Disseminated intravascular coagulation phenotype is regulated by the TRPM7 channel during sepsis

BACKGROUND: Sepsis is an uncontrolled inflammatory response against a systemic infection that results in elevated mortality, mainly induced by bacterial products known as endotoxins, producing endotoxemia. Disseminated intravascular coagulation (DIC) is frequently observed in septic patients and is associated with organ failure and death. Sepsis activates endothelial cells (ECs), promoting a prothrombotic phenotype contributing to DIC. Ion channel mediated calcium permeability participates in coagulation. The transient reception potential melastatin 7 (TRPM7) non-selective divalent cation channel that also contains an α-kinase domain, which is permeable to divalent cations including Ca2+, regulates endotoxin-stimulated calcium permeability in ECs and is associated with increased mortality in septic patients. However, whether endothelial TRPM7 mediates endotoxemia-induced coagulation is not known. Therefore, our aim was to examine if TRPM7 mediates coagulation during endotoxemia. RESULTS: The results showed that TRPM7 regulated endotoxin-induced platelet and neutrophil adhesion to ECs, dependent on the TRPM7 ion channel activity and by the α-kinase function. Endotoxic animals showed that TRPM7 mediated neutrophil rolling on blood vessels and intravascular coagulation. TRPM7 mediated the increased expression of the adhesion proteins, von Willebrand factor (vWF), intercellular adhesion molecule 1 (ICAM-1), and P-selectin, which were also mediated by the TRPM7 α-kinase function. Notably, endotoxin-induced expression of vWF, ICAM-1 and P-selectin were required for endotoxin-induced platelet and neutrophil adhesion to ECs. Endotoxemic rats showed increased endothelial TRPM7 expression associated with a procoagulant phenotype, liver and kidney dysfunction, increased death events and an increased relative risk of death. Interestingly, circulating ECs (CECs) from septic shock patients (SSPs) showed increased TRPM7 expression associated with increased DIC scores and decreased survival times. Additionally, SSPs with a high expression of TRPM7 in CECs showed increased mortality and relative risk of death. Notably, CECs from SSPs showed significant results from the AUROC analyses for predicting mortality in SSPs that were better than the Acute Physiology and Chronic Health Evaluation II (APACHE II) and the Sequential Organ Failure Assessment (SOFA) scores. CONCLUSIONS: Our study demonstrates that sepsis-induced DIC is mediated by TRPM7 in ECs. TRPM7 ion channel activity and α-kinase function are required by DIC-mediated sepsis-induced organ dysfunction and its expression are associated with increased mortality during sepsis. TRPM7 appears as a new prognostic biomarker to predict mortality associated to DIC in SSPs, and as a novel target for drug development against DIC during infectious inflammatory diseases.

Activated platelets in central venous catheters: a flow cytometry and numerical simulation approach

The central venous catheter that is inserted in patients undergoing hemodialysis can cause hemodynamic instability and trigger complications such as thrombus formation. The objective of this study was to investigate hemostatic and numerical influences on thrombus formation in patients undergoing hemodialysis with a central venous catheter. Participants were assigned to three groups: I: clinical and laboratorial healthy individuals matched by sex and age (controls); II: participants after one month of insertion of the catheter and III: participants after 4 months of insertion of the catheter. Platelet activation was investigated by GPIIb/IIIa and p-selectin expressions using flow cytometry. A three-dimensional model of the catheter was constructed in the numerical simulation for the calculation of partial differential equation of a platelet activation model. A significant difference was detected by the expression of p-selectin comparing the group I (33.42 ± 4.74), group II (40.79 ± 5.54) and group III(51.00 ± 7.21) (p < 0.0001). The median values for GPIIb/IIIa were 10426 (10029-10721), 13921 (13412-15652) and 19946 (18714-21815) after catheter insertion (p < 0.0001), for groups I, II and III, respectively. Excluding the first arterial orifice, venous orifices tend to have greater platelet activation when compared to the other arterial orifices. The results of this study showed the influence of arterial and venous lateral orifices in stimulating the development of thrombi associated with the activation of platelet markers the longer the catheter was used.

Effect of Vitamin D3 to Platelet Activation Mediated by Tumor Cell Culture Medium / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of vitamin D3 to platelet activation by tumor cell culture medium.@*METHODS@#The peripheral blood platelets of BALB/c mice were isolated. The platelets were activated in 4T1 culture fluid for 24 h. The platelets were divided into 7 groups: control group, activation group, 1 nmol/L vitamin D3 group, 10 nmol/L vitamin D3 group, 50 nmol/L vitamin D3 group, 100 nmol/L vitamin D3 group, and positive drug (0.1 μmol/L eptifibatide) group. CCK-8 assay was used to detect the platelet proliferation at 24, 48 and 72 h. Flow cytometry was used to detect the expression of CD61 and CD62p and receptor for advanced glycation end products (RAGE) at 24, 48 and 72 h. ELISA was used to detect the level of platelet-endothelial cell adhesion molecule-1 (PECAM-1) at 24, 48 and 72 h.@*RESULTS@#The CD41@*CONCLUSION@#Vitamin D3 shows antiplatelet effect and can inhibit platelet proliferation and activation.

