Paclitaxel exerts antiplatelet and antithrombotic activities: Additional benefit from use of paclitaxel-coated balloons and -eluting stents in coronary revascularization and prevention of in-stent restenosis.

INTRODUCTION: Paclitaxel is a microtubule-stabilizing drug used to treat several types of cancer, including ovarian and breast cancer. Because of its antiproliferative effect on vascular smooth muscle cells, balloons and stents are coated with paclitaxel for use in coronary revascularization and prevention of in-stent restenosis (ISR). However, mechanisms underlying ISR are complicated. Platelet activation is one of the major causes of ISR after percutaneous coronary intervention. Although the antiplatelet activity of paclitaxel was noted in rabbit platelets, the effect of paclitaxel on platelets remains unclear. This study investigated whether paclitaxel exhibits antiplatelet activity in human platelets. METHODS AND RESULTS: Paclitaxel inhibited platelet aggregation induced by collagen but not that induced by thrombin, arachidonic acid, or U46619, suggesting that paclitaxel is more sensitive to the inhibition of collagen-induced platelet activation. Moreover, paclitaxel blocked collagen receptor glycoprotein (GP) VI downstream signaling molecules, including Lyn, Fyn, PLCγ2, PKC, Akt, and MAPKs. However, paclitaxel did not directly bind to GPVI and cause GPVI shedding, as detected by surface plasmon resonance and flow cytometry, respectively, indicating that paclitaxel may interfere with GPVI downstream signaling molecules, such as Lyn and Fyn. Paclitaxel also prevented granule release and GPIIbIIIa activation induced by collagen and low convulxin doses. Moreover, paclitaxel attenuated pulmonary thrombosis and delayed platelet thrombus formation in mesenteric microvessels without significantly affecting hemostasis. CONCLUSION: Paclitaxel exerts antiplatelet and antithrombotic effects. Thus, paclitaxel may provide additional benefits beyond its antiproliferative effect when used in drug-coated balloons and drug-eluting stents for coronary revascularization and prevention of ISR.

Artesunate as a glycoprotein VI antagonist for preventing platelet activation and thrombus formation.

Platelets play a crucial role on hemostasis and are also involved in cardiovascular diseases, such as heart attack and stroke. Artesunate has been reported to possess multiple biological activities, including antitumor and anti-inflammatory activities. However, its effect on platelet activation remains unclear. Thus, we explored the detailed mechanisms underlying its antiplatelet effect. For the in vitro study, the data indicated that artesunate inhibited platelet aggregation induced by collagen, but not thrombin or U46619, indicating that artesunate may selectively inhibit collagen-mediated platelet activation Artesunate also blocked glycoprotein VI (GPVI) downstream signaling, including Syk, PLCγ2, PKC, Akt, and MAPKs. Moreover, artesunate could compete with collagen for binding to collagen receptor and bind to human recombinant GPVI with a high affinity (KD = 44 nM), indicating that it may directly interfere with GPVI. Artesunate also reduced collagen-induced granule release, calcium mobilization, and GPIIbIIIa activation. For the in vivo study, artesunate markedly prevented pulmonary thrombosis and delayed platelet thrombus formation in mesenteric veins and arteries but had minimal effects on hemostasis. In conclusion, we for the first time demonstrated that artesunate acts as a GPVI antagonist and effectively prevents platelet activation and thrombus formation with minimal risk of bleeding, highlighting its therapeutic potential in cardiovascular diseases.

Corrigendum to "Auraptene, a Monoterpene Coumarin, Inhibits LTA-Induced Inflammatory Mediators via Modulating NF-κB/MAPKs Signaling Pathways".

[This corrects the article DOI: 10.1155/2021/5319584.].

Auraptene, a Monoterpene Coumarin, Inhibits LTA-Induced Inflammatory Mediators via Modulating NF-κB/MAPKs Signaling Pathways.

