Objective To evaluate whether Vibrio vulnificus secreted exotoxin-hemolysin (VVH) can activate platelet, an important blood immune cell, and to explore the possible molecular mechanism of platelet activation by VVH. Methods Transcriptomics and immunohistochemistry were used to analyze whether Vibrio vulnificus infection caused platelet activation in mice. Then, flow cytometry was used to identify whether VVH was the main stimulator of platelet activation. Naturally expressed VVH toxin was purified and prepared. The effects of extracellular and intracellular Ca²⁺ signal inhibitors on VVH activated platelets were evaluated by flow cytometry and Western blotting. The immune activation effect of VVH in the early stage of Vibrio vulnificus infection was analyzed in vivo. Results VVH was the main stimulator of platelet activation in Vibrio vulnificus culture supernatant. Natural VVH can induce the increase of P-selectin (CD62P) on platelet surface, the formation of platelet-neutrophil complex (PNC), and the release of platelet microvesicles. The activation mechanism may be related to the VVH pore-dependent Ca²⁺-calmodulin (CaM) -myosin light chain kinase (MLCK) signaling pathway, which led to the release of platelet alpha particles and cascade activation of platelets. In a mouse model of ALD infected by Vibrio vulnificus gavage, VVH was strongly associated with platelet activation. Conclusion This study shows that VVH is an important platelet activating molecule in the early stage of Vibrio vulnificus infection, and its induction of platelet activation may be related to the pathogenic process.
Animals ; Platelet Activation/drug effects* ; Hemolysin Proteins/pharmacology* ; Vibrio vulnificus/metabolism* ; Mice ; Blood Platelets/drug effects* ; Vibrio Infections/immunology* ; P-Selectin/metabolism* ; Bacterial Proteins ; Female

OBJECTIVE: To investigate the effects of oridonin on platelet function and related mechanisms. METHODS: Washed platelets from healthy adults and mice were incubated with different concentrations of oridonin (2.5, 5 and 10 μmol/L) in vitro . The surface expression level of P-selectin and the activation of integrin αIIbβ3 in platelets were detected by flow cytometry, and the aggregation ability of platelets under the stimulation by various agonists was detected by light transmission aggregometry. The expression of P-AKT (Ser473) was detected by protein immunoblotting. Arterial thrombosis model was established in mice with mesenteric injury induced by ferric chloride, and tail hemorrhage model was established by cutting off the tail of mice. The effect of intraperitoneal injection of oridonin (10 mg/kg) on thrombosis and haemostasis was tested. RESULTS: Oridonin inhibited platelet P-selectin expression and integrin αIIbβ3 activation. In the presence of different stimulants, oridonin inhibited platelet aggregation in a concentration-dependent manner. The phosphorylation level of AKT Ser473 was reduced in the groups treated with different concentrations of oridonin. Oridonin significantly prolonged the time of mesenteric artery thrombosis in mice, but did not affect the tail bleeding time. CONCLUSION Oridonin inhibits platelet activation, aggregation, and thrombosis by inhibiting AKT phosphorylation, and may be used as a potential antiplatelet drug.
Diterpenes, Kaurane/pharmacology* ; Animals ; Mice ; Blood Platelets/drug effects* ; Platelet Aggregation/drug effects* ; P-Selectin/metabolism* ; Thrombosis ; Platelet Glycoprotein GPIIb-IIIa Complex/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Humans ; Phosphorylation ; Platelet Activation/drug effects*

