FeCl3-induced Carotid Artery Thrombosis Research Articles

Dual Inhibition of Factor XIIa and Factor XIa Produces a Synergistic Anticoagulant Effect.

Clinical practice shows that a critical unmet need in the field of thrombosis prevention is the availability of anticoagulant therapy without bleeding risk. Inhibitors against FXIa or FXIIa have been extensively studied because of their low bleeding risk. However, whether these compounds produce synergistic effects has not yet been explored. In this study, analyses of activated partial thromboplastin time in combination with the FXIa inhibitor PN2KPI and the FXIIa inhibitor Infestin4 at different proportions were performed using the SynergyFinder tool identifying synergistic anticoagulation effects. Both an FeCl 3 -induced carotid artery thrombosis mouse model and a transient occlusion of the middle cerebral artery mouse model showed that the combination of PN2KPI and Infestin4, which are 28.57% and 6.25% of the effective dose, respectively, significantly prevents coagulation, and furthermore, dual inhibition does not cause bleeding risk.

Bat-derived oligopeptide LE6 inhibits the contact-kinin pathway and harbors anti-thromboinflammation and stroke potential.

Thrombosis and inflammation are primary contributors to the onset and progression of ischemic stroke. The contact-kinin pathway, initiated by plasma kallikrein (PK) and activated factor XII (FXIIa), functions bidirectionally with the coagulation and inflammation cascades, providing a novel target for therapeutic drug development in ischemic stroke. In this study, we identified a bat-derived oligopeptide from Myotis myotis (Borkhausen, 1797), designated LE6 (Leu-Ser-Glu-Glu-Pro-Glu, 702 Da), with considerable potential in stroke therapy due to its effects on the contact kinin pathway. Notably, LE6 demonstrated significant inhibitory effects on PK and FXIIa, with inhibition constants of 43.97 μmol/L and 6.37 μmol/L, respectively. In vitro analyses revealed that LE6 prolonged plasma recalcification time and activated partial thromboplastin time. In murine models, LE6 effectively inhibited carrageenan-induced mouse tail thrombosis, FeCl 3-induced carotid artery thrombosis, and photochemically induced intracerebral thrombosis. Furthermore, LE6 significantly decreased inflammation and stroke injury in transient middle cerebral artery occlusion models. Notably, the low toxicity, hemolytic activity, and bleeding risk of LE6, along with its synthetic simplicity, underscore its clinical applicability. In conclusion, as an inhibitor of FXIIa and PK, LE6 offers potential therapeutic benefits in stroke treatment by mitigating inflammation and preventing thrombus formation.

ACT001 improved cardiovascular function in septic mice by inhibiting the production of proinflammatory cytokines and the expression of JAK-STAT signaling pathway

Sepsis is a life-threatening multiple organ dysfunction syndrome (MODS) caused by a microbial infection that leads to high morbidity and mortality worldwide. Sepsis-induced cardiomyopathy (SIC) and coagulopathy promote the progression of adverse outcomes in sepsis. Here, we reported that ACT001, a modified compound of parthenolide, improved the survival of sepsis mice. In this work, we used cecal ligation and puncture (CLP) model to induce SIC. Transthoracic echocardiography and HE staining assays were adopted to evaluate the influence of ACT001 on sepsis-induced cardiac dysfunction. Our results showed that ACT001 significantly improved heart function and reduced SIC. Coagulation accelerates organ damage in sepsis. We found that ACT001 decreased blood clotting in the FeCl3-induced carotid artery thrombosis experiment. ACT001 also reduced the production of neutrophil extracellular traps (NETs). RNA-sequencing of heart tissues revealed that ACT001 significantly downregulated the expression of pro-inflammatory cytokines and the JAK-STAT signaling pathway. These results were confirmed with real-time PCR and ELISA. In summary, we found ACT001 rescued mice from septic shock by protecting the cardiovascular system. This was partially mediated by inhibiting pro-inflammatory cytokine production and down-regulating the JAK-STAT signaling.

Design, expression and biological evaluation of DX-88mut as a novel selective factor XIa inhibitor for antithrombosis

Thrombotic diseases, such as myocardial infarction, stroke, and deep vein thrombosis, severely threaten human health, and anticoagulation is an effective way to prevent such illnesses. However, most anticoagulant drugs in the clinic have different bleeding risks. Previous studies have shown that coagulation factor XI is an ideal target for safe anticoagulant drug development. Here, we designed the FXIa inhibitory peptide DX-88mut by replacing Loop1 (DGPCRAAHPR) and Loop2 (IYGGC) in DX-88, which is a clinical drug targeting PKa for the treatment of hereditary angioedema, using Loop1 (TGPCRAMISR) and Loop2 (FYGGC) in the FXIa inhibitory peptide PN2KPI, respectively. DX-88mut selectively inhibited FXIa against a panel of serine proteases with an IC50 value of 14.840 ± 0.453 nM, dose-dependently prolonged APTT in mouse, rat and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1 µmol/kg. Additionally, DX-88mut did not show a significant bleeding risk at a dose of 5 µmol/kg. Taken together, these results show that DX-88mut is a potential candidate for the development of a novel antithrombotic agent.

