Safety of butylphthalide and edaravone in patients with ischemic stroke: a multicenter real-world study.

BACKGROUND: Butylphthalide (NBP) and edaravone (EDV) injection are common acute ischemic stroke medications in China, but there is a lack of large real-world safety studies on them. This study aimed to determine the incidence of adverse events, detect relevant safety signals, and assess the risk factors associated with these medications in real-world populations. METHODS: In this study, data of acute ischemic stroke patients were extracted from the electronic medical record database of six tertiary hospitals between January 2019 and August 2021. Baseline confounders were eliminated using propensity score matching. The drugs' safety was estimated by comparing the results of 24 laboratory tests standards on liver function, kidney function, lipid level, and coagulation function. The drugs' relative risk was estimated by logistic regression. A third group with patients who did not receive NBP or EDV was constructed as a reference. Prescription sequence symmetry analysis was used to evaluate the associations between adverse events and NBP and EDV, respectively. RESULTS: 81,292 patients were included in this study. After propensity score matching, the NBP, EDV, and third groups with 727 patients in each group. Among the 15 test items, the incidence of adverse events was lower in the NBP group than in the EDV group, and the differences were statistically significant. The multivariate logistic regression equation revealed that NBP injection was not a promoting factor for abnormal laboratory test results, whereas EDV had statistically significant effects on aspartate transaminase, low-density lipoprotein cholesterol and total cholesterol. Prescription sequence symmetry analysis showed that NBP had a weak correlation with abnormal platelet count. EDV had a positive signal associated with abnormal results in gamma-glutamyl transferase, alanine aminotransferase, aspartate aminotransferase, prothrombin time, and platelet count. CONCLUSIONS: In a large real-world population, NBP has a lower incidence of adverse events and a better safety profile than EDV or other usual medications.

SARS-CoV-2 vaccines are not associated with hypercoagulability in apparently healthy people.

Background: SARS-CoV-2 adenoviral vector DNA vaccines have been linked to the rare but serious thrombotic postvaccine complication vaccine-induced immune thrombotic thrombocytopenia. This has raised concerns regarding the possibility of increased thrombotic risk after any SARS-CoV-2 vaccines. Objectives: To investigate whether SARS-CoV-2 vaccines cause coagulation activation leading to a hypercoagulable state. Methods: This observational study included 567 health care personnel; 521 were recruited after the first dose of adenoviral vector ChAdOx1-S (Vaxzevria, AstraZeneca) vaccine and 46 were recruited prospectively before vaccination with a messenger RNA (mRNA) vaccine, either Spikevax (Moderna, n = 38) or Comirnaty (Pfizer-BioNTech, n = 8). In the mRNA group, samples were acquired before and 1 to 2 weeks after vaccination. In addition to the prevaccination samples, 56 unvaccinated blood donors were recruited as controls (total n = 102). Thrombin generation, D-dimer levels, and free tissue factor pathway inhibitor (TFPI) levels were analyzed. Results: No participant experienced thrombosis, vaccine-induced immune thrombotic thrombocytopenia, or thrombocytopenia (platelet count <100 × 109/L) 1 week to 1 month postvaccination. There was no increase in thrombin generation, D-dimer level, or TFPI level in the ChAdOx1-S vaccine group compared with controls or after the mRNA vaccines compared with baseline values. Eleven of 513 (2.1%) participants vaccinated with ChAdOx1-S had anti-PF4/polyanion antibodies without a concomitant increase in thrombin generation. Conclusion: In this study, SARS-CoV-2 vaccines were not associated with thrombosis, thrombocytopenia, increased thrombin generation, D-dimer levels, or TFPI levels compared with baseline or unvaccinated controls. These findings argue against the subclinical activation of coagulation post-COVID-19 vaccination.

FSAP Protects against Histone-Mediated Increase in Endothelial Permeability In Vitro.

Factor-VII-activating protease (FSAP) is involved in the regulation of hemostasis and inflammation. Extracellular histones play a role in inflammation and the conversion of latent pro-FSAP into active FSAP. FSAP has been shown to regulate endothelial permeability, but the mechanisms are not clear. Here, we have investigated the effects of FSAP on endothelial permeability in vitro. A mixture of histones from calf thymus stimulated permeability, and the wild-type (WT) serine protease domain (SPD) of FSAP blocked this effect. WT-SPD-FSAP did not influence permeability on its own, nor that stimulated by thrombin or vascular endothelial growth factor (VEGF)-A165. Histones induced a large-scale rearrangement of the junction proteins VE-cadherin and zona occludens-1 from a clear junctional distribution to a diffuse pattern. The presence of WT-SPD-FSAP inhibited these changes. Permeability changes by histones were blocked by both TLR-2 and TLR4 blocking antibodies. Histones upregulated the expression of TLR-2, but not TLR-4, in HUVEC cells, and WT-SPD-FSAP abolished the upregulation of TLR-2 expression. An inactive variant, Marburg I (MI)-SPD-FSAP, did not have any of these effects. The inhibition of histone-mediated permeability may be an important function of FSAP with relevance to sepsis, trauma, and stroke and the need to be investigated further in in vivo experiments.