Intercellular mediators in bone remodeling regulation in the experimental renal pathology / Mediadores intercelulares de la regulación de la remodelación ósea en un modelo experimental de patología renal

Bone metabolism disorders are characterized by an imbalance of bone resorption and formation in the bone remodeling process. Glucocorticoids that are used to treat kidney diseases exacerbate these disorders. P-selectin and galectin-3 are molecules involved in the sclerotic process in kidney, whereas bone resorption is regulated by the interaction between the nuclear factor activator kappa b receptor (RANK), its ligand (RANKL) and the RANKL decoy receptor osteoprotegerin (OPG). The aim of this study was to investigate the cellular and molecular mechanisms of disruption of bone remodeling regulation processes, reflected by intercellular mediators (RANKL, OPG, P-selectin and galectin-3) in chronic kidney disease experimental model treated with glucocorticoids. Rats were divided into four groups of 10 animals each. The first group, the control group, included intact animals. The second group consisted of rats with impaired bone remodeling resulting from chronic kidney disease (experimental group (CKD). The third group was a group of animals with impaired bone remodeling due to exposure to glucocorticoids (experimental group (GCs)). The fourth group consisted of rats with impaired bone remodeling in chronic kidney disease, followed by exposure to glucocorticoids (experimental group (CKD + GCs)). The effects of CKD and glucocorticoid were evaluated biochemically, histologically and by measuring bone density. An enzymelinked immunoassay was used to measure intercellular mediator levels in the serum. The bone density in the experimental groups was reduced compared to the control group. RANKL levels in animals of three experimental groups were higher than in intact animals. Serum levels of OPG were higher in CKD and GCs groups than in intact animals. At the same time, in the animals' blood serum of the CKD + GCs group, the levels of OPG were lower, than those in animals from the control group. The levels of galectin-3 in the serum of the experimental groups GCs and CKD + GCs were lower than in intact animals. The serum levels of galectin-3 in animals of the CKD group were higher than those in animals from the control group. The levels of P-selectin were lower in the serum of the GCs group than in intact animals. At the same time, the levels of P-selectin were higher in the CKD and CKD + GCs groups, than those in animals from the control group. In conclusion, the study of the complex system of bone remodeling regulation, which includes many factors and their interactions, may lead to the development of new methods for treating patients with chronic kidney disease in order to prevent osteoporosis in the future. (AU)

Las enfermedades metabólicas óseas se caracterizan por un desequilibrio en el proceso de remodelación ósea en los que participan mediadores tales como receptor del activador del factor nuclear- kappa- b (RANK), su ligando (RANKL) y la osteoprotegerina (OPG). Los glucocorticoides, recuentemente empleados en el tratamiento de la enfermedad renal crónica, exacerban este desequilibrio. En la enfermedad esclerótica renal, las moléculas de adhesión celular P-selectina and galectina-3 tienen un rol fundamental. El objetivo de esta trabajo fue estudiar las alteraciones en los mediadores de la remodelación ósea (RANKL, OPG, P-selectina and galectina-3) en un modelo de enfermedad renal crónica con tratamiento glucocorticoideo. Ratas Wistar hembras fueron divididos en 4 grupos: control (C); enfermedad renal crónica con afección de la remodelación ósea (ERC); animales con afección de la remodelación ósea expuestos a glucocorticoides (GC); enfermedad renal crónica con afección de la remodelación ósea tratados con glucocorticoides (ERC+GC). Los efectos de la ERC y los GC fueron evaluados bioquímicamente, histológicamente y por medición de la densidad ósea. RANKL, OPG, Pselectina and galectina-3 se cuantificaron en muestras de sangre venosa empleando enzimoinmuno análisis. En los 3 grupos experimentales la densidad ósea se evidenció reducida y los niveles séricos de RANKL elevados respecto al grupo control. Los niveles de OPG en los grupos ERC y GC fueron superiores mientras que en el grupo ERC+GC menores respecto a los animales controles. Galectina 3 plasmática en GC y ERC+GC se encontró reducida y aumentada en los animales ERC, en comparación con los animales controles. La concentración sérica de P-selectina sérica fue mayor en los grupos ERC y ERC+GC, y menor en los animales GC respecto a los niveles plasmáticos de los animales intactos. El avance del conocimiento sobre la regulación de la remodelación ósea a través de la interacción de mediadores sistémicos, en un futuro, puede conducir al desarrollo de nuevas estrategias terapéuticas para la prevención de la osteoporosis en pacientes con enfermedad renal crónica. (AU)