Isolation and Characterization of Poeciguamerin, a Peptide with Dual Analgesic and Anti-Thrombotic Activity from the Poecilobdella manillensis Leech

When Poecilobdella manillensis attacks its prey, the prey bleeds profusely but feels little pain. We and other research teams have identified several anticoagulant molecules in the saliva of P. manillensis, but the substance that produces the paralyzing effect in P. manillensis is not known. In this study, we successfully isolated, purified, and identified a serine protease inhibitor containing an antistasin-like domain from the salivary secretions of P. manillensis. This peptide (named poeciguamerin) significantly inhibited elastase activity and slightly inhibited FXIIa and kallikrein activity, but had no effect on FXa, trypsin, or thrombin activity. Furthermore, poeciguamerin exhibited analgesic activity in the foot-licking and tail-withdrawal mouse models and anticoagulant activity in the FeCl3-induced carotid artery thrombosis mouse model. In this study, poeciguamerin was found to be a promising elastase inhibitor with potent analgesic and antithrombotic activity for the inhibition of pain and thrombosis after surgery or in inflammatory conditions.

Mitochondrial pyruvate dehydrogenase kinases contribute to platelet function and thrombosis in mice by regulating aerobic glycolysis.

Resting platelets rely on oxidative phosphorylation (OXPHOS) and aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) for their energy requirements. In contrast, platelet activation exhibits an increased rate of aerobic glycolysis relative to OXPHOS. Mitochondrial enzymes pyruvate dehydrogenase kinases (PDKs) phosphorylate the pyruvate dehydrogenase (PDH) complex to inhibit its activity, thereby diverting the pyruvate flux from OXPHOS to aerobic glycolysis upon platelet activation. Of four PDK isoforms, PDK2 and PDK4 (PDK2/4) are predominantly associated with metabolic diseases. Herein, we report that the combined deletion of PDK2/4 inhibits agonist-induced platelet functions, including aggregation, integrin αIIbβ3 activation, degranulation, spreading, and clot retraction. Additionally, collagen-mediated PLCγ2 phosphorylation and calcium mobilization were significantly reduced in PDK2/4-/- platelets, suggesting impaired GPVI signaling. The PDK2/4-/- mice were less susceptible to FeCl3-induced carotid and laser-induced mesenteric artery thrombosis without any effect on hemostasis. In adoptive transfer experiments, thrombocytopenic hIL-4Rα/GPIbα-transgenic mice transfused with PDK2/4-/- platelets exhibited less susceptibility to FeCl3 injury-induced carotid thrombosis compared to hIL-4Rα/GPIbα-Tg mice transfused with WT platelets, suggesting a platelet-specific role of PDK2/4 in thrombosis. Mechanistically, the inhibitory effects of PDK2/4 deletion on platelet function were associated with reduced PDH phosphorylation and glycoPER in activated platelets, suggesting that PDK2/4 regulates aerobic glycolysis. Finally, using PDK2 or PDK4 single KO mice, we identified that PDK4 plays a more prominent role in regulating platelet secretion and thrombosis compared to PDK2. This study identifies the fundamental role of PDK2/4 in regulating platelet functions and identifies PDK/PDH axis as a potentially novel antithrombotic target.

Platelet ERO1α Is Crucial for Ca2+ Mobilization and Platelet Activation in Atherothrombosis

