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【Objective】 To compare the desalination effects of five desalination methods and their effects on the components for human coagulation factor Ⅷ(FⅧ), and provide reference for selection of protein desalination methods. 【Methods】 Sephadex G-25 Medium gel, Fractogel EMD BioSEC gel, ultrafiltration, room temperature dialysis and 4℃ dialysis were used to desalt human FⅧ. The desalination effect was evaluated by the removal rate of Na +, citrate ion and glycine. FⅧ protein recovery, FⅧ activity (FⅧ∶C), VWF antigen (VWF∶Ag), VWF activity(VWF∶Ac), VWF polymers and SDS-PAGE analysis before and after desalination were compared to evaluate the effect of desalination on FⅧ components. 【Results】 In terms of desalination effect, the removal rate of Na+ was the lowest in ultrafiltration desalination, while that of Fractogel EMD BioSEC gel was the highest [(97.90±0.06) % vs (99.82±0.07) %]. Except that there was no statistical significance between Sephadex G-25 Medium gel desalination and Fractogel EMD BioSEC gel desalination (P=0.90), the removal rates of the other four methods were statistically significant. The removal rate of glycine was the lowest in ultrafiltration desalination, wihle that of Fractogel EMD BioSEC gel desalination was the highest [(95.78±0.42) % vs (99.81±0.08) %]. Significant difference in glycine removal was noticed in ultrafiltration desalination, but not among the other four desalination methods. There was no significant difference in the removal rate of citrate ions among the five methods (P=0.85). For the effect of FⅧ components, FⅧ∶C, VWF∶Ag, VWF∶Ac and protein recovery rates of ultrafiltration desalination were the highest, with (18.34±1.99) IU/mL, (11.81±0.33) IU/mL, (12.26±0.58) IU/mL and (97.13±1.37) %, respectively. There was no significant change in VWF∶Ac/VWF∶Ag before and after desalination by the five methods. SDS-PAGE and VWF polymer analysis showed that different desalination methods had no significant impact on protein composition. 【Conclusion】 Although different desalination methods had no significant effect on the composition of FⅧ protein, the desalination effect was different. Moreover, different desalination methods had significant effects on protein recovery, FⅧ∶C, VWF∶Ag and VWF∶Ac. The selection of desalination methods should be more considered during protein processing,
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【Objective】 To explore the feasibility of tirofiban, a platelet surface glycoprotein (GP)Ⅱb/Ⅲa receptor antagonist intervene in transfusion-related acute lung injury (TRALI), by inhibiting platelet activation and by preventing platelet and neutrophil binding to form aggregates. 【Methods】 1) Fifty wild-type male Balb/c mice, aged 8 to 10 weeks, were randomly divided into TRALI, normal, tirofiban TRALI intervention, isotype control and tirofiban normal intervention groups. In the TRALI model, tirofiban TRALI intervention and isotype control groups, each mouse was injected intraperitoneally with lipopolysaccharide (LPS) 0.1 mg/kg, and after 18 h with 4.5 mg/kg anti-MHC-I or IgG2a isotype control antibody, in which 0.5 μg/g tirofiban was injected 30 min before anti-MHC-I injection, and was labeled as tirofiban TRALI intervention. The group without any treatment was set as normal group. The tirofiban normal intervention group was injected with only 0.5 μg/g tirofiban into the tail vein, 30 min before the injection of anti-MHC-I. 2) After antibody injection, the mice were observed for 2 h, then executed with their lungs removed, and the extent of lung injury and the intervention effect of tirofiban were analyzed by comparing the differences in lung dry to wet ratio, total protein, myeloperoxidase (MPO), inflammatory factors and quantitative results of HE staining. The platelet activation level in whole blood and immunofluorescence (IF) quantification of platelet and neutrophil fluorescence were detected by flow cytometry to analyze the mechanism of tirofiban on TRALI. 【Results】 1) The indexes of lung injury in the tirofiban TRALI intervention group and TRALI model group for HE staining were 0.663 3±0.141 9 vs. 0.173 3±0.120 4 (P<0.05), respectively; 2) Platelet activation levels(%)in whole blood in the TRALI group, normal group and tirofiban TRALI intervention group were 22.87±9.943 vs 5.070±2.234 vs 5.767±3.224(P<0.05), respectively. 3) The mean fluorescence density of platelet neutrophil aggregates for IF detection in the tirofiban intervention group and TRALI model group was 21.89±3.536 vs. 32.77±0.9624 (P<0.05). 【Conclusion】 The platelet GP Ⅱ b/Ⅲa-specific inhibitor tirofiban inhibited platelet-neutrophil binding in mice, thus could possibly intervene in TRALI.
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Transfusion-related acute lung injury (TRALI), with clinical manifestation, diagnosis and pathological mechanism consistent with acute lung injury(ALI), belongs to a sub-category of ALI. Excessive deposition of fibrin in lung is one of the characteristic of ALI, and reversing fibrin formation is of great significance to intervene ALI. The decrease of fibrinolytic activity is one of the important causes of excessive deposition of fibrin in lung, and also the important pathological feature of TRALI. This article discusses the potential of modulating fibrinolytic activity to intervene TRALI from the perspective of regulating the effectiveness of fibrinolytic activity to intervene ALI.