Análise da expressão de citocinas e quimiocinas, dos agregados de plaquetas-leucócitos e dos mediadores solúveis sCD40L e sCD62P no melanoma cutâneo / Analysis of the expression of cytokines and chemokines, platelet-leukocyte aggregates and soluble mediators sCD40L and sCD62P in cutaneous melanoma

Introdução: O melanoma cutâneo é uma neoplasia maligna dos melanócitos da pele com incidência variável no mundo e o prognóstico é desfavorável nos estágios mais avançados. O desenvolvimento do melanoma está associado ao sistema imunológico e a maior evolução no seu tratamento se deu a partir de medicamentos baseados no estímulo da resposta imune contra o tumor. Objetivo: Avaliar a expressão de citocinas e quimiocinas, dos agregados de plaquetas-leucócitos e de mediadores solúveis sCD40L e sCD62P no sangue de pacientes com melanoma cutâneo. Métodos: Foi realizado um estudo transversal em pacientes com melanoma cutâneo admitidos para tratamento cirúrgico no Hospital de Câncer de Pernambuco (HCP), entre os anos de 2015 e 2018. Foram incluídos 51 pacientes (média de 57,6 anos) com diagnóstico de melanoma cutâneo, e como grupo controle, 30 indivíduos saudáveis (média de 56,7 anos). As coletas de sangue foram realizadas antes da ressecção do melanoma. A determinação dos níveis de citocinas IL6, IL10, TNF, IL1 e IL12p70 e das quimiocinas CXCL10 (IP10), CCL2 (MCP-1), CXCL9 (MIG), CCL5 (RANTES) e CXCL8 (IL8), sCD40L e sCD62P, e agregados plaquetas-leucócitos foi realizada por citometria de fluxo. Foram realizadas análises entre os grupos de pacientes e controles, e pelos parâmetros como tipo histológico, estadio, espessura de Breslow e presença de metástases linfonodais. O teste de Mann-Whitney foi utilizado para comparação entre dois grupos, e de Kruskal-Wallis para as análises entre três ou mais grupos. Foi considerado significativo p<0,05. Resultados: Não houve detecção de IL1 e IL12 no sangue dos pacientes e controles. Verificou-se níveis elevados das citocinas IL10 e diminuídos de TNF nos pacientes comparados ao grupo controle, (p<0,0001). A IL6 esteve aumentada nos pacientes com estadio II em relação ao III (p=0,017) e em pacientes com linfonodos negativos (p<0,0001). Foram encontrados níveis reduzidos da quimiocina CCL5 (p=0,009) nos pacientes quando comparados ao grupo controle. O percentual de agregação plaquetária em linfócitos, monócitos e neutrófilos também foi elevado nos pacientes comparado ao grupo controle (p<0,0001, p=0,009 e p<0,0001 respectivamente). Foram encontrados níveis elevados de agregados plaquetas-monócitos nos pacientes com linfonodos positivos (p=0,008). Os níveis solúveis sCD40L e sCD62P foram elevados nos pacientes comparados aos controles (p=0,03 e p=0,006, respectivamente). Conclusão: Os dados obtidos das análises realizadas mostram que os pacientes com melanoma cutâneo apresentam um perfil imunossupressor com a participação de plaquetas e monócitos/macrófagos que favorecem a progressão tumoral

Cutaneous melanoma is a malignancy originated from the skin melanocytes, with a variable incidence worldwide and a poor prognosis in advanced stages. Melanoma growth is closely associated with the immune system and the most important treatment advances resulted from stimulation of immune response against the tumor. Objective: To evaluate the expression of cytokines and chemokines, platelet-leukocyte aggregates and soluble mediators sCD40L and sCD62P in the blood of patients with cutaneous melanoma. Methods: A cross-sectional study was performed in cutaneous melanoma patients admitted for surgical treatment at the Hospital de Câncer de Pernambuco (HCP) between 2015 and 2018. Fifty-one patients (mean age 57.6 years) with a diagnosis of melanoma were included, and 30 healthy individuals (mean age 56.7 years) were chosen as the control group. The blood samples were taken before resection of the melanoma. The determination of cytokines IL6, IL10, TNFα, IL1ß and IL12p70 and chemokines CXCL10 (IP10), CCL2 (MCP-1), CXCL9 (MIG), CCL5 (RANTES) and CXCL8 (IL8), sCD40L and sCD62P, and platelet-leukocyte aggregate was performed by flow cytometry. Analysis were performed between patient and control groups, and by parameters such as histological type, stage, Breslow thickness, and presence of lymph node metastases. Mann-Whitney test was used for comparison between the two groups, and Kruskal-Wallis test for analysis between three or more groups. It was considered significant p <0.05. Results: There was no detection of IL1ß and IL12 in the blood of patients and controls. Elevated levels of IL10 and decreased TNFα cytokines were found in patients compared to the control group (p <0.0001). IL6 was increased in patients with stage II compared to III (p=0.017) and in patients with negative lymph nodes (p <0.0001). Reduced CCL5 chemokine levels (p=0.009) were found in patients compared to the control group. The percentage of platelet aggregation in lymphocytes, monocytes and neutrophils was also high in patients when compared to the control group (p <0.05). High levels of platelet-monocyte aggregates were found in patients with positive lymph nodes (p=0.008). Soluble sCD40L and sCD62P levels were elevated in patients compared to controls (p=0.03 and p=0.006, respectively). Conclusion: The data obtained from the analysis performed show that patients with cutaneous melanoma have an immunosuppressive profile with platelet participation and monocytes/macrophages that favor tumor progression