Endoplasmic reticulum oxidoreductase 1α (ERO1α) is a key oxidase of protein disulfide isomerase (PDI) during protein folding in the ER. Platelet ERO1α was reported to interact with PDI and αIIbβ3 integrin and regulate platelet function. However, the regulatory mechanism and pathophysiological role remain unknown. In the present study, we demonstrate that platelet ERO1α orchestrates intracellular Ca2+ signaling during platelet activation and contributes to thrombogenesis. Intravital microscopy using megakaryocyte-specific Ero1α conditional knockout (CKO) and KO mice reveals that platelet ERO1α is crucial for platelet thrombus formation in mouse models of laser-induced cremaster arteriolar thrombosis and FeCl3-induced carotid arterial thrombosis. Deletion of Ero1α does not affect bleeding times and blood loss at the site of tail amputation, implying that ERO1α is not required for hemostasis. Studies using Ero1α-null platelets, novel function-blocking antibodies, and small-molecule inhibitors reveal that intracellular, but not extracellular, ERO1α promotes P-selectin exposure, αIIbβ3 integrin activation, and platelet aggregation induced by multiple agonists, such as thrombin and collagen-related peptide. Importantly, the regulatory function of ERO1α is independent of PDI activity. Mass spectrometry and immunoprecipitation assays identify stromal interaction molecule 1 (STIM1) and sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) as ERO1α binding partners. We have found that ERO1α-STIM1 binding is enhanced during platelet activation, whereas ERO1α-SERCA2 binding is reduced. Intriguingly, platelet ERO1α interacts with STIM1 through Cys49 and Cys56 residues and promotes its function by oxidizing the Cys49-Cys56 disulfide bond, contributing to Ca2+ store content and enhancing Ca2+ mobilization during platelet activation. Using a high throughput screen, we identify a small-molecule ERO1α inhibitor, B20, which inhibits ERO1α activity with an IC50 value of 10.0 μM but does not affect the activities of H2O2 and PDI. Treatment of platelets with B20 recapitulates the functional defects as seen in Ero1α-null platelets. Intravenous injection of B20 and eptifibatide, an αIIbβ3 integrin antagonist, into WT mice significantly inhibits platelet thrombus formation following laser- or FeCl3-induced vascular injury. While treatment of mice with eptifibatide markedly prolongs tail bleeding times and causes blood loss, B20 does not impair hemostatic function. Furthermore, treatment with B20 reduces the infarct volume in ischemic stroke. These results indicate that platelet ERO1α is a novel regulator of Ca2+ signaling and promotes platelet activation and aggregation in a PDI-independent manner, contributing to the pathogenesis of arterial thrombosis and thromboinflammation. Targeting ERO1α may be an attractive therapeutic strategy for treating patients with thrombotic disease.

Bifunctional fusion protein targeting both FXIIa and FXIa displays potent anticoagulation effects

AimsAnticoagulation in disease treatment has been wildly studied in recent years. The intrinsic coagulation pathway is attracting attention of research community due to its low bleeding risk, and inhibitors against intrinsic coagulation factor XIIa (FXIIa) or XIa (FXIa) have been extensively studied. However, studies to develop anticoagulant inhibitors simultaneous targeting FXIIa and FXIa have not been reported. Our study aimed to evaluate the anticoagulation effect of the dual targeting of FXIIa and FXIa. Main methodsA fusion protein Infestin-PN2KPI (IP) was designed by linking FXIIa inhibitor Infesin4 and FXIa inhibitor PN2KPI through a rigid linker, and was cloned, expressed and characterized. The binding of IP to FXIIa and FXIa was verified by SPR, and inhibitory ability of IP against FXIIa and FXIa was verified by chromogenic substrate method. And then, the anticoagulation and antithrombotic functions of IP were extensively evaluated by aPTT assay, FeCl3-induced carotid artery thrombosis model and transient occlusion of the middle cerebral artery model. Key findingsIP significantly prolonged aPTT, inhibited thrombosis and prevented stroke at a dose of at least 1/2 lower than the effective dose of its component Infestin4 or PN2KPI, and did not cause bleed risk. SignificanceThe bifunctional fusion protein IP showed good anticoagulation effects, and simultaneous targeting FXIIa and FXIa is a promising strategy for anticoagulation drug development.

Pharmacological Investigation of Asphodelus tenuifolius Cav. for its Potential Against Thrombosis in Experimental Models.

BackgroundThrombosis is a major disorder which is an outcome of an imbalance in the hemostatic system that develop undesirable blood clot and hinder blood circulation.PurposeThe current study was designed to verify the potential of aqueous methanolic crude extract of Asphodelus tenuifolius Cav. (At.Cr), used traditionally as remedy in circulatory problems.Research DesignAntioxidant activity, FTIR, and HPLC analysis were performed. In-vitro clot lysis assay was performed on human blood samples, and in-vivo acute pulmonary thromboembolism model was developed by administering the mixture of collagen and epinephrine in tail vein of mice. Carrageenan-induced thrombosis and FeCl3-induced carotid arterial thrombosis models were developed in rats.ResultsAt.Cr demonstrated significant increase in lysis of human blood clot. Bleeding and clotting times were increased dose-dependently. Lungs histology showed clear alveolar spaces with decreased red blood cells congestion. Reduction in infarcted tail length, augmentation in prothrombin time, and activated partial thromboplastin time with decrease in platelet count were observed. At.Cr also prolonged the arterial occlusion time and reduced the weight of thrombus and TXB2 levels dose-dependently.ConclusionsThe results demonstrated the antithrombotic and thrombolytic potential of At.Cr due to activation of coagulation factors through extrinsic and intrinsic pathways.

