BACKGROUND: High on-treatment platelet reactivity (HTPR) has been suggested as a risk factor for patients with ischemic vascular disease. We explored a predictive model of platelet reactivity to clopidogrel and the relationship with clinical outcomes. METHODS: A total of 441 patients were included. Platelet reactivity was measured by light transmittance aggregometry after receiving dual antiplatelet therapy. HTPR was defined by the consensus cutoff of maximal platelet aggregation >46% by light transmittance aggregometry. CYP2C19 loss-of-function polymorphisms were identified by DNA microarray analysis. The data were compared by binary logistic regression to find the risk factors. The primary endpoint was major adverse clinical events (MACEs), and patients were followed for a median time of 29 months. Survival curves were constructed with Kaplan-Meier estimates and compared by log-rank tests between the patients with HTPR and non-HTPR. RESULTS: The rate of HTPR was 17.2%. Logistic regression identified the following predictors of HTPR: age, therapy regimen, body mass index, diabetes history, CYP2C192, or CYP2C193 variant. The area under the curve of receiver operating characteristic for the HTPR predictive model was 0.793 (95% confidence interval: 0.738-0.848). Kaplan-Meier analysis showed that patients with HTPR had a higher incidence of MACE than those with non-HTPR (21.1% vs. 9.9%; χ = 7.572, P = 0.010). CONCLUSIONS Our results suggest that advanced age, higher body mass index, treatment with regular dual antiplatelet therapy, diabetes, and CYP2C192 or CYP2C193 carriers are significantly associated with HTPR to clopidogrel. The predictive model of HTPR has useful discrimination and good calibration and may predict long-term MACE.
Aged ; Blood Platelets ; drug effects ; Clopidogrel ; pharmacology ; therapeutic use ; Coronary Artery Disease ; metabolism ; prevention & control ; Cytochrome P-450 CYP2C19 ; metabolism ; Female ; Genotype ; Glycated Hemoglobin A ; metabolism ; Humans ; Kaplan-Meier Estimate ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Myocardial Ischemia ; metabolism ; prevention & control ; Regression Analysis

BACKGROUNDPlatelet function tests are widely used in clinical practice to guide personalized antiplatelet therapy. In China, the thromboelastography (TEG) test has been well accepted in clinics, whereas VerifyNow, mainly used for scientific research, has not been used in routine clinical practice. The aim of the current study was to compare these two point-of-care platelet function tests and to analyze the consistency between the two tests for evaluating on-clopidogrel platelet reactivity in Chinese acute myocardial infarction patients undergoing percutaneous coronary intervention (PCI).

METHODSA total of 184 patients admitted to Fuwai Hospital between August 2014 and May 2015 were enrolled in the study. On-clopidogrel platelet reactivity was assessed 3 days after PCI by TEG and VerifyNow using adenosine diphosphate as an agonist. Based on the previous reports, an inhibition of platelet aggregation (IPA) <30% for TEG or a P2Y12 reaction unit (PRU) >230 for VerifyNow was defined as high on-clopidogrel platelet reactivity (HPR). An IPA >70% or a PRU <178 was defined as low on-clopidogrel platelet reactivity (LPR). Correlation and agreement between the two methods were analyzed using the Spearman correlation coefficient (r) and kappa value (κ), respectively.

RESULTSOur results showed that VerifyNow and TEG had a moderate but significant correlation in evaluating platelet reactivity (r = -0.511). A significant although poor agreement (κ = 0.225) in identifying HPR and a significantly moderate agreement in identifying LPR (κ = 0.412) were observed between TEG and VerifyNow. By using TEG as the reference for comparison, the cutoff values of VerifyNow for the Chinese patients in this study were identified as PRU >205 for HPR and PRU <169 for LPR.

CONCLUSIONSBy comparing VerifyNow to TEG which has been widely used in clinics, VerifyNow could be an attractive alternative to TEG for monitoring on-clopidogrel platelet reactivity in Chinese patients.

Adenosine Diphosphate ; therapeutic use ; Aged ; Aspirin ; therapeutic use ; Blood Platelets ; drug effects ; China ; Female ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; drug therapy ; surgery ; Percutaneous Coronary Intervention ; methods ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; therapeutic use ; Point-of-Care Systems ; Receptors, Purinergic P2Y12 ; metabolism ; Thrombelastography ; Ticlopidine ; analogs & derivatives ; therapeutic use

OBJECTIVETo evaluate the long-term clinical effect of Tangyiping Granules (, TYP) on patients with impaired glucose tolerance (IGT) to achieve normal glucose tolerance (NGT) and hence preventing them from conversion to diabetes mellitus (DM).