OBJECTIVE: Oxidative stress-mediated inflammatory events involve in the progress of several diseases such as asthma, cancers, and multiple sclerosis. Auraptene (AU), a natural prenyloxycoumarin, possesses numerous pharmacological activities. Here, the anti-inflammatory effects of AU were investigated in lipoteichoic acid- (LTA-) induced macrophage cells (RAW 264.7). METHODS: The expression of cyclooxygenase (COX-2), tumor necrosis factor (TNF-α), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase (iNOS) and the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, c-Jun N-terminal kinase (JNK), heme oxygenase (HO-1), p65, and IκBα were all identified by western blotting assay. The level of nitric oxide (NO) was measured by spectrometer analysis. The nuclear translocation of p65 nuclear factor kappa B (NF-κB) was assessed by the confocal microscopic staining method. Native polyacrylamide gel electrophoresis was performed to perceive the activity of antioxidant enzyme catalase (CAT). RESULTS: AU expressively reduced NO production and COX-2, TNF-α, IL-1 ß, and iNOS expression in LTA-stimulated cells. AU at higher concentration (10 µM) inhibited ERK and JNK, but not p38 phosphorylation induced by LTA. Moreover, AU blocked IκB and p65 phosphorylation, and p65 nuclear translocation. However, AU pretreatment was not effective on antioxidant HO-1 expression, CAT activity, and reduced glutathione (GSH, a nonenzymatic antioxidant), in LTA-induced RAW 264.7 cells. CONCLUSION: The findings of this study advocate that AU shows anti-inflammatory effects via reducing NF-κB/MAPKs signaling pathways.

Influence of Vincristine, Clinically Used in Cancer Therapy and Immune Thrombocytopenia, on the Function of Human Platelets.

Vincristine is a clinically used antimicrotubule drug for treating patients with lymphoma. Due to its property of increasing platelet counts, vincristine is also used to treat patients with immune thrombocytopenia. Moreover, antiplatelet agents were reported to be beneficial in thrombotic thrombocytopenic purpura (TTP). Therefore, we investigated the detailed mechanisms underlying the antiplatelet effect of vincristine. Our results revealed that vincristine inhibited platelet aggregation induced by collagen, but not by thrombin, arachidonic acid, and the thromboxane A2 analog U46619, suggesting that vincristine exerts higher inhibitory effects on collagen-mediated platelet aggregation. Vincristine also reduced collagen-mediated platelet granule release and calcium mobilization. In addition, vincristine inhibited glycoprotein VI (GPVI) signaling, including Syk, phospholipase Cγ2, protein kinase C, Akt, and mitogen-activated protein kinases. In addition, the in vitro PFA-100 assay revealed that vincristine did not prolong the closure time, and the in vivo study tail bleeding assay showed that vincristine did not prolong the tail bleeding time; both findings suggested that vincristine may not affect normal hemostasis. In conclusion, we demonstrated that vincristine exerts antiplatelet effects at least in part through the suppression of GPVI signaling. Moreover, this property of antiplatelet activity of vincristine may provide additional benefits in the treatment of TTP.

A novel naphthalimide derivative reduces platelet activation and thrombus formation via suppressing GPVI.

Naphthalimide derivatives have multiple biological activities, including antitumour and anti-inflammatory activities. We previously synthesized several naphthalimide derivatives; of them, compound 5 was found to exert the strongest inhibitory effect on human DNA topoisomerase II activity. However, the effects of naphthalimide derivatives on platelet activation have not yet been investigated. Therefore, the mechanism underlying the antiplatelet activity of compound 5 was determined in this study. The data revealed that compound 5 (5-10 µM) inhibited collagen- and convulxin- but not thrombin- or U46619-mediated platelet aggregation, suggesting that compound 5 is more sensitive to the inhibition of glycoprotein VI (GPVI) signalling. Indeed, compound 5 could inhibit the phosphorylation of signalling molecules downstream of GPVI, followed by the inhibition of calcium mobilization, granule release and GPIIb/IIIa activation. Moreover, compound 5 prevented pulmonary embolism and prolonged the occlusion time, but tended to prolong the bleeding time, indicating that it can prevent thrombus formation but may increase bleeding risk. This study is the first to demonstrate that the naphthalimide derivative compound 5 exerts antiplatelet and antithrombotic effects. Future studies should modify compound 5 to synthesize more potent and efficient antiplatelet agents while minimizing bleeding risk, which may offer a therapeutic potential for cardiovascular diseases.

Platelet autophagic machinery involved in thrombosis through a novel linkage of AMPK-MTOR to sphingolipid metabolism.