Tissue factor pathway inhibitor upregulates CXCR7 expression and enhances CXCL12-mediated migration in chronic lymphocytic leukemia.

The infiltration of chronic lymphocytic leukemia (CLL) cells into lymphoid organs correlates with disease severity. CXCL12 is a key chemotactic factor for the trafficking of CLL. Tissue factor pathway inhibitor (TFPI) is a serine protease inhibitor and plays a role in CXCL12-mediated hematopoietic stem cell homing. We aim to explore the role of TFPI in CXCL12-mediated migration of CLL cells. In this study, plasma TFPI concentrations were measured by ELISA. CLL cells were isolated from patients and used for trans-endothelial migration (TEM) assays. Quantitative RT-PCR and Western blotting were used to detect the expression of CXCR7, CXCR4 and ß-catenin. Immunofluorescence and co-immunoprecipitation was used to detect the binding of TFPI and glypican-3 (GPC3). We found that plasma TFPI levels in CLL patients were higher than in healthy controls, particularly in the patients with advanced disease. TFPI enhanced CXCL12-mediated TEM of CLL cells by increasing the expression of the CXCL12 receptor CXCR7, but not of the CXCL12 receptor CXCR4. The effect of TFPI on TEM was abolished by the CXCR7 inhibitor, CCX771, while the CXCR4 inhibitor AMD3100 strongly increased TEM. TFPI co-localized with GPC3 on the cell surface. An antibody to GPC3, HS20, decreased CXCR7 expression and abolished the effect of TFPI on TEM. TFPI activated ß-catenin and the Wnt/ß-catenin inhibitor IWP4 repressed the effect of TFPI on CXCR7 expression and TEM. We conclude that TFPI may contribute to organ infiltration in CLL patients.

A novel hypoxia response element regulates oxygen-related repression of tissue factor pathway inhibitor in the breast cancer cell line MCF-7.

BACKGROUND: Hypoxia is one of the most pervasive physiological stresses in solid tumors. We have previously demonstrated that tissue factor (TF) pathway inhibitor (TFPI) expression was transcriptionally repressed by the activation of hypoxia inducible factor (HIF)-1α under hypoxic conditions. However, the role of HIF-2α, also known as endothelial PAS domain-containing protein 1 (EPAS1), on TFPI expression remains unclear. AIM: To explore the role of HIF-2α/EPAS1 in the regulation of TFPI expression under hypoxia in breast cancer cells. METHODS AND RESULTS: Quantitative RT-PCR showed that total TFPI mRNA and protein levels were decreased by the overexpression of HIF-2α/EPAS1 in MCF7 cells. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay indicated a HIF-2α/EPAS1 responsive region located in the TFPI promoter region at -170 to +21 relative to the transcriptional start site. Subsequent mutagenesis demonstrated a functional hypoxia response element (HRE) 5'-AAACAGGA-3' for HIF-2α/EPAS1 within the TFPI promoter located at -45 to -38. In breast cancer patients, a positive correlation between HIF-2α/EPAS1 and total TFPI mRNA expression was observed by using gene expression analysis. CONCLUSIONS: This study provides evidence that HIF-2α/EPAS1 is involved in the regulation of TFPI gene expression in breast cancer cells, suggesting that the activation of coagulation and the increased risk of thrombosis observed in breast cancer patients may correlate with local hypoxic regulation of coagulation factors and their inhibitors.

EPAS1/HIF-2 alpha-mediated downregulation of tissue factor pathway inhibitor leads to a pro-thrombotic potential in endothelial cells.

Neovascularization and hemorrhaging are evident in advanced atherosclerotic plaques due to hypoxic conditions, and mediate the accumulation of metabolic substrates, inflammatory cells, lipids, and other blood born factors inside the plaque. Tissue factor (TF) pathway inhibitor (TFPI) is mainly expressed by endothelial cells and is the endogenous inhibitor of the coagulation activator TF, which together with the downstream product thrombin can drive plaque progression and atherogenesis. We aimed to investigate the effect of hypoxic conditions on endothelial cell expression and activity of TFPI and TF that are important in coagulation initiation. Hypoxia was induced in primary human umbilical vein endothelial cells using chemicals or 1% oxygen tension, and mRNA and protein expressions were measured using qRT-PCR, ELISA, and Western blot analysis. Microscopy of fluorescence-labeled cells was used to visualize cell-associated TFPI. Cell-surface factor Xa (FXa) activity was measured using a two-stage chromogenic substrate method. We found that hypoxia reduced the TFPI mRNA and protein levels and increased the TF mRNA expression in a dose-dependent manner. The effect on TFPI was apparent on all the protein pools of TFPI, i.e., secreted TFPI, cell-surface associated TFPI, and intracellular TFPI, and seemed to be dependent upon hypoxia inducible factor-2α (HIF-2α). An increase in FXa activity was also observed on the endothelial cell surface, reflecting an increase in pro-thrombotic potential of the cells. Our findings indicate that hypoxic conditions may enhance the pro-coagulant activity of endothelial cells, which may promote atherogenesis in addition to clinical events and thus the severity of atherosclerotic disorders.