S298: CS585 IS A FIRST-IN-CLASS COMPOUND TARGETING THE IP RECEPTOR FOR PREVENTION OF THROMBOSIS WITHOUT INCREASED RISK OF BLEEDING

Background: Uncontrolled platelet activation leads to the formation of occlusive thrombi resulting in myocardial infarction, stroke, VTE and critical limb ischemia. There is an unmet need for more efficacious anti-thrombotics with less bleeding in this area. Our group recently identify a novel oxidized lipid in the blood that potently and selectively inhibits platelet activation through activation of the prostacyclin receptor. Aims: Develop a first-in-class antiplatelet drug, CS585, that potently and selectively inhibits platelet reactivity and thrombosis without altering hemostasis and bleeding risk. Methods: We developed a mimetic of the oxidized lipid shown to selectively bind to and activate the prostacyclin receptor. Using human platelets, we assessed the ability of CS585 to inhibit platelet activation by assessing 1)aggregometry, 2)adhesion under arterial flow, and 3) granule secretion and integrin activation using flow cytometry. We additionally assessed thrombosis in vivo in 2 different mouse models as well as bleeding. Finally, we assessed potential off-target effects using thromboelastography (TEG). Results: CS585 potently inhibited both collagen and thrombin-induced platelet aggregation. This inhibition was confirmed by measuring inhibition of αIIbβ3 activation, α-granule secretion, and dense-granule secretion by flow cytometry. Selectivity was confirmed in human platelets by demonstrating a full reversal of inhibition when the IP receptor was pharmacologically blocked or genetically eliminated in IP receptor knockout mice. In vivo inhibition of injury-induced thrombosis in the small (laser-induced cremaster thrombosis model) and large (FeCl3-induced carotid artery thrombosis model) vessels by CS585 was demonstrated and no increased risk for bleeding was observed using the tail vein bleeding model. Finally, TEG experiments in human blood spiked with CS585 demonstrated no delay or decrease in clot strength confirming the tail vein bleeding assay experiments in the mouse. Summary/Conclusion: We have shown for the first time that CS585, a first-in-class analog of an oxidized lipid in the blood potently and selectively activates the prostacyclin receptor resulting in inhibition of human and mouse platelet activation and thrombosis without an increased risk of bleeding. This discovery represents a new class of inhibitors for prevention of platelet activation and thrombosis and protection from myocardial infarction, stroke, VTE, and critical limb ischemia.

CT-001 is a rapid clearing factor VIIa with enhanced clearance and hemostatic activity for the treatment of acute bleeding in non-hemophilia settings

IntroductionAcute bleeding leads to significant morbidity and mortality. Recombinant wildtype Factor VIIa (WT FVIIa) had been reported to have some therapeutic effects in some clinical trials, however, its use was associated with thromboembolic events. We sought to develop a novel FVIIa molecule (CT-001) with enhanced activity and lowered thrombogenicity risk. Methods and methodsCT-001 has 4 N-glycans (T106N/N145/V253N/N322) with terminal sialic acid residues removed to promote active clearance via the asialoglycoprotein receptor, and P10Q/K32E substitutions introduced to its gamma-carboxyglutamic acid (Gla) domain for enhanced phospholipid affinity and activity. ResultsIn mice, CT-001 had a half-life of 5 min and a clearance of 467 mL/h/kg at 3 mg/kg, significantly faster than WT FVIIa (t1/2 = 1.8 h, Cl = 39 mL/h/kg). Interestingly, CT-001 was efficacious in reducing blood loss even with its rapid clearance. In a severe hemorrhage mouse model with tail amputated 5 cm from the tip, 1 mg/kg CT-001 provided efficacy comparable to 3 mg/kg WT FVIIa. The fast clearance of CT-001 resulted in significantly reduced thrombogenicity in comparison to WT FVIIa in a FeCl3-induced carotid artery thrombosis mouse model, and further confirmed in a soluble tissue factor-induced thrombosis model. ConclusionsThe data on CT-001 demonstrate that a short duration of highly active FVIIa procoagulant activity has the potential to be an optimal paradigm for the treatment of acute bleeds.

WPK5, a Novel Kunitz-Type Peptide from the Leech Whitmania pigra Inhibiting Factor XIa, and Its Loop-Replaced Mutant to Improve Potency.

Kunitz-type proteins or peptides have been found in many blood-sucking animals, but the identity of them in leeches remained elusive. In the present study, five Kunitz-type peptides named WPK1-WPK5 were identified from the leech Whitmania pigra. Recombinant WPK1-WPK5 were expressed in Pichia pastoris GS115, and their inhibitory activity against Factor XIa (FXIa) was tested. WPK5 showed inhibitory activity against FXIa with an IC50 value of 978.20 nM. To improve its potency, the loop replacement strategy was used. The loop 1 (TGPCRSNLER) and loop 2 (QYGGC) in WPK5 were replaced by loop 1 (TGPCRAMISR) and loop 2 (FYGGC) in PN2KPI, respectively, and the resulting peptide named WPK5-Mut showed an IC50 value of 8.34 nM to FXIa, which is about 100-fold the potency of FXIa compared to that of WPK5. WPK5-Mut was further evaluated for its extensive bioactivity in vitro and in vivo. It dose-dependently prolonged APTT on both murine plasma and human plasma, and potently inhibited FeCl3-induced carotid artery thrombosis in mice at a dose of 1.5 mg/kg. Additionally, WPK5-Mut did not show significant bleeding risk at a dose of 6 mg/kg. Together, these results showed that WPK5-Mut is a promising candidate for the development of an antithrombotic drug.