Predictive Effect of Platelet Activation Index Expression before and after Adenosine Bisphosphate Activation on Bleeding Risk in ITP Patients / 中国实验血液学杂志

OBJECTIVE@#To investigate the predictive effect of platelet activation index expression before and after adenosine bisphosphate activation on bleeding risk in patients with primary immune thrombocytopenia (ITP).@*METHODS@#Eighty-nine patients with ITP admitted in our hospital from January 2017 to October 2018 were selected and inrolled in ITP group, the bleeding scoreing and grading were performed by using the ITP-BAT for ITP patients, then 89 ITP patients were divided into 4 subgroups: nothing bleeding symptom group, mild bleeding symprom group, mode rate bleeding symptom group and severe bleeding symptom group according to bleeding scores and grades obtained from ITP-BAT detection. At the same time, 22 persons underwent the health physical examination were selected and enrolled in control group. The adenosine diphosphate (ADP) was used as activator for all patients and controls. The flow cytonetry was used to analyze the expression of platelet membranc glyco protein (GPⅠb, GPⅡb /Ⅲ a) and P-selectin before and after ADP activation, the multiple linear person's correlation analysis was used to analyze the correlation of bleeding degree of ITP patients before and after ADP acbivation with the expression levels of GPⅠb, GPⅡb/Ⅲa and P-selectin.@*RESULTS@#After the ADP activation, the expression level of GPⅠb significantly decreased, while the expression levels of GPⅠb, GPⅡb/Ⅲ a and P-selectin significantly increased in control group, nothing bleeding symptom group and mild bleeding symptom group; but the expression level of GPⅠb significantly increased, while the expression level of GPⅡb/Ⅲ a significantly decreased in moderate and severe bleeding symptom group, the both differences were statistically significant (P＜0.05). however, the expression level of P-selectin in moderate and severe bleeding symptom groups before and after ADP activation was not statistivally significant (P＞0.05). Before ADP activation, the expression level of GPⅠb in ITP subgroups was lower than that in control group, the expression level of GPⅡb/Ⅲ a in ITP subgroups was higher than that in control group, the expression level of P-selectin in moderate and severe bleeding symptom groups was higher than that in control group (P＜0.05). After ADP activation, the expression levels of GPⅠb and P-selectin in ITP subgroups both were lower than those in control group, the expression level of GPⅡb/Ⅲa in ITP subgroups was higher than that in control group (P＜0.05). The comparison among ITP subgroups showed that before ADP activation, the expression level of GPⅠb in moderate and severe bleeding symptom groups was lower than that in nothing bleeding symotom and mild bleeding symptom groups, while the expression levels of GPⅡb/Ⅲa and P-selectin were higher than those in nothing bleeding symptom and mild bleeding symptom groups (P＜0.05), however, after ADP activation, the expression level of GPⅠb in moderate and severe bleeding symptom groups was higher than that in nothing bleeding symptom and mild bleeding symptom groups, while the expression levels of GPⅡb/Ⅲ a and P-selection in moderate and severe bleeding symptom groups were lower than those in nothing and mild bleeding symptom groups (P＜0.05). The correlation analysis showed that before ADP activation, the expression levels of GPⅠb and GPⅡb/Ⅲa positivdy correlated with the bleeding risk (r=0.483, 0.504), and the P-selectin not correlated with the bleeding risk (r=0.000); however, after ADP activation, the expression level of GPⅠb and GPⅡb/Ⅲ a negatively correlated with the bleeding risk (r=-0.627, -0.406, -0.108).@*CONCLUSION@#The expression level of platelet activation indicators before and after ADP activation is of certain value for prevention of bleeding risk in ITP patients and can be used as a reference indicator for the treatment and efficacy evaluation.