OBJECTIVE: To investigate the effect of zedoarondiol on neovascularization of atherosclerotic (AS) plaque by exosomes experiment. METHODS: ApoE^-/- mice were fed with high-fat diet to establish AS model and treated with high- and low-dose (10, 5 mg/kg daily) of zedoarondiol, respectively. After 14 weeks, the expressions of anti-angiogenic protein thrombospondin 1 (THBS-1) and its receptor CD36 in plaques, as well as platelet activation rate and exosome-derived miR-let-7a were detected. Then, zedoarondiol was used to intervene in platelets in vitro, and miR-let-7a was detected in platelet-derived exosomes (Pexo). Finally, human umbilical vein endothelial cells (HUVECs) were transfected with miR-let-7a mimics and treated with Pexo to observe the effect of miR-let-7a in Pexo on tube formation. RESULTS: Animal experiments showed that after treating with zedoarondiol, the neovascularization density in plaques of AS mice was significantly reduced, THBS-1 and CD36 increased, the platelet activation rate was markedly reduced, and the miR-let-7a level in Pexo was reduced (P<0.01). In vitro experiments, the platelet activation rate and miR-let-7a levels in Pexo were significantly reduced after zedoarondiol's intervention. Cell experiments showed that after Pexo's intervention, the tube length increased, and the transfection of miR-let-7a minics further increased the tube length of cells, while reducing the expressions of THBS-1 and CD36. CONCLUSION Zedoarondiol has the effect of inhibiting neovascularization within plaque in AS mice, and its mechanism may be potentially related to inhibiting platelet activation and reducing the Pexo-derived miRNA-let-7a level.
Animals ; MicroRNAs/genetics* ; Exosomes/drug effects* ; Plaque, Atherosclerotic/genetics* ; Neovascularization, Pathologic/genetics* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Humans ; Blood Platelets/drug effects* ; Apolipoproteins E/deficiency* ; Thrombospondin 1/metabolism* ; CD36 Antigens/metabolism* ; Platelet Activation/drug effects* ; Male ; Mice ; Mice, Inbred C57BL

The effect of triptolide( TP) on VEGFA,SDF-1,CXCR4 pathway were investigated in vitro to explore the mechanism in improving platelet activation in patients with ankylosing spondylitis( AS). Peripheral blood mononuclear cells( PBMC) were used for the experiment and divided into 4 groups: normal group( NC),model group( MC),triptolide group( TP),and AMD3100 group. The optimal concentration of TP was measured by the MTT method. The expressions of TNF-α,IL-1β,IL-4,IL-10,VEGFA and VEGFR were detected by ELISA. The expressions of SDF-1,CXCR4 and VEGFA were detected by real-time quantitative PCR( RT-qPCR).The expressions of SDF-1,CXCR4,VEGFA and VEGFR were detected by Western blot. The expression levels of CD62 p,CD40 L and PDGFA were detected by immunofluorescence. MTT results showed that medium-dose TP had the strongest inhibitory effect on cells at24 h. The results of ELISA and PCR showed that TP inhibited mRNA expressions of IL-1β,TNF-α,VEGFA,VEGFR and SDF-1,CXCR4 and VEGFA. The results of Western blot indicated that TP inhibited SDF-1,CXCR4 and VEGFA,VEGFR protein expressions; immunofluorescence results indicate that TP can inhibit the expressions of CD62 p,CD40 L,PDGFA. TP may regulate platelet activation by down-regulating SDF-1,CXCR4,VEGFA and VEGFR mRNA expressions,thereby down-regulating IL-1β and TNF-αexpressions,and up-regulating the expressions of IL-4 and IL-10 cytokines.
Cells, Cultured ; Chemokine CXCL12 ; metabolism ; Cytokines ; metabolism ; Diterpenes ; pharmacology ; Epoxy Compounds ; pharmacology ; Heterocyclic Compounds ; pharmacology ; Humans ; Leukocytes, Mononuclear ; drug effects ; Phenanthrenes ; pharmacology ; Platelet Activation ; Receptors, CXCR4 ; metabolism ; Spondylitis, Ankylosing ; Vascular Endothelial Growth Factor A ; metabolism