Basal macroautophagy/autophagy has recently been found in anucleate platelets. Platelet autophagy is involved in platelet activation and thrombus formation. However, the mechanism underlying autophagy in anucleate platelets require further clarification. Our data revealed that LC3-II formation and SQSTM1/p62 degradation were noted in H2O2-activated human platelets, which could be blocked by 3-methyladenine and bafilomycin A1, indicating that platelet activation may cause platelet autophagy. AMPK phosphorylation and MTOR dephosphorylation were also detected, and block of AMPK activity by the AMPK inhibitor dorsomorphin reversed SQSTM1 degradation and LC3-II formation. Moreover, autophagosome formation was observed through transmission electron microscopy and deconvolution microscopy. These findings suggest that platelet autophagy was induced partly through the AMPK-MTOR pathway. In addition, increased LC3-II expression occurred only in H2O2-treated Atg5f/f platelets, but not in H2O2-treated atg5-/- platelets, suggesting that platelet autophagy occurs during platelet activation. atg5-/- platelets also exhibited a lower aggregation in response to agonists, and platelet-specific atg5-/- mice exhibited delayed thrombus formation in mesenteric microvessles and decreased mortality rate due to pulmonary thrombosis. Notably, metabolic analysis revealed that sphingolipid metabolism is involved in platelet activation, as evidenced by observed several altered metabolites, which could be reversed by dorsomorphin. Therefore, platelet autophagy and platelet activation are positively correlated, partly through the interconnected network of sphingolipid metabolism. In conclusion, this study for the first time demonstrated that AMPK-MTOR signaling could regulate platelet autophagy. A novel linkage between AMPK-MTOR and sphingolipid metabolism in anucleate platelet autophagy was also identified: platelet autophagy and platelet activation are positively correlated.Abbreviations: 3-MA: 3-methyladenine; A.C.D.: citric acid/sod. citrate/glucose; ADP: adenosine diphosphate; AKT: AKT serine/threonine kinase; AMPK: AMP-activated protein kinase; ANOVA: analysis of variance; ATG: autophagy-related; B4GALT/LacCS: beta-1,4-galactosyltransferase; Baf-A1: bafilomycin A1; BECN1: beclin 1; BHT: butylate hydrooxytoluene; BSA: bovine serum albumin; DAG: diacylglycerol; ECL: enhanced chemiluminescence; EDTA: ethylenediamine tetraacetic acid; ELISA: enzyme-linked immunosorbent assay; GALC/GCDase: galactosylceramidase; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GBA/GluSDase: glucosylceramidase beta; GPI: glycosylphosphatidylinositol; H2O2: hydrogen peroxide; HMDB: human metabolome database; HRP: horseradish peroxidase; IF: immunofluorescence; IgG: immunoglobulin G; KEGG: Kyoto Encyclopedia of Genes and Genomes; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MPV: mean platelet volume; MTOR: mechanistic target of rapamycin kinase; ox-LDL: oxidized low-density lipoprotein; pAb: polyclonal antibody; PC: phosphatidylcholine; PCR: polymerase chain reaction; PI3K: phosphoinositide 3-kinase; PLS-DA: partial least-squares discriminant analysis; PRP: platelet-rich plasma; Q-TOF: quadrupole-time of flight; RBC: red blood cell; ROS: reactive oxygen species; RPS6KB/p70S6K: ribosomal protein S6 kinase B; SDS: sodium dodecyl sulfate; S.E.M.: standard error of the mean; SEM: scanning electron microscopy; SGMS: sphingomyelin synthase; SM: sphingomyelin; SMPD/SMase: sphingomyelin phosphodiesterase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; UGT8/CGT: UDP glycosyltransferase 8; UGCG/GCS: UDP-glucose ceramide glucosyltransferase; ULK1: unc-51 like autophagy activating kinase 1; UPLC: ultra-performance liquid chromatography; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; WBC: white blood cell; WT: wild type.

Phospholipase D1 and D2 Synergistically Regulate Thrombus Formation.

Previously, we reported that phospholipase D1 (PLD1) and PLD2 inhibition by selective PLD1 and PLD2 inhibitors could prevent platelet aggregation in humans, but not in mice. Moreover, only the PLD1 inhibitor, but not PLD2 inhibitor, could effectively prevent thrombus formation in mice, indicating that PLD might play different roles in platelet function in humans and mice. Although PLD1 and PLD2 were reported to be implicated in thrombotic events, the role of PLD in mice remains not completely clear. Here, we investigated the role of PLD1 and PLD2 in acute pulmonary thrombosis and transient middle cerebral artery occlusion-induced brain injury in mice. The data revealed that inhibition of PLD1, but not of PLD2, could partially prevent pulmonary thrombosis-induced death. Moreover, concurrent PLD1 and PLD2 inhibition could considerably increase survival rate. Likewise, inhibition of PLD1, but not PLD2, partially improved ischemic stroke and concurrent inhibition of PLD1, and PLD2 exhibited a relatively better protection against ischemic stroke, as evidenced by the infarct size, brain edema, modified neurological severity score, rotarod test, and the open field test. In conclusion, PLD1 might play a more important role than PLD2, and both PLD1 and PLD2 could act synergistically or have partially redundant functions in regulating thrombosis-relevant events.