Correlations of inhaled NO with the cTnI levels and the plasma clotting factor in rabbits with acute massive pulmonary embolism

Abstract Purpose: To investigate the correlation of inhaled nitric oxide (NO) on plasma levels of cardiac troponin I (cTnI) and von Willebrand factor (vWF), glycoprotein (GP) IIb/IIIa, granule membrane protein 140 (GMP-140) in rabbits with acute massive pulmonary embolism (PE). Methods: Thirty apanese white rabbits were divided into 3 groups, thrombus were injected in model group (n = 10), NO were inhalated for 24 h after massive PE in NO group (n = 10), saline were injected in control group (n = 10). The concentrations of vWF, GP IIb/IIIa, GMP-140 and cTnI were tested at 4, 8, 12, 16, 20, and 24 h, Correlation analyses were conducted between cTnI and vWF, GP IIb/IIIa, and GMP-140 by Pearson's correlation. Results: The concentration of cTnI and vWF, GP IIb/IIIa, and GMP-140 was increased in the model group, compared to control group. In the inhaled group, the concentrations of cTnI, vWF, GP IIb/IIIa, and GMP-140 were reduced compared to model group. There was a positive correlation between cTnI and vWF, GP IIb/IIIa, and GMP-140. Conclusion: Inhaled nitric oxide can lead to a decrease in levels of cardiac troponin I, von Willebrand factor, glycoprotein, and granule membrane protein 140, after an established myocardial damage, provoked by acute massive pulmonary embolism.

The effect of mesenchymal stem cell use on intra-abdominal adhesions in a rat model

PURPOSE: Intra-abdominal adhesions (IAA) are among the most frequently seen pathologies in general surgery practice with an increased morbidity and mortality. In the present study, we investigated the effect of locally applied mesenchymal stem cells (MSCs) on IAA. METHODS: Twenty-four Wistar Albino rats were used in the study. The rats were divided into three groups including: Sham, control, and MSCs group. On day 0, cecum was reached under anesthesia in all groups, except the Sham group. Scraping with a sponge was performed until petechial bleeding occurred. The control group received no treatment. In the stem cell group, MSCs were applied topically immediately after surgery on adhesions. The rats were sacrificed on day 10 and colon tissues and blood samples were collected for macroscopic, histopathological, and biochemical analysis. RESULTS: In our study, E-selectin, P-selectin, TNF-α and IL-1 levels were statistically significantly lower in the MSC group than the control group, while the sham group has the lowest levels. In both the macroscopic and histopathological analyses (Zühlke's scale), the least amount of adhesion was observed in the Sham group. In addition, although there was less adhesion in the MSC group than the control group, the difference did not reach statistical significance. CONCLUSION: Topical MSC application immediately after surgery suppresses the inflammatory process. However it was found to be ineffective in histopathological and macroscopic examinations performed on the 10th day.

Anti-platelet effects of epigallocatechin-3-gallate in addition to the concomitant aspirin, clopidogrel or ticagrelor treatment

BACKGROUND/AIMS: Although epigallocatechin-3-gallate (EGCG), which is found in high contents in the dried leaves of green tea, has been reported to have an anti-platelet effect, synergistic effects of EGCG in addition to current anti-platelet medications remains to be elucidated. METHODS: Blood samples were obtained from 40 participants who took aspirin (ASA, n = 10), clopidogrel (CPD, n = 10), ticagrelor (TCG, n = 10) and no anti-platelet medication (Control, n = 10). Ex vivo platelet aggregation and adhesion under various stimulators were analyzed by multiple electrode aggregometry (MEA) and Impact-R systems. PAC-1 and P-selectin expressions in human platelets were analyzed by flow cytometry. RESULTS: In MEA analysis, adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP)-induced platelet aggregations were lower in the CPD and the TCG groups; arachidonic acid (AA)-induced platelet aggregation was lower in the ASA group, whereas collagen (COL)-induced platelet aggregations were comparable among four groups. EGCG significantly reduced ADP- and COL-induced platelet aggregation in dose-dependent manner (ADP, p = 0.04; COL, p < 0.01). There were no additional suppressions of platelet aggregation stimulated by AA in the ASA group, and by ADP in the CPD and TCG groups. Moreover, EGCG suppressed shear stress-induced platelet adhesion on Impact-R, and had no effect on P-selectin and PAC-1 expressions. CONCLUSIONS: Ex vivo treatment of EGCG inhibited platelet adhesion and aggregation without changes in P-selectin and PAC-1 expression. There was no additional suppressions in platelet aggregation stimulated by AA in the ASA group and ADP in the CPD and TCG groups.