Investigating the Roles of Platelet PAR4 in Hemostasis, Thrombosis and Viral Infection Using a Newly Generated PAR4 Floxed Mouse

Introduction: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes and lung epithelial cells, and activated by multiple ligands including thrombin and cathepsin G. Importantly, PAR4 is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and protection from thrombosis which are attributed to loss of platelet PAR4, but this has not been specifically demonstrated in mice. Additionally, global PAR4 deficiency increases mortality after influenza A virus infection, but the cell type/s responsible for the enhanced mortality have not been determined. Here, we describe the generation of PAR4 floxed (PAR4 fl/fl) mice that can be used to delete PAR4 in a cell type-specific manner, and examine the effect of megakaryocyte/platelet-specific deletion of PAR4 on hemostasis, thrombosis and viral infection using PAR4 fl/fl;PF4Cre + mice.Methods: PAR4global knockout (PAR4 -/-), MK/platelet-specific knockout (PAR4 fl/fl;PF4Cre +) and appropriate littermate control mice were used for experiments. Platelet function was determined by light transmission aggregometry and flow cytometry. Hemostasis was assessed in the saphenous vein laser injury model. Platelet plug formation was visualized by intravital microscopy following saphenous vein laser ablation (~50 μm diameter injury), followed by 2 subsequent ablations to reinjure the same site unless on-going bleeding was occurring. Mice were treated with ibrutinib (12.5 mg/kg) to inhibit GPVI signaling, or dabigatran etexilate (chow containing 10 mg/g) or recombinant hirudin (50 mg/kg) to inhibit thrombin activity. Thrombosis was assessed in the carotid artery FeCl 3 model. The carotid artery was exposed and 8% FeCl 3 applied for 3 mins. Blood flow was observed for 30 mins and occlusion was defined as no blood flow for 2 mins. To study susceptibility to viral infection, mice were challenged intranasally with a mouse-adapted H1N1 influenza A virus (H1N1 IAV PR8; 0.02 hemagglutination assay units), which induces mortality in 20% of WT mice. Mortality was defined as body weight loss greater than 25%, which required euthanasia.Results: As expected, PAR4 fl/fl;PF4Cre + platelets were unresponsive to thrombin or PAR4-specific stimulation, while the response to other agonists was retained. In the saphenous vein laser injury hemostasis model, PAR4 fl/fl;PF4Cre + mice were able to rapidly form a hemostatic platelet plug, but the majority of plugs (7/8) were unstable and re-opened after several minutes, leading to severely prolonged total bleeding times. We observed similar findings in global PAR4 -/- mice with 8/12 plugs re-opening. To investigate the mechanism mediating initial platelet plug formation, we inhibited GPVI signaling in PAR4 fl/fl;PF4Cre + mice using the Btk inhibitor ibrutinib. Ibrutinib administration shortened time to plug re-opening in PAR4 fl/fl;PF4Cre + mice but plugs were still able to form, which is likely mediated by GPIbα/VWF. We observed a similar phenotype to PAR4 fl/fl;PF4Cre + mice in mice treated with the direct thrombin inhibitor hirudin, suggesting thrombin is the primary activator of PAR4 during hemostatic plug formation. In the FeCl 3-induced carotid artery thrombosis model, both PAR4 fl/fl;PF4Cre + and PAR4 -/- mice were significantly protected compared to controls. Finally, when challenged with the mouse-adapted H1N1 IAV PR8, PAR4 fl/fl;PF4Cre + mice demonstrated similar body weight loss and survival as littermate controls.Conclusions: Our results in mice demonstrate that 1) platelet PAR4 is not required for initial hemostatic plug formation but is necessary for maintaining hemostatic plug stability, 2) loss of platelet PAR4 protects from arterial thrombosis, and 3) platelet PAR4 does not alter the course of H1N1 IAV infection, at least at the virus dose used in this study. In summary, we generated a novel mouse line carrying a floxed PAR4 allele which can be used to investigate cell-specific roles of PAR4 in disease. DisclosuresNo relevant conflicts of interest to declare.