[Corrigendum] Nobiletin, a citrus flavonoid, activates vasodilator-stimulated phosphoprotein in human platelets through non­cyclic nucleotide­related mechanisms.

Following the publication of the above paper, the authors noted that the first author affiliation was presented incorrectly. Essentially, 'School of Medicine' had been omitted from the address. Therefore, the author and affiliation details for this paper should have been presented as follows (the changes are highlighted in bold): THANASEKARAN JAYAKUMAR1*, KAO­CHANG LIN1,2*, WAN-JUNG LU1,3, CHIA­YING LIN4, GERALDINE PITCHAIRAJ5, JIUN­YI LI4,6 and JOEN­RONG SHEU1,4. 1Department of Pharmacology, School of Medicine, College of Medicine, Taipei Medical University, Taipei; 2Department of Neurology, Chi Mei Medical Center, Tainan; 3Department of Medical Research, Taipei Medical University Hospital; 4Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei, Taiwan, R.O.C.; 5Department of Animal Science, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, India; Department of Cardiovascular Surgery, Mackay Memorial Hospital, and Mackay Medical College, Taipei, Taiwan, R.O.C. The authors regret that the error with the first author affiliation was not noticed prior to the publication of their paper, and apologize for any inconvenience caused. [The original article was published in International Journal of Molecular Medicine 39: 174­182, 2017; DOI: 10.3892/ijmm.2016.2822].

[Corrigendum] Structure­activity relationship of three synthesized benzimidazole­based oligosaccharides in human platelet activation.

Following the publication of the above paper, the authors noted that the third author affiliation was presented incorrectly. The third author affiliation should have been written as 'Department of Pharmacology, School of Medicine, College of Medicine, and Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan'. Therefore, the author and affiliation details for this paper should have been presented as follows (the changes are highlighted in bold): YI CHANg1­3*, WEN­HsIEN HsU2,4*, WEN­BIN YANg5, THANAsEKARAN JAYAKUMAR3, TZU­YIN LEE3, JOEN­RONg sHEU3, WAN­JUNg LU3,6 and JIUN­YI LI3,7. 1Department of Anesthesiology, Shin Kong Wu Ho­Su Memorial Hospital, Taipei 111; 2School of Medicine, Fu­Jen Catholic University, Xin Zhuang, New Taipei City 242; 3Department of Pharmacology, School of Medicine, College of Medicine, and Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110; 4Department of Surgery, Wan­Fang Hospital, Taipei Medical University, Taipei 116; 5Genomics Research Center, Academia Sinica, Taipei 115; 6Department of Medical Research and Translational Laboratory, Research Department, Taipei Medical University Hospital, Taipei 110; 7Department of Cardiovascular Surgery, Mackay Memorial Hospital, and Mackay Medical College, Taipei 104, Taiwan, R.O.C.. The authors regret that the error with the third author affiliation was not noticed prior to the publication of their paper, and apologize for any inconvenience caused. [The original article was published in International Journal of Molecular Medicine 40: 1520­1528, 2017; DOI: 10.3892/ijmm.2017.3133].

Author Correction: A novel ruthenium (II)-derived organometallic compound, TQ-6, potently inhibits platelet aggregation: Ex vivo and in vivo studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC.

NADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKß and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

Suppression of Human Platelet Activation via Integrin αIIbß3 Outside-In Independent Signal and Reduction of the Mortality in Pulmonary Thrombosis by Auraptene.