Effect of Coagulation and Platelet Activation on Tumor-Associated Hypercoagulable State in Lymphoma-Bearing Mice / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the coagulation abnormality and tumor-associated hypercoagulable state in lymphoma-bearing mice by measuring the changes in coagulation indices (D-D, vWF, TF) and platelet activation indices (P-selectin, GPIIbIIIa).</p><p><b>METHODS</b>The mouse model with lymphoma was established by the subcutaneous injection of 38B9 lymphoma cells into BALB/c mice, and the tumor formation was evaluated by using MRI and B ultrasonography. The D-D, vWF and TP levels of blood samples from inner canthal vein of tumor-bearing mice on 1 d, 14 d and 21 d were detected by using ELISA, the platelet activation indices (P-selectin, GPIIbIIIa) were detected by using flow cytometry.</p><p><b>RESULTS</b>The lymphoma-bearing mouse model was successfully established. The levels of D-D, vWF and TF as well as platelet activation indices P-selectin and GPIIbIIIa in the peripheraI blood were significantly higher than those of control group (P<0.05).</p><p><b>CONCLUSION</b>Lymphoma-bearing mice showed abnormalities of coagulation and platelet activation, which relates with the tumor hypercoagulable state in lymphoma-bearing mice.</p>

Effect of ulinastatin on serum levels of tumor necrosis factor-α, P-selectin, and thrombin-antithrombin complex in young rats with sepsis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the effect of ulinastatin (UTI) for early drug intervention on the serum levels of tumor necrosis factor-α (TNF-α), P-selectin, and thrombin-antithrombin complex (TAT) in young rats with sepsis.</p><p><b>METHODS</b>A total of 120 male rats aged 4 weeks were randomly divided into normal control group, sham-operation group, sepsis group, low-dose UTI group (50 000 U/kg), and high-dose UTI group (200 000 U/kg), with 24 rats in each group. Modified cecal ligation and puncture was performed to establish a rat model of sepsis, and the rats in the low- and high-dose UTI groups were given caudal vein injection of UTI after model establishment. ELISA was used to measure the serum levels of TNF-α, P-selectin, and TAT at 6, 12, and 24 hours after model establishment.</p><p><b>RESULTS</b>The sepsis group had significant increases in the serum levels of TNF-α, P-selectin, and TAT at 6 hours, and the serum levels of TNF-α and TAT continued to increase by 24 hours (P<0.05); P-selectin reached the peak at 12 hours and decreased slightly at 24 hours (P<0.05). The UTI groups had similar change patterns in the levels of P-selectin and TAT as the sepsis group. The UTI groups had significant increases in the level of TNF-α at 6 hours, but gradually decreased over time. The changes in serum levels of TNF-α, P-selectin, and TAT in the UTI groups were significantly smaller than in the sepsis group (P<0.05). The high-dose UTI group had significantly smaller changes in serum levels of TNF-α, P-selectin, and TAT than the low-dose UTI group (P<0.05).</p><p><b>CONCLUSIONS</b>Early intervention with UTI can significantly improve coagulation function and inhibit the production of TNF-α, P-selectin, and TAT in young rats with sepsis. High-dose UTI has a significantly greater effect than low-dose UTI.</p>

Association between platelet-activating factor acetylhydrolase gene polymorphisms and gastrointestinal bleeding in children with Henoch-Schönlein purpura / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the association between the single nucleotide polymorphisms (SNPs) of the ninth exon Val279Phe of platelet-activating factor acetylhydrolase (PAF-AH) gene and gastrointestinal bleeding in children with Henoch-Schönlein purpura (HSP).</p><p><b>METHODS</b>A total 516 children with HSP were enrolled, among whom 182 had gastrointestinal bleeding and 334 had no gastrointestinal bleeding. PCR was used to investigate the distribution of genotypes and alleles in the SNPs of Val97Phe. The plasma PAF-AH activity was measured, as well as the levels of platelet-activating factor (PAF), granular membrane protein-140 (GMP-140), β-thromboglobulin (β-TG), and platelet factor 4 (PF4).</p><p><b>RESULTS</b>The Val279Phe genotype and allele frequencies were in Hardy-Weinberg equilibrium, and the homozygous genotype TT and heterozygotes accounted for 0.97% and 6.05% respectively. The gastrointestinal bleeding group had a significantly higher allele frequency than the control group (5.22% vs 3.33%; P<0.01). The HSP patients with GG genotype in the gastrointestinal bleeding group had significantly higher levels of plasma PAF and GMP-140 than those in the non-gastrointestinal bleeding group (P<0.05), while the non-gastrointestinal bleeding group had a significantly higher PAF-AH activity than the gastrointestinal bleeding group (P<0.05). There were no significant differences in β-TG and PF4 between the two groups (P>0.05).</p><p><b>CONCLUSIONS</b>Val279Phe gene polymorphisms in PAF-AH are associated with PAF-AH activity and PAF and GMP-140 levels and may be a risk factor for HSP with gastrointestinal bleeding.</p>

Force-dependent calcium signaling and its pathway of human neutrophils on P-selectin in flow

P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.