Pyruvate Dehydrogenase Kinase Modulates Platelet Function and Thrombosis

Background and objective: Pyruvate dehydrogenase kinases (PDK 1-4) are mitochondrial enzymes that regulate the activity of pyruvate dehydrogenase (PDH) complex, which converts pyruvate (end product of glycolysis in cytosol) to acetyl coenzyme A that is further oxidized to produce energy (oxidative phosphorylation; OXPHOS). Like most normal cells, resting platelets rely primarily on OXPHOS to generate ATP, whereas activated platelets exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen, a phenomenon referred to as the Warburg effect in tumor cells). It seems paradoxical that why platelets prefer aerobic glycolysis instead of OXPHOS. The rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which is well suited for high-energy demand of activated platelets to form blood clot. We tested the hypothesis that manipulating aerobic glycolysis (metabolic reprogramming), by down regulating PDK activity in the mitochondria of platelets, will inhibit platelet function and thereby thrombosis.Methods: Global knock out or floxed PDK mice does not exists, therefore we used dichloroacetic acid (DCA), a potent inhibitor of PDKs, known to divert metabolism back from aerobic glycolysis to OXPHOS. DCA has been successfully used in clinical trials to treat several type of cancer. In human and murine platelets, using standardized platelet in vitro assays, we determined the mechanistic role of PDK in regulating platelet function. Rate of glycolysis and mitochondrial respiration was measured using the Seahorse XF24 extracellular flux analyzer. Using intravital microscopy, thrombus growth was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid artery thrombosis) models.Results: Using, extracellular flux analysis, we first confirmed that activated platelets have higher aerobic glycolysis. With both human and mice platelets, we found that DCA (10 and 25 mM) significantly inhibited in vitro platelet aggregation to collagen and convulxin-induced signaling, but not thrombin or ADP-induced signaling in a dose-dependent manner (P<0.05 vs. vehicle treated). DCA also inhibited static adhesion of washed platelets to collagen in dose-dependent manner. Furthermore, we found that DCA significantly inhibited tyrosine phosphorylation of Syk and PLCγ2 in the GPVI signaling pathway with both mice and human platelets (P<0.05 vs. vehicle treated). Metabolic profiling, using extracellular flux analysis, showed that DCA significantly inhibited extracellular acidification rate in convulxin-stimulated platelets in dose-dependent manner. Using microfluidics flow chamber, we found that both mouse and human whole blood treated with DCA formed small thrombi when perfused over collagen for 10 min at a arterial shear rate of 1500s-1 (P<0.05 vs. vehicle control), suggesting that inhibiting PDK with DCA limits thrombus growth. Finally, wild-type mice pre-treated with DCA (600 mg/Kg body weight) were less susceptible to thrombosis in FeCl3-induced arterial thrombosis model (P<0.05 vs. control, N=10 mice/group), without altered hemostasis (tail-bleeding assay). The precise mechanism how aerobic glycolysis modulates GPVI signaling pathway is unclear and is currently under investigation.Conclusion: Our results suggest that PDK present in mitochondria regulates platelet function and thrombus growth, most likely via GPVI signaling pathway. DisclosuresNo relevant conflicts of interest to declare.

Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation.

Sanguinarine, a benzophenanthridine alkaloid, has been described to have an antiplatelet activity. However, its antithrombotic effect and the mechanism of platelet inhibition have not thoroughly been explored. The current study found that sanguinarine had an inhibitory effect on thrombus formation. This inhibitory effect was quite evident both in the flow-chamber assays as well as in a murine model of FeCl3-induced carotid artery thrombosis. Further investigations also revealed that sanguinarine inhibited the collagen-induced human platelet aggregation and granule release. At the same time, it also prevented platelet spreading and adhesion to immobilized fibrinogen. The molecular mechanisms of its antiplatelet activity were found to be as follows: 1. Reduced phosphorylation of the downstream signaling pathways in collagen specific receptor GPVI (Syk-PLCγ2 and PI3K-Akt-GSK3β); 2. Inhibition of collagen-induced increase in the intracellular Ca2+ concentration ([Ca2+]i); 3. Inhibition of integrin αIIbβ3 outside-in signaling via reducing β3 and Src (Tyr-416) phosphorylation. It can be concluded that sanguinarine inhibits collagen-induced platelet activation and reduces thrombus formation. This effect is mediated via inhibiting the phosphorylation of multiple components in the GPVI signaling pathway. Current data also indicate that sanguinarine can be of some clinical value to treat cardiovascular diseases involving an excess of platelet activation.

Anti-thrombotic effects mediated by dihydromyricetin involve both platelet inhibition and endothelial protection

Classical antithrombotics and antiplatelets are associated with high frequencies of bleeding complications or treatment failure when used as single agents. The platelet-independent fibrin generation by activated endothelium highlights the importance of vascular protection in addition to platelet inhibition in thrombosis prevention. Dihydromyricetin (DHM), the most abundant flavonoid in Ampelopsis grossedentata, has unique vasoprotective effects. This study aims to characterize the antithrombotic potential of DHM. The effects of DHM on the activation of platelets and endothelial cells were evaluated in vitro. Calcium mobilization and activation of mitogen-activated protein kinases (MAPKs) were examined as the potential targets of DHM based on molecular docking analysis. The in vivo effects of DHM were determined in FeCl3-injured carotid arteries and laser-injured cremasteric arterioles. The results showed that DHM suppressed a range of platelet responses including aggregation, secretion, adhesion, spreading and integrin activation, and inhibited exocytosis, phosphatidylserine exposure and tissue factor expression in activated endothelial cells. Mechanistically, DHM attenuated thrombin-induced calcium mobilization and phosphorylation of ERK1/2 and p38 both in platelets and endothelial cells. Intravenous treatment with DHM delayed FeCl3-induced carotid arterial thrombosis. Furthermore, DHM treatment inhibited both platelet accumulation and fibrin generation in the presence or absence of eptifibatide in the laser injury-induced thrombosis model, without prolonging ex vivo plasma coagulation or tail bleeding time. DHM represents a novel antithrombotic agent whose effects involve both inhibition of platelet activation and reduction of fibrin generation as a result of endothelial protection.

