Effect of thrombin on malignant biological behavior of esophageal cancer cell line Eca109 / 肿瘤研究与临床

Objective To study the effect of thrombin on proliferation and invasion of esophageal cancer cell line Eca109, and to explore its possible mechanism. Methods The proliferation and invasion of Eca 109 cells treated with thrombin were detected by MTT and Transwell assay, respectively. The activity of matrix metalloproteinase 2 (MMP-2) and MMP-9 in the supernatant of Eca109 cells was detected by gelatin zymography. Reverse transcription polymerase chain reaction (PCR) and immunocytochemistry were used to study the mRNA expression of protease-activated receptor 1 (PAR-1), the important receptor of thrombin, and subcellular localization of PAR-1 protein in Eca109 cells, respectively. Results Thrombin could promote Eca109 cells proliferation in a dose-dependent manner. Cell proliferative rates of 0.5 U/ml and 1.0 U/ml thrombin were 34.38 % and 57.19 %, respectively (P< 0.05). Compared to that of control group, the number of Eca109 cells incubated with 1.0 U/ml thrombin invading through the basement membrane of Transwell was increased (303.33 ±6.66 vs. 116.33 ±11.51, P< 0.05). When treated with various concentrations of thrombin for 24 h, the activities of MMP-2 and MMP-9, especially MMP-9, in the supernatant of Eca109 cells were increased in a dose-dependent manner. Eca109 cells expressed PAR-1 mRNA, and PAR-1 protein was mainly located on the cellular membrane. Conclusion Thrombin increases proliferation and invasion of esophageal cancer Eca109 cells and enhances the activities of MMP-2 and MMP-9 in cells supernatant, which might be induced by activation of PAR-1 located on cellular membrane.

Role of Coagulation Factor 2 Receptor during Respiratory Pneumococcal Infections

Coagulation factor 2 receptor (F2R), also well-known as a protease-activated receptor 1 (PAR1), is the first known thrombin receptor and plays a critical role in transmitting thrombin-mediated activation of intracellular signaling in many types of cells. It has been known that bacterial infections lead to activation of coagulation systems, and recent studies suggest that PAR1 may be critically involved not only in mediating bacteria-induced detrimental coagulation, but also in innate immune and inflammatory responses. Community-acquired pneumonia, which is frequently caused by Streptococcus pneumoniae (S. pneumoniae), is characterized as an intra-alveolar coagulation and an interstitial neutrophilic inflammation. Recently, the role of PAR1 in regulating pneumococcal infections has been proposed. However, the role of PAR1 in pneumococcal infections has not been clearly understood yet. In this review, recent findings on the role of PAR1 in pneumococcal infections and possible underlying molecular mechanisms by which S. pneumoniae regulates PAR1-mediated immune and inflammatory responses will be discussed.

Change in endogeous hydrogen sulfide in patients with acute pancreatitis and its relationship to coagulation function / 中华危重病急救医学

Objective To study the change in endogenous hydrogen sulfide (H2S) in patients with acute pancreatitis and its relationship to coagulation function. Methods A prospective case control study was conducted. Forty patients with mild acute pancreatitis (MAP group) and 40 with severe acute pancreatitis (SAP group) admitted to Yiwu Central Hospital in Zhejiang Province from December 2002 to March 2015 were enrolled. Forty healthy persons served as control (healthy control group). Blood was collected to determine the levels of H2S, blood coagulation factor Ⅷ (FⅧ), von Willebrand factor (vWF), plasminogen (PLG), antithrombin (AT), platelet count (PLT), tissue factor (TF), tumor necrosis factor-α (TNF-α), and protease activated receptor-1 (PAR-1). The correlations among the above parameters were analyzed. Results There was no statistical significance in sex, age, body weight and time of disease among three groups, indicating it was comparable among the groups. Compared with healthy control group, the levels of H2S, FⅧ, vWF, TF, TNF-α, and PAR-1 in MAP and SAP groups were significantly elevated [H2S (μmol/L): 67.42±6.34, 112.47±12.69 vs. 42.57±4.18, FⅧ: (67.5±5.8)%, (82.3±4.7)% vs. (57.2±6.4)%, vWF: (112.6±9.7)%, (142.5±12.5)% vs. (76.4±8.2)%, TF (ng/L): 45.27±4.34, 64.76±6.25 vs. 18.15±1.89, TNF-α (ng/L): 197.67±13.62, 324.72±25.54 vs. 20.08±2.57, PAR-1 (fluorescence intensity): 32.16±4.43, 56.12±7.07 vs. 12.27±2.12, all P < 0.01], and PLG and AT activity were significantly decreased [PLG: (52.4±4.7)%, (36.7±3.2)% vs. (62.1±5.6)%, AT: (43.2±6.9)%, (35.5±5.4)% vs. (53.6±6.1)%, all P < 0.01]. The changes in the parameters in SAP group were more remarkable than those in MAP group (all P < 0.01). PLT in SAP group was significantly lower than that in healthy control and MAP groups (×109/L: 8.5±1.1 vs. 15.7±2.8, 12.4±1.9, both P < 0.01). H2S was positively correlated with FⅧ, vWF, TF, TNF-α, and PAR-1 (r value was 0.56, 0.61, 0.72, 0.66, 0.64, respectively, all P < 0.01), and it was negatively correlated with PLG and AT (r value was -0.64, -0.57, both P < 0.01). Conclusion As an inflammatory factor, endogenous H2S deteriorates coagulation function in patients with acute pancreatitis by up-regulating TF, TNF-α, and PAR-1.