METHODSIn total, 127 participants with IGT were randomly assigned to the control (63 cases, 3 lost to follow-up) and treatment groups (64 cases, 4 lost to follow-up) according to the random number table. The control group received lifestyle intervention alone, while the patients in the treatment group took orally 10 g of TYP twice daily in addition to lifestyle intervention for 12 weeks. The rates of patients achieving NGT or experiencing conversion to DM as main outcome measure were observed at 3, 12, and 24 months after TYP treatment. The secondary outcome measures included fasting plasma glucose (FPG), 2-h postprandial plasma glucose (2hPG), glycosylated hemoglobin (HbA1c), fasting insulin (FINS), 2-h insulin (2hINS), homeostatic model assessment of insulin resistance (HOMA-IR), blood lipid and patients' complains of Chinese medicine (CM) symptoms before and after treatment.

RESULTSA higher proportion of the treatment group achieved NGT compared with the control group after 3-, 12- and 24-month follow-up (75.00% vs. 43.33%, 58.33% vs. 35.00%, 46.67% vs. 26.67%, respectively, P<0.05). The IGT to DM conversion rate of the treatment group was significantly lower than that of the control group at the end of 24-month follow-up (16.67% vs. 31.67%, P<0.05). Before treatment, FPG, 2hPG, HbA1c, FINS, 2hINS, HOMA-IR, triglyceride (TG), total cholesterol, low- and high-density lipoprotein cholesterol levels had no statistical difference between the two groups (P>0.05). After treatment, the 2hPG, HbA1c, HOMA-IR, and TG levels of the treatment group decreased significantly compared with those of the control group (P<0.05). CM symptoms such as exhaustion, irritability, chest tightness and breathless, spontaneous sweating, constipation, and dark thick and greasy tongue were significantly improved in the treatment group as compared with the control group (P<0.05). No severe adverse events occurred.

CONCLUSIONTYP administered at the IGT stage with a disciplined lifestyle delayed IGT developing into type 2 DM.

Blood Glucose ; metabolism ; Blood Platelets ; drug effects ; metabolism ; Case-Control Studies ; Diabetes Mellitus, Type 2 ; blood ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Erythrocytes ; drug effects ; metabolism ; Female ; Glucose Intolerance ; blood ; drug therapy ; Humans ; Insulin ; blood ; Kidney ; drug effects ; physiopathology ; Leukocytes ; drug effects ; metabolism ; Lipids ; blood ; Liver ; drug effects ; physiopathology ; Male ; Middle Aged ; Time Factors

OBJECTIVETo investigate the effect and mechanism of Factor Xa on the differentiation of Meg-01 cells into platelet-like particles.

METHODSThe Meg-01 cells were used as experimental object, Factor Xa was used as agonist. Cell proliferation was detected by CCK-8 assay. The viability of platelet-like particles was analyzed by AlamaBlue kit. MAPK/ERK pathway and PI3K/AKT pathway were assayed by Western blot. The expression of CD41b was analyzed by Western blot and flow cytometry. Cell cycle and apoptosis were detected by flow cytometry.

RESULTSThe Factor Xa (1 µg/ml) inhibited cell viability, induced apoptosis. Factor Xa triggered cell arrest at the G(2)/M stage and down-regulated the expression of SKP2. After Meg-01 cells were stimulated by Factor Xa, the expression of CD41b was up-regulated and the MAPK/ERK pathway and PI3K/AKT pathway were activated. The platelets-like particles stimulated by FXa activation were viable.

CONCLUSIONThe Factor Xa maybe display some effect on the differentiation of megakaryocytes into platelets.