Atherothrombosis is the major cause of acute coronary syndromes and cardiovascular deaths. Platelets participate in the processes of forming and extending atherosclerotic plaques. Therefore, antiplatelet therapy is a milestone in the primary and second prevention of atherothrombotic diseases. Along with the longterm use of antiplatelet agents, the safety and drug resistance has become a big concern in clinic and new drugs possessing higher effectiveness and fewer adverse effects are needed. Abundant recent data support that traditional Chinese herbs may be a good alternative and complementary choice of new antiplatelet drugs. This review highlights the progress of antiplatelet effect of active components derived from traditional Chinese herbs based on their chemical structures.
Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Platelet Activation ; drug effects ; Platelet Aggregation Inhibitors ; chemistry ; pharmacology ; Receptors, G-Protein-Coupled ; metabolism ; Signal Transduction ; drug effects

OBJECTIVETo explore the effects of exogenous carbon monoxide-releasing molecules 2 (CORM-2) on LPS-induced abnormal activation of platelets in peripheral blood of healthy human donors and its possible molecular mechanism.

METHODSVenous blood samples were collected from a healthy volunteer, and platelet-rich plasma (PRP) from the blood were isolated by differential centrifugation. The PRP was subpackaged into siliconized test tubes and then divided into control group, LPS group, inactive CORM-2 (iCORM-2) group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group according to the random number table, with 3 tubes in each group. The PRP in control group did not receive any treatment. The PRP in LPS group received LPS (20 mL, 10 µg/mL) stimulation, and the PRP in iCORM-2 group, 10 µmol/L CORM-2 group, and 50 µmol/L CORM-2 group underwent the same LPS stimulation and treatment of 50 µmol/L iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2, respectively, with the dosage of 20 mL. After being cultured for 30 min, the platelet adhesion rate was determined by glass bottle method, the number of platelet spreading on fibrinogen was determined with immunofluorescent method, and the platelet aggregation rate was measured by turbidimetric method. The platelet poor plasma (PPP) was prepared from PRP, the levels of ATP in PPP and platelets were determined by chemical fluorescein method. The expressions of platelet glycoprotein I bα (GPIbα) and GPVI were analyzed by flow cytometer. The expressions of glycogen synthase kinase 3β (GSK-3β) and phosphorylated GSK-3β were determined by Western blotting and immunoprecipitation, respectively. Measurement of the above indices was repeated for 3 times. Data were processed with one-way analysis of variance and SNK test.

RESULTSCompared with those in control group, the platelet adhesion rates, numbers of platelets spreading on fibrinogen, platelet aggregation rates, expressions of GPIbα and GPVI in PRP, levels of ATP in PPP in LPS and iCORM-2 groups were significantly increased, while levels of ATP in platelets were significantly decreased (with P values below 0.05). Compared with those in LPS group, the former 7 indices in iCORM-2 group showed no significant differences (with P values above 0.05), while the levels of ATP in platelets in the 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly increased, and the other 6 indices in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased (with P values below 0.05). The expression levels of GSK-3β of the platelets in PRP in control, LPS, iCORM-2, 10 µmol/L CORM-2, and 50 µmol/L CORM-2 groups were 0.550 ± 0.060, 1.437 ± 0.214, 1.210 ± 0.108, 0.720 ± 0.010, and 0.670 ± 0.010, respectively, and the expression levels of the phosphorylated GSK-3β of the platelets in PRP in the above 5 groups were 0.950 ± 0.070, 1.607 ± 0.121, 1.420 ± 0.040, 1.167 ± 0.015, and 0.513 ± 0.122, respectively. Compared with those in control group, both the expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in LPS and iCORM-2 groups were significantly increased (with P values below 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP between LPS group and iCORM-2 group were similar (with P values above 0.05). The expression levels of GSK-3β and phosphorylated GSK-3β of the platelets in PRP in 10 µmol/L CORM-2 and 50 µmol/L CORM-2 groups were significantly decreased compared with those in LPS group (with P values below 0.05).

