenase-1 (COX-1)-deficient animals. The ω-3 PUFA eicosapentaenoic acid (EPA) dose-dependently reduced platelet adhesion, degranulation, and aggregation in vitro. Moreover, arterial thrombus formation in wild-type mice was inhibited by oral EPA administration before thrombus formation. Photoaffinity labeling and in silico docking analyses suggested a direct, competitive interaction of EPA and arachidonic acid at the level of COX-1. The COX-1 dependency of EPA's inhibitory effects was confirmed by platelet-specific COX-1-deficient animals that had no reduction of thrombus burden by EPA. In patients with cardiovascular disease, switching from 2 grams of EPA twice daily to 1 gram of docosahexaenoic acid (DHA) (460 milligrams of EPA and 380 milligrams of DHA) once daily completely blunted the platelet inhibition achieved by EPA. Our results may partially explain contradictory results with different ω-3 PUFA formulations in clinical trials.
Serine-threonine kinase 10 (STK10) is a member of Ste20 family of serine/threonine kinases. Our previous study showed STK10 is expressed in platelets and regulates platelet function in arterial thrombosis. Whether it plays a role in venous thrombosis remains unclear. In this study, we aim to investigate the role of platelet STK10 in deep vein thrombosis (DVT) by using platelet-specific STK10 knockout mice. A DVT model was constructed via ligation of the inferior vena cava, and 24 hours later, an inferior vena cava sample was obtained for analysis of thrombi length and weight, the accumulation of platelets, neutrophils, platelet-neutrophil interaction, neutrophil extracellular traps (NET) formation, and platelet procoagulant function by immunofluorescence staining. During the development of DVT in mouse models, a significantly increased STK10 phosphorylation along with an increase of integrin-linked protein kinase phosphorylation (Ser343) was observed in the platelets. Deletion of platelet STK10 reduced the incidence and severity of DVT and inhibited platelet activation, platelet-neutrophil interaction, the recruitment and accumulation of platelets and neutrophils, and NET formation in the venous thrombin. In addition, absence of platelet STK10 inhibits NET formation induced by thrombin-stimulated platelets in vitro. Moreover, deficiency of platelet STK10 decreased platelet procoagulant activity in the peripheral blood and venous thrombi. Our study shows a novel regulatory role of platelet STK10 in the development and pathogenesis of DVT, implying that targeting platelet STK10 might be a novel approach for the prevention and treatment of venous thrombosis.
Platelet activation processes begin when injury to blood vessels exposes the subendothelial matrix, leading platelets to attach to it, where they become activated and exert their hemostatic function. Excessive platelet aggregation is associated with thrombotic disorders such as arterial thrombosis. To manage such diseases, medications that inhibit thrombosis are continuously sought, despite potential drawbacks that include hemorrhage. This study described the development of a novel peptide-based vaccine that targets the purinergic ADP P2Y1 receptor (abbreviated EL2Vac) and its pharmacological characterization. Thus, we designed and developed an EL2Vac that targets the ligand-binding domain of the P2Y1 receptor protein, which is located in its second extracellular loop (EL2). We then evaluated the vaccine's ability to trigger an immune response (antibody production) in immunized mice, modulate platelet function, its antithrombotic activity, and any effects on hemostasis, by employing a thrombosis model and the tail bleeding time assay. Results showed significant levels of antibody production in mice treated with EL2Vac, in comparison with the random peptide vaccine control (EL2rVac), which persisted at least up to six months post vaccination. Moreover, we observed significant inhibition of the ADP-induced aggregation response in platelets from EL2Vac-treated mice, relative to those from EL2rVac controls. This inhibition was selective for ADP, as other agonists, such as the thromboxane A2 receptor (TPR) agonist U46619 or high-dose collagen, had no detectable effect on aggregation. As for its capacity to protect against thrombosis, our data showed a significant delay in the occlusion time of the EL2Vac mice when compared with the random peptide control vaccine, which was also observed (at least) six months post vaccination. Interestingly, EL2Vac did not appear to prolong the tail bleeding time, supporting the notion that it is devoid of a bleeding diathesis. As a conclusion, this study documents the design and evaluation of a novel peptide-based vaccine, EL2Vac, which appears to selectively target the P2Y1 receptor and protect against thrombus formation without impairing hemostasis. Thus, EL2Vac may provide a promising clinical option to treat thromboembolic disorders.