Auraptene is the most abundant coumarin derivative from plants. The pharmacological value of this compound has been well demonstrated, especially in the prevention of cancer and neurodegenerative diseases. Platelet activation is a major factor contributing to arterial thrombosis. Thus, this study evaluated the influence of auraptene in platelet aggregation and thrombotic formation. Auraptene inhibited platelet aggregation in human platelets stimulated with collagen only. However, auraptene was not effective in inhibiting platelet aggregation stimulated with thrombin, arachidonic acid, and U46619. Auraptene also repressed ATP release, [Ca2+]i mobilization, and P-selectin expression. Moreover, it markedly blocked PAC-1 binding to integrin αIIbß3. However, it had no influence on properties related to integrin αIIbß3-mediated outside-in signaling, such as the adhesion number, spreading area of platelets, and fibrin clot retraction. Auraptene inhibited the phosphorylation of Lyn-Fyn-Syk, phospholipase Cγ2 (PLCγ2), protein kinase C (PKC), Akt, and mitogen-activated protein kinases (MAPKs; extracellular-signal-regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK1/2), but not p38 MAPK). Neither SQ22536, an adenylate cyclase inhibitor, nor ODQ, a guanylate cyclase inhibitor, reversed the auraptene-mediated inhibition of platelet aggregation. Auraptene reduced mortality caused by adenosine diphosphate (ADP)-induced pulmonary thromboembolism. In conclusion, this study provides definite evidence that auraptene signifies a potential therapeutic agent for preventing thromboembolic disorders.

Embelin as a Novel Inhibitor of PKC in the Prevention of Platelet Activation and Thrombus Formation.

Embelin is a quinone derivative and found in the fruits of Embelia ribes Burm.f. Embelin has been identified as a small molecular inhibitor of X-chromosome-linked inhibitor of apoptosis proteins, and has multiple biological activities, including antioxidation, anti-inflammation, and antitumor effects. However, the effect of embelin in platelets remains unclear. Thus, this study investigated the antiplatelet mechanism of embelin. Our data revealed that embelin could inhibit platelet aggregation induced by various agonists, including the protein kinase C (PKC) activator phorbol 12,13-dibutyrate (PDBu). Embelin, as well as the PKC inhibitor Ro 31-8220, markedly reduced PDBu-mediated phosphorylation of the PKC substrate, suggesting that embelin may be a PKC inhibitor for platelets. Embelin could block PKC downstream signaling and events, including the inhibition of protein kinase B and mitogen-activated protein kinase activation, granule release, and glycoprotein IIbIIIa activation. Moreover, embelin could delay thrombus formation in the mesenteric microvessels of mice, but did not significantly affect the tail bleeding time. In conclusion, we demonstrated that embelin is a PKC inhibitor and possesses antiplatelet and antithrombotic effects. The further analysis is necessary to more accurately determine clinical therapeutic potential of embelin in all clinical thromboembolic events with disturbance of thrombocyte function.

New therapeutic strategy of hinokitiol in haemorrhagic shock-induced liver injury.

Haemorrhagic shock and resuscitation (HS/R) may cause global ischaemia-reperfusion injury, which can result in systemic inflammation, multiorgan failure (particularly liver failure) and high mortality. Hinokitiol, a bioactive tropolone-related compound, exhibits antiplatelet and anti-inflammatory activities. Targeting inflammatory responses is a potential strategy for ameliorating hepatic injury during HS/R. Whether hinokitiol prevents hepatic injury during HS/R remains unclear. In the present study, we determined the role of hinokitiol following HS/R. The in vivo assays revealed that hinokitiol markedly attenuated HS/R-induced hepatic injury. Hinokitiol could inhibited NF-κB activation and IL-6 and TNF-α upregulation in liver tissues. Moreover, hinokitiol reduced caspase-3 activation, upregulated Bax and downregulated Bcl-2. These findings suggest that hinokitiol can ameliorate liver injury following HS/R, partly through suppression of inflammation and apoptosis. Furthermore, the in vitro data revealed that hinokitiol significantly reversed hypoxia/reoxygenation (H/R)-induced cell death and apoptosis in the primary hepatocytes. Hinokitiol prevented H/R-induced caspase-3 activation, PPAR cleavage, Bax overexpression and Bcl-2 downregulation. Moreover, hinokitiol attenuated H/R-stimulated NF-κB activation and reduced the levels of IL-6 and TNF-α mRNAs, suggesting that hinokitiol can protect hepatocytes from H/R injury. Collectively, our data suggest that hinokitiol attenuates liver injury following HS/R, partly through the inhibition of NF-κB activation.

An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets.

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

Licochalcone A attenuates glioma cell growth in vitro and in vivo through cell cycle arrest.

Licochalcone A (LA), an active ingredient of licorice, has multiple biological activities, including antioxidative and anti-inflammatory activities. Although LA exerts antitumor effects in various cancer cells, its role in gliomas remains unclear. Therefore, this study determined whether LA inhibits glioma cell growth in vitro and in vivo. The present data revealed that LA effectively inhibited the growth of U87 glioma cells by inducing cell cycle arrest in the G0/G1 and G2/M phases; cell cycle arrest was attributed to the LA-mediated reduction of mRNA and protein levels of cyclins and cyclin-dependent kinases. Moreover, subcutaneous (flank) and orthotopic (brain) tumor models were used to determine the role of LA in gliomas. LA significantly alleviated tumor growth in both models. These findings indicate that LA exerts antitumor effects in gliomas in vitro and in vivo and that it is a potential agent for treating glioblastoma multiforme.