Neuroprotective effects of protease-activated receptor-1 antagonist, SCH79797, on early stage of brain injury and expression patterns of brain-derived neuro trophic factor and nerve growth factor after subarachnoid hemorrhage in rats / 中华神经医学杂志

Objective To investigate the neuroprotective effects of protease-activated receptor (PAR)-1 antagonist SCH79797 on early stage of brain injury and expression patterns of brain-derived neuro trophic factor (BDNF) and nerve growth factor (NGF) after subarachnoid hemorrhage (SAH) in rats. Methods Fifty-four clean male SD rats were randomly divided into three groups:sham-operative group, SAH group and treatment group (n=18). SAH rat models in the later two groups were induced by single cisterna magna blood injection. Rats in the treatment group were given SCH79797 (25μg/kg) after SAH modeling. Neurologic function was assessed and scored after SAH for 24 h. Then the rats were sacrificed, and then, the brains were collected for determination of brain water content. The expression patterns of BDNF and NGF in cerebral tissues were tested by enzyme linked immunosorbent assay and immunofluorescence method. Results As compared with rats in the SAH group, rats in the treatment group had significantly increased neurological deficit scale scores (12.11 ±2.62 vs. 14.59 ±2.24), statistically decreased water content in brain tissues (83.01%±0.38%vs. 79.79%±0.44%), with significant difference (P<0.05). As compared with those in the SAH group, the expressions of BDNF and NGF in treatment group were significantly higher ([100.15±59.13] pg/mL vs. [368.00±137.52] pg/mL; [33.44± 2.21] pg/mL vs. [37.49±2.29] pg/mL, P<0.05). As compared with those in the SAH group, the immunofluorescent staining intensities of BDNF and NGF in treatment group were significantly higher (41.65±1.50 vs. 91.40±1.30;23.50±1.70 vs. 30.65±1.80, P<0.05). Conclusion SCH79797 given early may reduce the degree of brain edema and increase the expressions of BDNF and NGF, which has obvious neuroprotective effect.

Vorapaxar, a novel oral antiplatelet drug.

Vorapaxor is first in the class of protease activated receptor 1 (PAR 1) antagonists. It acts by inhibiting the binding of thrombin to PAR 1 and thereby prevents platelet aggregation. USFDA approved it in May 2014 as the results of clinical trials showed that the benefit: risk ratio was high. It is to be used in a dose of 2.5 mg once daily as triple antiplatelet therapy with aspirin and clopidogrel for reduction of thrombotic cardiovascular events in patients with a history of myocardial infarction or peripheral arterial disease. Increase in the incidence of intracranial hemorrhage is the major side-effect seen.

Protective effects of activated protein C on neurovascular unit in a rat model of intrauterine infection-induced neonatal white matter injury / 华中科技大学学报（医学）（英德文版）