Apoptosis ; Blood Platelets ; cytology ; drug effects ; Cell Cycle Checkpoints ; Cell Differentiation ; drug effects ; Cell Line ; Cell Proliferation ; Cell Survival ; Factor Xa ; pharmacology ; Humans ; MAP Kinase Signaling System ; Megakaryocytes ; cytology ; drug effects ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism

This study was designed to investigate inhibitory effects and possible mechanisms of snake venom tripeptide (pENW) on platelet adhesion in order to promote the development of a novel anti-platelet therapy. To study the inhibitory effects of pENW on platelet adhesion, washed platelets pre-incubated with pENW (116.5-466.2 μmol x L(-1)) were used to test the ability of platelet adhesion to fibrinogen. Effect of pENW on fibrin clot retraction was also tested. Effect of pENW on platelets viability was tested by MTT assay. Effect of pENW on reactive-oxygen species (ROS) levels of platelet was studied by flow cytometry assay. Calcium mobilization in Fura-2/AM-loaded platelets was monitored with a spectrofluorimeter. Cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP), thromboxane A2 (determined as its metabolite thromboxane B2) were measured using enzyme immunoassay kits. Akt, ERK and p38 phosphorylation were tested by Western blot. The results showed that pENW inhibited platelet adhesion and fibrin clot retraction in a concentration-dependent manner without cytotoxicity. Intracellular cGMP and cAMP in both resting and thrombin-activated platelets were increased by pENW. In addition, pENW attenuated intracellular Ca2+ mobilization and TXA2 production in platelets stimulated by thrombin. As shown by Western blot assay, Akt, ERK and p38 phosphorylation in thrombin-induced platelet were attenuated by pENW. However, inhibitory effects of pENW had nothing to do with ROS. Thus, pENW exhibited a significant inhibition on platelet adhesion to fibrinogen, which means pENW could block the first step of thrombosis as while as retard the more stable clot formation. The mechanisms of pENW on inhibition platelet adhesion might be related to instant regulations, such as protein kinases.
Blood Platelets ; drug effects ; Blotting, Western ; Calcium ; metabolism ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Flow Cytometry ; Phosphorylation ; Platelet Aggregation ; drug effects ; Reactive Oxygen Species ; metabolism ; Snake Venoms ; chemistry ; Thromboxane A2 ; metabolism ; Thromboxane B2 ; metabolism

OBJECTIVETo study the effect of aqueous extract of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro.

METHODSBlood was collected from volunteers. Effects of the prepared water extracts of herbs on platelet aggregation were monitored on a Packs-4 aggregometer. The fluorescence intensity of water extracts of Caulis Spatholobi, Flos Carthami and Rhizoma Curcumae on the expression of P-selectin in human platelets of healthy persons was measured with flow cytometry.

RESULTSOut of several herbs investigated, Flos Carthami and Rhizoma Curcumae potently inhibited platelet aggregation after incubation with platelet-rich plasma (PRP) for 15 min. Caulis Spatholobi Flos Carthami and Rhizoma Curcumae inhibited adenosine-5'-diphosphate (ADP) or platelet activating factor (PAF)-induced platelet aggregation in PRP in a dose-dependent manner. In contrast to Flos Carthami and Rhizoma Curcumae, Caulis Spatholobi could not inhibit thrombin-induced platelet aggregation. Despite its inability to inhibit thrombin-induced platelet aggregation in PRP, Caulis Spatholobi had a greater anti-aggregating activity in PRP induced by ADP or PAF. Caulis Spatholobi and Flos Carthami showed significant inhibitory effects on the expression of P-selectin.

CONCLUSIONSCaulis Spatholobi, Flos Carthami and Rhizoma Curcumae have potent anti-platelet properties, and their inhibitory actions are mediated via different mechanisms. Caulis Spatholobi inhibited ADP-induced platelet aggregation but not by thrombin, indicating that its mechanism of action might be independent of the thromboxane pathway. The effect of Caulis Spatholobi and Flos Carthami were associated with suppressing the expression of P-selectin.

Adult ; Blood Platelets ; drug effects ; metabolism ; Curcuma ; chemistry ; Fabaceae ; chemistry ; Humans ; P-Selectin ; metabolism ; Plant Extracts ; chemistry ; pharmacology ; Plants, Medicinal ; chemistry ; Platelet Aggregation ; drug effects ; Platelet Function Tests ; Water ; chemistry ; Young Adult

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 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 explore the protein expression of heme oxygenase-1 (HO-1) and platelet endothelial cell adhesion molecules-1 (PECAM-1) in human coronary artery endothelial cells induced with Zinc Oxide Nanoparticle (ZnO-NPs).

METHODSMTT assay was used to determine the cell viability of ZnO-NPs. Levels of HO-1 and PECAM-1 protein in culture supernatants were measured using ELISA after human coronary artery endothelial cells were treated with different concentrations (0, 10, 20, 40µg/ml) of ZnO-NPs for 24 h.