Prevention of platelet aggregation and arterial thrombosis using a modified Shenzhu Guanxin Formula.

ObjectiveModified Shenzhu Guanxin Formula (mSGF) has beneficial effects in coronary artery disease. Previously, we found that mSGF inhibited platelet aggregation in rats. In the present study we further investigated the antiplatelet and antithrombotic activities of mSGF in rats.MethodsRats were orally administered mSGF (4.2, 8.4, or 16.8 g crude drug/kg), the adenosine 5′-diphosphate (ADP) receptor antagonist clopidogrel (7.875 mg/kg), or saline once a day for 7 days. The effects of mSGF on platelet aggregation were measured. Levels of cyclic adenosine monophosphate (cAMP) and phosphoinositide 3-kinase (PI3K) signaling were analyzed by ELISA and western blotting, respectively. The antithrombotic effect of mSGF was investigated using a FeCl3-induced carotid arterial thrombosis model and effects on bleeding time were assessed in a rat tail transection model.ResultsmSGF significantly inhibited ADP-induced platelet aggregation in a dose-dependent manner, elevated cAMP levels and inhibited phosphorylation of extracellular signal-regulated kinase (ERK) and PI3K/protein kinase B (Akt). Moreover, mSGF dose-dependently inhibited thrombosis in a FeCl3-induced carotid arterial thrombus model and had a significantly smaller effect on bleeding time compared with clopidogrel.ConclusionsmSGF represents a potent and safe antithrombotic agent whose antiplatelet activity is probably mediated through blockade of PI3K/Akt signaling and increased cAMP generation.

Antiplatelet Activity of Tussilagone via Inhibition of the GPVI Downstream Signaling Pathway in Platelets.

Tussilagone is a sesquiterpenoid extracted from Tussilago farfara and is used as an oriental medicine for asthma and bronchitis. Although previous studies have shown that tussilagone has an inhibitory effect on platelet aggregation, no studies have been performed to investigate its precise effect on platelets, and the underlying mechanism remains unclear. In the present study, we showed that tussilagone inhibited platelet aggregation induced by collagen, thrombin and ADP, as well as platelet release induced by collagen and thrombin, in mice. Tussilagone decreased P-selectin expression and αIIbβ3 activation (JON/A binding) in activated platelets, which indicated that tussilagone inhibited platelet activation. Moreover, tussilagone suppressed platelet spreading on fibrinogen and clot retraction. The levels of phosphorylated Syk, PLCγ2, Akt, GSK3β, and MAPK (ERK1/2 and P38) and molecules associated with GPVI downstream signaling were downregulated in the presence of tussilagone. In addition, tussilagone prolonged the occlusion time in a mouse model of FeCl3-induced carotid artery thrombosis and had no effect on mouse tail bleeding time. These results indicate that tussilagone inhibits platelet function in vitro and in vivo and that the underlying mechanism involves the Syk/PLCγ2-PKC/MAPK and PI3K-Akt-GSK3β signaling pathways downstream of GPVI. This research suggests that tussilagone is a potential candidate antiplatelet drug for the prevention of thrombosis.

Targeting Metabolic Enzyme Pyruvate Kinase M2: A Novel Strategy to Inhibit Platelet Function and Arterial Thrombosis