Norcantharidin, a clinical used chemotherapeutic agent, acts as a powerful inhibitor by interfering with fibrinogen-integrin αIIb ß3 binding in human platelets.

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin αIIb ß3 , thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1-1.0 µM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P-selectin expression and only slight inhibition on ATP-release reaction in activated platelets. Moreover, NCTD markedly hindered integrin αIIb ß3 activation by interfering with the binding of FITC-labelled PAC-1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin ß3 , Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin αIIb ß3 -mediated outside-in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.

Structure-activity relationship of three synthesized benzimidazole-based oligosaccharides in human platelet activation.

Antiplatelet agents have considerable benefits in the treatment of thromboembolic diseases; however, these agents still have substantial limitations due to their severe side-effects. In this study, the antiplatelet activity of three newly synthesized saccharide based benzimidazole derivatives, M3BIM, Malto-BIM and Melibio-BIM, in collagen and thrombin-stimulated human platelets in vitro was examined. Among the compounds tested, only compound M3BIM exerted concentration (20-60 µM)-dependent inhibitory effects against collagen (1 µg/ml) and thrombin (0.01 U/ml)-induced washed human platelet aggregation. Moreover, at a concentration of 60 µM, M3BIM distinctly abolished collagen-induced adenosine triphosphate (ATP) release and intracellular Ca2+ mobilization. Additionally, this compound attenuated the collagen-induced phosphorylation of p47, a marker of the activation of protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK). However, Malto-BIM and Melibio-BIM were not effective in this regard. Moreover, the toxic effects of these compounds were evaluated using zebrafish embryo toxicity (ZET) assay, and the results revealed that all three compounds had no comparative cytotoxicity within the range of 25-200 µM. Overall, the results of this study provide evidence for the inhibitory effects of M3BIM on collagen-induced platelet aggregation in vitro compared to other imidazole derivatives. The presence of 1-imidazolyl moiety at one end with a longer chain length (three sugar moieties) may be mainly responsible for the observed effects of M3BIM. These results suggest that compound M3BIM may be used as a potential candidate for the treatment of aberrant platelet activation-related diseases as it inhibits the activation of p47 and p38 MAPK, and reduces ATP release and Ca2+ mobilization.

Development of Benzimidazole Derivatives as Novel Anti-platelet Drugs.

BACKGROUND: Benzimidazoles are privileged biomolecules which form an integral part of vitamin B12 and have been attracting numerous researchers all over the world to assess their potential therapeutic significance. OBJECTIVES: The comparative in vitro antiplatelet activity of newly synthesized benzimidazole derivatives, M3BIM, C2BIM, and L2BIM in thrombin, adenosine diphosphate (ADP) and epinephrineinduced washed human platelets was investigated. METHOD: Reversed-phase silica gel column chromatography, Aggregometry, Flow cytometry and Immunoblotting were used in this study. RESULTS: M3BIM exhibited a concentration (25-100 µM) dependent inhibitory effect on platelet aggregation induced by thrombin (0.01 U/mL) in washed human platelets and by epinephrine (10 µM) only at a maximum concentration of 500 µM in platelet-rich plasma (PRP); however, C2BIM and L2BIM had no response even at 500 µM against thrombin and 1mM against epinephrine-induced platelet aggregation. Moreover, all these three compounds were not inhibited platelet aggregation induced by ADP (20 µM). Additionally, these compounds showed no effects in thrombin-induced P-selectin expression and αIIbß3 activation, as evidenced by flow cytometry and clot reaction assays, respectively. Besides, M3BIM (100 µM) significantly abolished thrombin-induced Akt and mitogen-activated protein kinases (MAPKs) phosphorylation; whereas 200 µM C2BIM and L2BIM were not effective on these proteins. CONCLUSION: This study affords confirmation for the inhibitory effect of M3BIM in a low dose thrombin and epinephrine-induced platelet aggregation in vitro compared to other imidazole derivatives, C2BIM and L2BIM. These outcomes may recommend that M3BIM can be appraised as a prospective benzeimidazole compound for the treatment of thrombin -induced platelet defect and its related diseases.