Hypoxia influences stem cell-like properties in multidrug resistant K562 leukemic cells.

OBJECTIVES: The present study investigates the potential role of hypoxia in maintaining stem cell-like properties and therapeutic resistance in K562 leukemic cell. METHODS: Western blot, flow cytometry and cell viability assays were used to investigate the effects of hypoxia (1% O2) on cell proliferation, drug resistance and expression of the hypoxia inducible factor-2α (HIF-2α), the octamer-binding transcription factor 4 (Oct4), CD133, CD34 and the ATP-binding cassette sub-family G member 2 (ABCG2) as well as Smad2 phosphorylation in the drug resistant cell line K562/DOX and its parental cell line. RESULTS: Hypoxia induced growth inhibition and significantly upregulated HIF-2α, CD133, Oct4, CD34 and ABCG2 expression in the wild type K562 cells (p<0.05). The IC50 of doxorubicin was also enhanced about 2.5-fold in hypoxia. In contrast, the K562/DOX cells, which showed significantly higher ABCG2 expression and IC50 for various drugs, no significant difference in cell proliferation was observed between hypoxia and normoxia. The hypoxia-induced upregulation of HIF-2α, CD133, Oct4, CD34 and ABCG2 expression was significantly lower than in the wild type cells (p<0.05). Moreover, hypoxia induced the phosphorylation of Smad2 and additional treatment with SD-208, an inhibitor of the TGF-ß receptor I kinase, resulted in a dose-dependent downregulation of CD133 and Oct4 in the K562/DOX cells. CONCLUSIONS: Hypoxia plays an important role in enhancing the stem cell-like properties and to induce multidrug resistance of leukemia cells. The activation of the TGF-ß/Smad2 signaling pathway may be involved in the regulation of this pathophysiological process.

[Effect of proteasome inhibitor bortezomib on proliferation, apoptosis and XIAP expression in K562 cells].

OBJECTIVE: To investigate the effect of proteasome inhibitor bortezomib on proliferation, apoptosis of K562 cells and the expression of XIAP. METHODS: K562 cells were treated with bortezomib at different concentration. Cell proliferation was analyzed by WST-1 assay, cell apoptosis by flow cytometry and TUNEL, XIAP mRNA expression from 5 - 100 nmol/L by RT-PCR, and XIAP protein expression by SP immunohistochemistry. RESULTS: K562 cells were treated with bortezomib at different concentrations for 24 h respectively, the cells growth was significantly inhibited with inhibition rates from (13. 6 ± 0. 2)% to (81. 4 ± 0. 1)%, respectively, being markedly higher than that of control (1. 2 ± 0. 1)% (P < 0.05). IC(50) was 24. 6 nmol/L of bortezomib treated for 24 h. When K562 cells were treated with 30 nmol/L of bortezomib for 12 - 48 h, the inhibition rates were (29. 1 ± 0. 9)% to (59. 8 ± 1. 2)%, respectively, the differences being statistically significant (P < 0.05) between 12 h group and 24 h group, while there was no statistical difference between 24 h, 36 h and 48 h groups. K562 cells treated with 30 nmol/L bortezomib for 24 h showed nuclear condensation, nuclear margination, nuclear fragmentation, cytoplasmic vacuoles and a large number of apoptotic body formation. The apoptotic cells rate was 83. 67% in bortezomib treated group, and 2. 33% in untreated group (P < 0.05). The expression of XIAP mRNA was decreased in a dose-dependent manner, and the expression of its protein was down-regulated. CONCLUSION: Bortezomib can inhibit the proliferation of K562 cells, and induce apoptosis by down-regulating the expression of XIAP, providing the laboratory evidence for the targeted therapy in acute leukemia.

Simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine in human plasma by high-performance liquid chromatography.

A simple and sensitive high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method has been developed and validated for simultaneous quantification of five local anesthetics in human plasma: procaine, lidocaine, ropivacaine, tetracaine and bupivacaine. In an ice-water bath, 500 microL plasma sample, containing 100 microg/mL neostigmine methylsulfate as anticholinesterase, was spiked with carbamazepine as internal standard and alkalized by sodium hydroxide. Liquid-liquid extraction with ethyl ether was used for plasma sample preparation. The chromatographic separation was achieved on a Kromosil ODS C18 column with a mobile phase consisting of 30 mM potassium dihydrogen phosphate buffer (0.16% triethylamine, pH adjusted to 4.9 with phosphoric acid) and acetonitrile (63/37, v/v). The detection was performed simultaneously at wavelengths of 210 and 290 nm. The chromatographic analysis time was 13 min per sample. The calibration curves of all five analytes were linear between 0.05 and 5.0 microg/mL (r(2) > or = 0.998). Precision ranged from 1.4% to 7.9% and accuracy was between 91.7% and 106.5%. The validated method is applicable for simultaneous determination of procaine, lidocaine, ropivacaine, tetracaine and bupivacaine for therapeutic drug monitoring and pharmacokinetic study.