Activated protein C (APC), a natural anticoagulant, has been reported to exert direct vasculoprotective, neural protective, anti-inflammatory, and proneurogenic activities in the central nervous system. This study was aimed to explore the neuroprotective effects and potential mechanisms of APC on the neurovascular unit of neonatal rats with intrauterine infection-induced white matter injury. Intraperitoneal injection of 300 μg/kg lipopolysaccharide (LPS) was administered consecutively to pregnant Sprague-Dawley rats at embryonic days 19 and 20 to establish the rat model of intrauterine infection- induced white matter injury. Control rats were injected with an equivalent amount of sterile saline on the same time. APC at the dosage of 0.2 mg/kg was intraperitoneally injected to neonatal rats immediately after birth. Brain tissues were collected at postnatal day 7 and stained with hematoxylin and eosin (H&E). Immunohistochemistry was used to evaluate myelin basic protein (MBP) expression in the periventricular white matter region. Blood-brain barrier (BBB) permeability and brain water content were measured using Evens Blue dye and wet/dry weight method. Double immunofluorescence staining and real-time quantitative PCR were performed to detect microglial activation and the expression of protease activated receptor 1 (PAR1). Typical pathological changes of white matter injury were observed in rat brains exposed to LPS, and MBP expression in the periventricular region was significantly decreased. BBB was disrupted and the brain water content was increased. Microglia were largely activated and the mRNA and protein levels of PAR1 were elevated. APC administration ameliorated the pathological lesions of the white matter and increased MBP expression. BBB permeability and brain water content were reduced. Microglia activation was inhibited and the PAR1 mRNA and protein expression levels were both down-regulated. Our results suggested that APC exerted neuroprotective effects on multiple components of the neurovascular unit in neonatal rats with intrauterine infection- induced white matter injury, and the underlying mechanisms might involve decreased expression of PAR1.

Virtual and experimental screening of protease activated receptor 1 antagonist in plant ingredients / 中草药

Objective: To find the new type of structures with protease activated receptor 1 (PAR-1) inhibition from plant ingredients. Methods: Thirty ingredients were docked into PAR-1, and then, docking score, occupied space, hydrogen bonding, and other indicators were used for virtual screening. In vitro platelet aggregation experiments in guinea pig were performed to screen the activities of all ingredients. Results: Virtual screening suggested that T30 and T21 had the prospects to inhibit PAR-1. Experiment screening showed that T21, T5, T28, and T29 have the real inhibitory effects on PAR-1. Combination analyses of virtual and experimental screening suggested the following results. Residue 258 and area III had the key effects. Hydrogen matching was required at area II. Area IV and V regions mainly need hydrophobic match. The hydrogen bonding played an important role in improving the activity. Conclusion: According to the binding mode of control drug, T21 is found. To examine the binding mode of T5, T28, and T29, their experimental activities suggest a novel action mode which provides a new direction to find the PAR-1 antagonist.

The hemostatic effect of hemocoagulase in lung cancer radical surgery analysis and its effect on PAR-1 / 中国生化药物杂志

Objective To investigate hemocoagulase hemostatic effect in lung cancer radical surgery and its effect on protease-activated receptor-1 (PAR-1 ).Methods 86 cases of pathologically confirmed lung cancer patients were randomly divided into control group and observation group,which controls were used etamsylate,observation group were used hemocoagulase,then to measure the amount of bleeding,clotting system-related factors before and after surgery,using ELISA assay to detect P-selectin,flow cytometry to measure platelet PAR-1 ,Western Blot to determine lung tissue expression of PAR-1,and the resulting data were statistically analyzed. Results The blood loss of observation group during surgery and postoperative 2d compared with the control group,had a significant difference (P<0.05 );flow cytometry results showed that,PAR-1-positive rate and P-selectin in different time periods in observation group were no statistically significant,while had a statistically significant compared with control group(P<0.05 );Western Blot detected hemocoagulase of lung tissue in patients with PAR-1 had no obvious effect,while control group had significant difference (P<0.05).Conclusion Venin hemocoagulase in lung cancer radical surgery can reduce the amount of bleeding,and be more stable for the role of the coagulation system,as well as less effect on the P-selectin and PAR-1.The results suggest that vonin hemocoagulase might not make patients with systemic hypercoagulable state in patients undergoing radical process with lung cancer,in other words it might not lead to PAR-1 highly expression to cause tumor cell metastasis.

Roles of hirudin in protease activated receptor 1 expression changes in basal artery of rat subarachnoid hemorrhage models / 中华神经医学杂志