Construction and identification of human p-selectin promotor luciferase reporter gene vector / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a luciferase reporter gene vector of p-selectin gene promoter and determine its transcriptional activity for screening the effect of drugs on the transcriptional activity of p-selectin promoter.</p><p><b>METHODS</b>Primers were designed based on human p-selectin promoter sequence from UCSC software. The p-selectin promoter from human genome DNA was then amplified. After digestion of pGL3-Basic vector and p-selectin promoter with Kpn I and Xho I, p-selectin promoter was inserted into pGL3-basic vector. The recombinant plasmid, namely pGL3-p-selectin-promoter, was transiently cotransfected into 293F cells with pRL-SV40 as the control vector, and the activity of the dual luciferase was detected. The transcription activity of serially truncated segments of the p-selectin promoter reporter gene was quantified by luciferase expression. 293F cells transfected with pGL3-p-selectin-promoter reporter gene and dual luciferase were stimulated with LPS, TNF-α and As2O3, and the transcriptional activity of p-selectin promoter were assessed.</p><p><b>RESULTS</b>pGL3-p-selectin-promoter was constructed successfully as verified by restriction digestion and sequence analysis. The luciferase activity was higher in pGL3-p-selectin-promoter/pRL-SV40 group than in pGL3-basic/pRL-SV40 group (0.8573±0.4703 vs 0.03955±0.05894). pGL3- 1826 bp was actively transcribed compared with pGL3-1092 bp and pGL3-3738 bp. LPS, TNF-α and As2O3 significantly enhanced the transcriptional activity of p-selectin promoter.</p><p><b>CONCLUSION</b>pGL3-p-selectin-promoter can be transcribed and activated in 293F cells. This study provided an important basis for acquiring transcriptional factors and screening inflammatory factors and drugs.</p>

Effects of Angiotensin-II Receptor Blocker on Inhibition of Thrombogenicity in a Canine Atrial Fibrillation Model

BACKGROUND AND OBJECTIVES: Angiotensin-II receptor blockers (ARBs) are known to reduce the development of atrial fibrillation (AF) through reverse-remodeling. However, the effect of ARBs on thrombogenicity in AF remains unknown. MATERIALS AND METHODS: Twelve dogs were assigned to control (n=4), ARB (candesartan cilexitil 10 mg/kg/day p.o., 12 weeks; n=4), or sham (n=4) groups. Sustained AF was induced by rapid atrial pacing. Both arterial and venous serum levels of tissue inhibitor of matrix metalloproteinase-1, von Willebrand factor, P-selectin, and vascular cell adhesion molecule-1 (VCAM-1) were measured at baseline and during AF (0, 4, and 12 weeks) with enzyme-linked immunosorbent assay. Biopsies from both atria including the appendages were performed to semi-quantitatively assess endocardial and myocardial fibrosis after 12 weeks. RESULTS: The serum levels of bio-markers were not significantly different at baseline or during AF between the control and the candesartan groups. The levels were not significantly different over time, but there was a trend toward a decrease in arterial VCAM-1 from 4 to 12 weeks in the candesartan group compared to the control group. The grades of endocardial fibrosis after 12 weeks but not those of myocardial fibrosis were slightly reduced in the candesartan group compared to the control group. CONCLUSION: This study did not show that the ARB candesartan significantly reverses thrombogenicity or fibrosis during AF. Future studies using a larger number of subjects are warranted to determine the therapeutic effect of renin-angiotensin-aldosterone system blockade on prothrombogenic processes in AF.

IL-17 Induces MPTP opening through ERK2 and P53 signaling pathway in human platelets / 华中科技大学学报（医学）（英德文版）

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.

Prethrombotic status and long-time thromboembolic events in primary hypertensive patients with or without elevated homocysteine level / 中华心血管病杂志