CONCLUSIONSLPS stimulation can abnormally activate the platelets in peripheral blood of healthy human, but the abnormal activation can be inhibited by CORM-2 intervention, and the mechanism of the latter may involve the phosphorylation of GSK-3β mediated by GP.

Blood Platelets ; drug effects ; metabolism ; Carbon Monoxide ; metabolism ; Glycogen Synthase Kinase 3 ; Glycogen Synthase Kinase 3 beta ; Humans ; Lipopolysaccharides ; pharmacology ; Organometallic Compounds ; pharmacology ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Platelet-Rich Plasma

OBJECTIVETo investigate the effect of the anticoagulants on PRP quality, so as to clarify the appropriate anticoagulant used in PRP production.

METHODSThe microstructure change of platelets collected via heparin, citrate, acid citrate dextrose (ACD) and citrate-theophylline-adenosine-dipyridamole ( CTAD) was observed by TEM following time course. The extent of spontaneous activation of platelets in four groups was detected by measuring sP-selectin in plasma. The TGF-β1 release amount of activated PRP of four groups was measured.

RESULTSCTAD is superior to other anticoagulants in maintaining the integrity of platelet structures for a long time and preventing platelet spontaneous activation. ACD slightly surpassed heparin and citrate in above two aspects. ACD-PRP and CTAD-PRP released significantly more TGF-β1 compared with heparin and citrate.

CONCLUSIONSThe PRP quality and biological effects were strongly associated with the type of Anticoagulants. ACD and CTAD are optimal anticoagulants in PRP production for they can maintain platelet viability at a high level.

Adenosine ; pharmacology ; Anticoagulants ; pharmacology ; Blood Platelets ; drug effects ; physiology ; Citrates ; pharmacology ; Citric Acid ; pharmacology ; Dipyridamole ; pharmacology ; Drug Combinations ; Glucose ; analogs & derivatives ; pharmacology ; Heparin ; pharmacology ; Platelet Activation ; drug effects ; Platelet-Rich Plasma ; Theophylline ; pharmacology ; Transforming Growth Factor beta1 ; metabolism

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Cell Separation ; Cyclosporine ; pharmacology ; Dual Specificity Phosphatase 2 ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Interleukin-17 ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; P-Selectin ; genetics ; metabolism ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Primary Cell Culture ; Signal Transduction ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo observe the effect of activating blood circulation drugs or activating blood circulation and detoxication drugs on indices of platelet activation, inflammation, and coagulation status correlated with blood-stasis and toxin in acute myocardial infarction rats.

METHODSTotally 100 male SD rats were randomly divided into the sham-operation group, the model group, the activating blood circulation group, the activating blood circulation and detoxication group, and the metoprolol group, 20 in each group. Rats in the activating blood circulation group were administered with Xiongshao Capsule at the daily dose of 0.39 g/kg. Rats in the activating blood circulation and detoxication group were administered with Xiongshao Capsule (at the daily dose of 0.39 g/kg) and Huanglian Capsule (at the daily dose of 0.135 g/kg). Rats in the metoprolol group received metoprolol at the daily dose of 2.25 mg/kg. And rats in the rest two groups were administered with normal saline. All medication lasted for 3 successive weeks. After the last administration, the rat model of acute myocardial infarction was prepared by ligation of left anterior descending artery. No ligation was given to rats in the sham-operation group. Animals were sacrificed 24 h after modeling. Tumor necrosis factor-α (TNF-α), β-thromboglobulin (β-TG), platelet α granule membrane protein-140 (GMP-140), 11 dehydro-thromboxane B2 (11-DH-TXB2), fibrinopeptide A (FPA), antithrombin III (AT-III), and D-dimer (DD) were detected by ELISA. The mRNA expression of TNF-α was tested by RT-PCR.