Objective To discuss the influence of direct thrombin inhibitor hirudin in the expression ofprotease activated receptor 1 (PAR-1) in basilar artery of the rat model with subarachnoid hemorrhage (SAH).Methods Thirty-six Sprague Dawley rats (7 weeks old,clearanimal) were divided into 3 groups randomly:control group (n=12),SAH group (n=12) and SAH+hirudin treatment group (n=12).The rat models of SAH in the later two groups were induced by twice injection blood into the cisterna magna; rats in the SAH+hirudin treatment group also accepted blood with 200 U/mL hirudin.Animal behavior changes were recorded.Rats of different groups were killed on the 7th d of model making,and samples of basilar artery were performed HE staining for histological observation under microscope; vascular changes of cross-sectional area were measured by Image-Pro Plus6.0 imaging analysis software; and PAR-1 expression was detected by immunohistochemistry.Results Larger vascular lumen,thinner wall,smoother lining and no fold in the basal artery of control group were found as compared with thick wall,narrow lumen and endothelial fold in SAH model rats; the changes of basal artery in SAH+hirudin treatment group enjoyed in the medium ranking.The vascular changes of cross-sectional area in the SAH+hirudin treatment group were more obvious than those in the SAH group (P＜0.05); the cross-sectional area in the SAH+hirudin treatment group and SAH group was significantly smaller than that in the control group (P＜0.05).The immunohistochemistry results showed there was no PAR-1 expression in the rats of control group,and PAR-1 positively expressed in the basilar artery of rats in the SAH group and weakly expressed in that of SAH+hirudin treatment group.The PAR-1 absorbance value in the SAH+hirudin treatment group and SAH group was significantly higher than that in the control group; and that in the SAH+hirudin treatment group was significantly lower than that in the SAH group (P＜0.05).Conclusion Hirudin can reduce PAR-1 expression in the basilar artery of SAH rat models and relieve artery vasospasm,which indicates that thrombin may play important roles in the pathological process of cerebral vasospasm after SAH.

Effect of activated PAR-1 on the expression of CXCR4 mRNA,proliferation and migration of mouse endothelial progenitor cells / 重庆医学

Objective To explore the effect of protease-activated receptor 1(PAR-1) activation on the expression of CXCR4 mR-NA of mouse endothelial progenitor cells (EPCs) and proliferation ,migration of EPCs .Methods Mouse EPCs were activated by different concentration of SFLLRN (one agonist of PAR-1) ,or transfected by small interfering RNA of PAR-1 .The expressions of PAR-1 and CXCR4 mRNA of EPCs were detected by fluorescent quantitative real-time PCR .Proliferation ,migration of EPCs were detected by MTT ,Transwell chambers respectively .Results The expressions of PAR-1 and CXCR4 mRNA in SFLLRN group were higher than that in other groups(P< 0 .05) .The expression of CXCR4 mRNA was highly positive correlation with PAR-1 mRNA(r=0 .991) .The proliferation ,migration of mouse EPCs were induced by activation of PAR-1 .Conclusion Activation of PAR-1 promotes cell proliferation and migration of mouse EPCs ,this effect may be depended on CXCR4 .

MMP-1/PAR-1 signal transduction axis and its prognostic impact in esophageal squamous cell carcinoma

The matrix metalloprotease-1 (MMP-1)/protease-activated receptor-1 (PAR-1) signal transduction axis plays an important role in tumorigenesis. To explore the expression and prognostic value of MMP-1 and PAR-1 in esophageal squamous cell carcinoma (ESCC), we evaluated the expression of two proteins in resected specimens from 85 patients with ESCC by immunohistochemistry. Sixty-two (72.9 percent) and 58 (68.2 percent) tumors were MMP-1- and PAR-1-positive, respectively, while no significant staining was observed in normal esophageal squamous epithelium. MMP-1 and PAR-1 overexpression was significantly associated with tumor node metastasis (TNM) stage and regional lymph node involvement. Patients with MMP-1- and PAR-1-positive tumors, respectively, had poorer disease-free survival (DFS) than those with negative ESCC (P = 0.002 and 0.003, respectively). Univariate analysis showed a significant relationship between TNM stage [hazard ratio (HR) = 2.836, 95 percent confidence interval (CI) = 1.866-4.308], regional lymph node involvement (HR = 2.955, 95 percentCI = 1.713-5.068), MMP-1 expression (HR = 2.669, 95 percentCI = 1.229-6.127), and PAR-1 expression (HR = 1.762, 95 percentCI = 1.156-2.883) and DFS. Multivariate analysis including the above four parameters identified TNM stage (HR = 2.035, 95 percentCI = 1.167-3.681), MMP-1 expression (HR = 2.109, 95 percentCI = 1.293-3.279), and PAR-1 expression (HR = 1.967, 95 percentCI = 1.256-2.881) as independent and significant prognostic factors for DFS. Our data suggest for the first time that MMP-1 and PAR-1 were both overexpressed in ESCC and are novel predictors of poor patient prognosis after curative resection. The MMP-1/PAR-1 signal transduction axis might be a new therapeutic target for future therapies tailored against ESCC.