<p><b>OBJECTIVE</b>To evaluate the association between homocysteine level and prethrombotic status and long-term thromboembolic events in patients with primary hypertension.</p><p><b>METHODS</b>Results between 110 hypertensive patients with elevated homocysteine (HCY) level were compared with 110 hypertensive patients with normal HCY level which were enrolled from October 2003 to November 2009. Fibrinogen (FIB), viscosity, thrombomodulin (TM), granule membrane protein (GMP-140), prethrombin F1+2 fragment (F1+2), D-dimer fragment (D-Dimer) and antithrombin III (AT-III) were measured and correlated to HCY and prethrombotic state. The endpoints of the study were arterial and venous thromboembolic events. The variables linked with arterial and venous thromboembolic events were included in Cox proportional hazard models. The event-free survival was illustrated with Kaplan-Meier survival curves and compared by the Log-rank test.</p><p><b>RESULTS</b>The patients were followed up for 8-122 months (median follow-up time was 85 months). Compared with hypertensive patients with normal HCY, the plasma level of TM ((4.8±1.2) µg/L vs. (4.5±1.0) µg/L, P = 0.045), GMP-140 ((18.8±3.2) µg/L vs. (17.1±4.3) µg/L, P = 0.001), F1+2 ((1.2±0.4) nmol/L vs. (1.0±0.6) nmol/L, P = 0.004) were significantly higher while the plasma level of AT-III ((95.3±10.4) % vs. (98.6±10.6)%, P = 0.021) was significantly lower in hypertensive patients with elevated HCY level. FIB, viscosity of plasma and D-dimer were similar between the two groups. Multiple regression analyses indicated that HCY level was negatively correlated with AT-III (β = -0.199, P = 0.011) and positively correlated with age (β = 0.217, P = 0.04), female gender (β = 5.667, P = 0.001) and TM (β = 2.341, P = 0.003). Cox multivariate analysis revealed that age and HCY level were independent prognostic risk factors of thromboembolic events (OR 1.046, 95% CI 1.013-1.082, OR 1.052, 95% CI 1.027-1.078, respectively) (all P < 0.05). Kaplan-Meier curves showed that there was a significant difference in the event-free survival between the two groups (Log-rank test, P = 0.027).</p><p><b>CONCLUSIONS</b>Compared with normal HCY hypertensive patients, the levels of plasma prothrombin activators such as TM, GMP-140 and F1+2 were significant increased and anti-thrombin factor such as AT-III was significant decreased in hypertensive patients with elevated HCY. Old age and high HCY level were independent prognostic risk factors of thromboembolic events. The event-free survival in hypertensive patients with elevated HCY is lower than in hypertensive patients with normal HCY level.</p>

Angiotensin II induces expression of inflammatory mediators in vascular adventitial fibroblasts / 生理学报

Vascular adventitial fibroblasts (AF) may play an important role in vascular inflammation. This study was aimed to investigate the expression pattern of inflammatory mediators in AF induced by angiotensin II (AngII) and to explore the effects of AF-derived inflammatory mediators on the adhesion and migration of macrophages both in vitro and in vivo. We used real-time RT-PCR to detect the mRNA expression of inflammatory mediators in cultured AF. The results showed that AngII (1 × 10(-7) mol/L) up-regulated mRNA expression of 4 inflammatory mediators, including P-selectin, ICAM-1, IL-6 and MCP-1, in cultured AF. Western blot analysis or ELISA revealed that AngII up-regulated P-selectin and ICAM-1 protein expression and IL-6 secretion in cultured AF, but did not alter MCP-1 secretion. We further detected the effects of AF-derived inflammatory mediators on the adhesion and chemotaxis of RAW264.7, a macrophage cell line. We found that AF stimulated with AngII could enhance the adhesion of RAW264.7 and the conditioned medium from AngII-stimulated AF could enhance the migration of RAW264.7. Immunofluorescence study showed an enhanced accumulation of CD68 positive cells and the up-regulation of P-selectin, ICAM-1, IL-6 and MCP-1 in aortic adventitia of AngII-infused (200 ng/kg per min for 2 weeks) rats. We concluded that AF may contribute to vascular inflammation via expression of certain inflammatory mediators and the subsequent adhesion and chemotaxis of macrophages.

Effect of aqueous extracts of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro / 中国结合医学杂志

<p><b>OBJECTIVE</b>To study the effect of aqueous extract of several kinds of herbs on human platelet aggregation and expression of P-selectin in vitro.</p><p><b>METHODS</b>Blood 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.</p><p><b>RESULTS</b>Out 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.</p><p><b>CONCLUSIONS</b>Caulis 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.</p>

Expression and significance of adhesion molecules CD62P and CD44 in peripheral blood of infants with bronchiolitis / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To explore the expression and significance of the adhesion molecules CD62P and CD44 in the peripheral blood of infants with bronchiolitis.</p><p><b>METHODS</b>Thirty-three infants with bronchiolitis in the acute phase and 19 infants with bronchiolitis in the recovery phase, who were hospitalized between November 2014 and May 2015, were enrolled. Thirty infants with bronchopneumonia and 26 infants without infection were enrolled as the bronchopneumonia group and the control group, respectively. The CD62P expression in the peripheral blood of each group was measured by flow cytometry, and the CD44 level in serum was determined using ELISA.</p><p><b>RESULTS</b>The levels of the adhesion molecules CD62P and CD44 in the bronchiolitis group in the acute phase were significantly higher than those in the bronchiolitis group in the recovery phase, the bronchopneumonia group, and the control group (P<0.05). The levels of the adhesion molecules CD62P and CD44 in the bronchiolitis group in the recovery phase were also significantly higher than those in the control group (P<0.05). In the bronchiolitis group in the acute phase, there was a positive correlation between CD62P expression and serum CD44 level (r=0.91; P<0.05).</p><p><b>CONCLUSIONS</b>The adhesion molecules CD62P and CD44 play an important role in the pathogenesis of bronchiolitis, and their levels can reflect the severity of inflammatory response in infants with bronchiolitis.</p>