RESULTSThe cell viability of human coronary artery endothelial cells in each group was 89.76%, 83.61%, 63.10%, 53.20%, 48.11%, 42.35%, 38.06%, 25.44% respectively when treated with different concentrations of ZnO-NPs (12.5, 25, 50, 70, 80, 90, 100, 200µg/ml). Protein levels of HO-1 (ng/L) in each group were 0.041±0.011, 0.512±0.076, 0.906±0.059, 1.062±0.089 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40µg/ml). Comparisons in each group were statistically significant (P < 0.05). Protein levels of PECAM-1 (µg/L) in each group were 7.966 ± 0.046, 7.993 ± 0.036, 8.629 ± 0.052, 8.811 ± 0.039 respectively after the stimulation of different concentrations of ZnO-NPs (0, 10, 20, 40 µg/ml). Compared with the control group, protein levels of PECAM-1 increased (P < 0.05) when the concentration of ZnO-NPs was 20µg/ml or 40 µg/ml.

CONCLUSIONZnO-NPs stimulation could inhibit the viability of human coronary artery endothelial cells and upregulate the protein expression of HO-1 and PECAM-1.

Blood Platelets ; Cell Survival ; Coronary Vessels ; Endothelial Cells ; drug effects ; Heme Oxygenase-1 ; metabolism ; Humans ; Nanoparticles ; toxicity ; Platelet Endothelial Cell Adhesion Molecule-1 ; metabolism ; Zinc Oxide ; toxicity

OBJECTIVETo evaluate efficacies of three commonly used oral drugs including Berbamine Hydrochloride Tablet (B), Qijiao Shengbai Capsule (Q), and Leucogen Tablet (L) (by single drug, two drugs or three drugs) combined with granulocyte colony-stimulating factor (G-CSF) for treat ment of chemotherapy related leukocytopenia in mice.

METHODSTotally 156 Kunming male mice were divided into the normal control group (A, n=24), the model group (B, n=24), the G-CSF group (C, n =24), the G-CSF+Q group (D, n=12), G-CSF+ B (E, n=12), the G-CSF+L group (F, n=12), the G-CSF + Q + B group (G, n=12), the G-CSF + Q + L group (H, n=12), the G-CSF + L + B group (I, n=12), and the G-CSF + L + Q + B (J, n=12). Mouse models of chemotherapy related leukocytopenia were established by intraperitoneal injection of cyclophosphamide (CTX). A G-CSF group was set up as a positive control. Mice were treated by a single oral drug, a single oral drug combined with G-CSF, and two or three drugs combined with G-CSF respectively, and the death rate calculated. Hemocytes [such as white blood cells (WBC) and its classification, red blood cells (RBC), platelet (PLT), hemoglobin (Hb)] were calculated by hematology analyzer. Mice were anatomized and important organs weighed. Organ indices were calculated.

RESULTSThere was no statistical difference in the mortality rate among all groups (P > 0.05). Compared with Group B, WBC was elevated in all other groups (P < 0.01). WBC and PLT were elevated most in Group J, Hb and RBC were also increased at the same time (P < 0.05, P < 0. 01). Compared with Group B, RBC increased in Group E, F, G, I, and J (P < 0.01); Hb obviously increased in Group C, E, F, H, I, and J (P<0.01). Compared with Group B and D, the promotion of erythroid hematopoiesis by G-CSF could be elevated in any group contained drug B and L (P < 0.05, P < 0.01). The spleen index of model mice could be significantly improved in Group C, D, and G (P < 0.01). The thymus index of model mice could be significantly improved in Group H (P < 0.05).

CONCLUSIONSThe best scheme to treat mice with chemotherapy related leukopenia or decreased three blood series was to administrate three commonly oral drugs combined with G-CSF. Authors speculated that G-CSF and Q might have a certain effect on CTX induced immune inhibition.

Administration, Oral ; Animals ; Blood Platelets ; Cyclophosphamide ; Drug-Related Side Effects and Adverse Reactions ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Erythrocyte Count ; Granulocyte Colony-Stimulating Factor ; metabolism ; Hematopoiesis ; Hemoglobins ; Leukocyte Count ; Leukocytes ; Leukopenia ; chemically induced ; drug therapy ; Male ; Mice ; Pharmaceutical Preparations