RESULTSPlatelet activation parameters were significantly increased in the model group, when compared with the sham-operation group (P < 0.01). Compared with the model group, all indices (except GMP-140 in the metoprolol group) obviously decreased in each medicated group (P < 0.01, P < 0.05). Besides, β-TG and 11-DH-TXB2 were superior in the activating blood circulation and detoxication group to that of the metoprolol group (P < 0.05). But 11-DH-TXB2 was also obviously superior in the activating blood circulation and detoxication group to that of the activating blood circulation group (P < 0.05). Compared with the sham-operation group, an obviously hypercoagulable state was obviously shown in the AMI model group, with significantly increased FPA and DD (P < 0.05 or 0.01) and significantly decreased AT III (P < 0.01). Compared with the model group, the FPA level significantly decreased in each medicated group (P < 0.01), and the AT III level significantly increased in the activating blood circulation group and the activating blood circulation and detoxication group (both P < 0.01). The level of DD obviously decreased in the activating blood circulation and detoxication group (P < 0.01). Besides, the 3 indices were superior in the activating blood circulation and detoxication group to those of the metoprolol group (P < 0.05). Compared with the sham-operation group, the serum TNF-α level and myocardial TNF-α mRNA expression were significantly increased in the model group (P < 0.05, P < 0.01). Compared with the model group, not only the serum TNF-α level was significantly decreased, but also the TNF-α gene expression in the myocardial tissue was improved in the activating blood circulation and detoxication group (P < 0.01).

CONCLUSIONCombined use of activating blood circulation and detoxication drugs could play an effective role in treatment of coronary heart disease by fighting against platelet activation, improving the hypercoagulable state, and inhibiting inflammation, which was significantly better than using activating blood circulation and removing stasis drugs alone.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Fibrin Fibrinogen Degradation Products ; Inflammation ; metabolism ; Male ; Medicine, Chinese Traditional ; Myocardial Infarction ; physiopathology ; Myocardium ; metabolism ; Platelet Activation ; drug effects ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate and compare the effects and mechanisms of three functional parts of Dahuang Zhechong Pill (DHZCP), including drugs with the function of removing blood stasis and promoting blood circulation (FP-I), drugs with the function of expelling heat and moistening dryness (FP-II), and drugs with the function of nourishing yin and replenishing blood (FP-III) of DHZCP, on platelet-derived growth factor (PDGF)-stimulated vascular smooth muscle cells (VSMCs) proliferation with the method of serum pharmacology.

METHODSVSMCs proliferation of rat was assayed by measuring the cell viability with the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) method. DNA synthesis in VSMCs was examined by detecting 5'-bromo-2'-deoxyuridine incorporation with the immunocytochemical method. Cycle of VSMCs was evaluated with flow cytometry. Expression of cyclin D1, p27, PKCα, and phosphorylated extracellular signal regulated kinase 1/2 (ERK1/2) was quantified by the Western blotting method.

RESULTSThe FP-I and FP-III containing serum was capable of inhibiting PDGF-stimulated proliferation and DNA synthesis of VSMCs, arrested VSMCs in G phase, downregulated cyclin D1, and upregulated p27 expression (P <0.01 or P <0.05). The FP-I and FP-III containing serum also inhibited the PDGF-induced phosphorylation of tyrosine of ERK1/2 and PKCα expression (P <0.01 or P <0.05).

CONCLUSIONSFP-I and FP-III of DHZCP are able to inhibit VSMCs proliferation via interrupting PKCα-ERK1/2 signaling, modulating the expression of cell cycle proteins to result in arresting the cells in G phase. The inhibitory effect is mainly related to the function of removing blood stasis and promoting blood circulation, slightly to the function of nourishing yin and replenishing blood, but not to the function of expelling heat and moistening dryness.

Animals ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; DNA ; biosynthesis ; Drugs, Chinese Herbal ; pharmacology ; Enzyme Activation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; MAP Kinase Signaling System ; drug effects ; Male ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; drug effects ; enzymology ; Platelet-Derived Growth Factor ; pharmacology ; Protein Kinase C-alpha ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum ; metabolism