Background: The cellular responses initiated upon platelet activation are energy consuming. Activated platelets, in comparison to their resting state, exhibit a high level of aerobic glycolysis (conversion of glucose to lactate in the presence of oxygen) relative to oxidative phosphorylation (OXPHOS), suggesting that metabolic plasticity exists in platelets. Although aerobic glycolysis yields less total ATP when compared to OXPHOS, the rate of ATP generation is faster in aerobic glycolysis compared to OXPHOS, which we hypothesize is well suited for high-energy requirement during platelet activation. The glycolytic enzyme pyruvate kinases (PKs) catalyzes the final step of glycolysis and contributes to net ATP production. Four PK isoforms (L, R, M1 and M2) exist in mammals: L and R isoforms are expressed in the liver and red blood cells; the M1 isoform is expressed in most adult tissues that have high catabolic demands including muscle and brain; M2 is expressed in cells including activated platelets and leukocytes. Unlike other isoforms of PK that function only as tetramers, PKM2 can exist in either a tetrameric state or a dimeric state. PKM2 is allosterically regulated by the upstream metabolite fructose-1, 6 biphosphate. While PKM1 and tetrameric PKM2 favor ATP production from OXPHOS through the TCA cycle, dimeric PKM2 drives aerobic glycolysis. The glycolytic and non-glycolytic functions of PKM2 in platelets have not investigated yet. Objective: We tested an innovative concept that whether targeting metabolic enzyme PKM2 will inhibit platelet function and arterial thrombosis. Methods: Using a specific inhibitor of PKM2 (that prevents PKM2 dimerization and stabilizes tetramers) and a range of standardized platelet in vitro assays, we determined the mechanistic role of PKM2 in modulating platelet function in human and mice. To provide definitive evidence, we generated a megakaryocyte or platelet-specific PKM2-/- mouse (PKM2fl/flPF4Cre). Susceptibility to thrombosis was evaluated in vitro (microfluidics flow chamber) and in vivo (FeCl3-induced carotid and laser-injury induced mesenteric artery thrombosis models) by utilizing intravital microscopy. Susceptibility to hemostasis was evaluated in tail bleeding assay. Results: Human and mouse platelets pretreated with PKM2 inhibitor significantly decreased platelet aggregation to sub-optimal doses of collagen, convulxin, thrombin, and ADP. Consistent with this, inhibiting PKM2 dimerization reduced αIIbβ3 activation, alpha and dense granule secretion, clot retraction that was concomitant with decreased glucose uptake. Furthermore, treatment with PKM2 inhibitor reduced Akt and GSK3β phosphorylation, that are predominantly involved in PI3K/Akt signaling, suggesting a non-glycolytic role of the PKM2 in regulating platelet function. In microfluidics flow chamber assay, human and whole mouse blood pretreated with PKM2 inhibitor formed small thrombi when perfused over collagen for 5 minutes at an arterial shear rate of 1500s-1 (P&lt;0.05 vs. vehicle). In agreement with PKM2 inhibitor studies, platelets from PKM2fl/flPF4Cre mice exhibited decreased agonist-induced platelet aggregation, which was in agreement with decreased alpha and dense granule secretion, αIIbβ3 activation, clot retraction, lactate production, and Akt and GSK3β phosphorylation (P&lt;0.05 vs. PKM2fl/fl littermate controls). Wild-type mice-treated with PKM2 inhibitor and/or PKM2fl/flPF4Cre were less susceptible to thrombosis in the FeCl3-induced carotid and laser injury-induced mesenteric artery thrombosis models. Lack of effect on tail bleeding time suggested normal hemostasis in PKM2fl/flPF4Cre mice and PKM2 inhibitor-treated wild-type mice. No sex-based differences were observed. Currently, we are performing platelet metabolomics to determine the effect of targeting PKM2 on metabolic pathways. Conclusions: Our results suggest that manipulating metabolic plasticity by targeting dimeric PKM2 may be explored as a novel strategy to inhibit platelet function and arterial thrombosis. Disclosures Lentz: Novo Nordisk Inc.: Consultancy, Honoraria, Research Funding.

A bacterial metabolite, trimethylamine N-oxide, disrupts the hemostasis balance in human primary endothelial cells but no coagulopathy in mice.

: The gut microbial metabolite, trimethylamine N-oxide (TMAO), was previously reported to induce platelet hypersensitivity, which leads to thrombotic risk. However, the molecular mechanism underlying the effects of TMAO on endothelial cells (EC), which is the primary vessel wall contact with the lumen, remains unclear. Here, we investigated the impact of TMAO on procoagulant activity (PCA) in EC and mice, for a possible link between microbiota and coagulation. To test the PCA of TMAO in EC, we performed one-stage clotting assays and converted into PCA. Antitissue factor (TF) antibody was used to test the TF role in PCA. Quantitative PCR was performed to measure the TF, thrombomodulin, IL-6, TF pathway inhibitor and IL-1b expressions at mRNA levels. To test the PCA and thrombotic risk by TMAO in mice, we challenged the mice with TMAO (8 mg/kg; 3 h) and measured the thrombin-anti-thrombin complex (TAT) and D-dimer levels as well as ferric chloride (FeCl3)-induced carotid artery thrombosis model. TMAO-induced TF expression in EC at mRNA and protein levels, dose-dependently. TF blocking experiment confirmed that the increased PCA by TMAO is TF-dependent. Also, mitogen-activated protein kinase pathway inhibitors abolished TMAO-induced TF expression. However, TMAO challenged mice failed to develop systemic activation of coagulation (TAT and D-dimer), as well as a FeCl3-induced carotid arterial thrombosis model. Our results indicated that TMAO triggered TF-dependent PCA via activation of nuclear factor-κB and downregulated thrombomodulin expression in human EC, but failed to develop systemic activation of coagulation in mice.