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【Objective】 To obtain the quality information of von Willebrand factor (vWF) in coagulation factor Ⅷ (FⅧ) concentrates in China. 【Methods】 FⅧ concentrates produced by 7 domestic blood product manufactures and 1 foreign manufacture were collected, then FⅧ and vWF contained in FⅧ concentrates were evaluated. 【Results】 The activity loss of vWF was more than 25% in 2 of the 7 domestic FⅧ concentrates. The ratio of vWF activity to FⅧ activity in FⅧ concentrates from different domestic manufactures was significantly different (P<0.05). The ratio in FⅧ concentrates prepared by C, D, F manufacturer was greater than 1, which was similar to that in willate@ approved abroad for the treatment of vWD. The ratio in FⅧ concentrates prepared by E manufacturer was greater than 0.7 and less than 1, and by A, B, G manufacturers was less than 0.5. In addition, the specific activities of FⅧ and vWF were significantly different among different FⅧ concentrates in China (P<0.05), and the specific activities of FⅧ and vWF were much lower than that of willate@. 【Conclusion】 The variation of vWF quality between domestic FⅧ concentrates and willate@ is mainly due to the different in vWF content. After the comprehensive consideration of various indicators, the FⅧ concentrates made by C and D manufacturers may be used in the treatment of vWD.
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【Objective】 To explore the efficacy and possible mechanisms of activation of Death receptor 3 (DR3) signaling pathway in the prevention of antibody-mediated transfusion-related acute lung injury (TRALI) via DR3 agonistic (αDR3) antibody. 【Methods】 8-10-week-old male wild-type Balb/c mice (40) were randomly divided into Naïve group, isotype control group, TRALI model group, and intervention group. Mice without any treatment served as Naïve group. Isotype and TRALI model were established by intraperitoneally priming 8-10-week Balb/c mice with LPS 18 h prior to injection of an IgG2a isotype antibody and anti-MHC-Ⅰ antibody via tail vein, respectively. Intervention group: mice were intraperitoneally injected with a single dose of αDR3 antibody (1 mg/kg) on day 1; after 3 days, the mice were challenged with LPS 18 h prior to injection of an anti-MHC-I antibody. The lung tissues and spleens of mice in each group were collected at the mice died or 2 hours after TRALI modeling for the lung injury severity. Spleens were collected to measure the proportion of Treg by flow cytometry. Foxp3, iNOS, and CD206 immunohistochemical staining combining with optical density analysis of lung tissues were used to represent Treg, M1 macrophages and M2 macrophages, respectively. The concentration of IL-6, IL-1β, TNF-α, and IL-10 cytokines in lung tissues was detected via Cytometric Beads Array. 【Results】 Compared with TRALI group, 1) the lung injury of mice were significantly alleviated in intervention group; 2) the proportion of Treg(%) in the spleens (9.295±1.349 vs 2.257±0.610, P<0.05), Foxp3 expression of Treg in the lungs (0.302 6±0.052 6 vs 0.230 2±0.016 3, P<0.05), and the concentration of Treg derived cytokines IL-10 in the lungs (29.52±8.885 vs 8.045±1.911, P<0.05) increased significantly in intervention group; 3) the iNOS expression of M1 macrophages (0.209 6±0.013 9 vs 0.279 6±0.045 2) and the concentration of M1 macrophage derived cytokines IL-6 (23.22±19.35 vs 301.1±157.7), IL-1β (46.76±25.34 vs 307.6±183.8), and TNF-α (45.99±14.16 vs 143.9±44.43) in the lungs was significantly reduced(P<0.05), while CD206 expression of M2 macrophages (0.291 2±0.032 1 vs 0.221 5±0.012 7) and the concentration of M2 macrophage derived IL-10 cytokines (29.52±8.885 vs 8.045±1.911) in the lungs increased significantly in intervention group(P<0.05). 【Conclusion】 Activation of DR3 signaling pathway by αDR3 antibody prevents antibody-mediated TRALI via expanding Treg, which regulates macrophage polarization by IL-10 derived from Treg.
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Objective To concentrate coagulation Factor Ⅺ(FⅪ)from human plasma.Methods FⅪwas isolated and purified from human plasma via two-step chromatography,including CM-Sepharose fast flow ion exchange and Heparin CL-6B affinity chromatography.Results The recovery rate of FⅪ was(21.02?5.04)%,the specific clotting activity of purified FⅪ was(17.59?1.96) U/mg,and the purification factor was(1162.29?129.64) fold(n=7).Conclusion The two-step chromatography is effective in concentrating FⅨ.
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Objective To isolate and purify human coagulation factor Ⅶ from Cohn fraction Ⅲ precipitate.Methods The purification procedure of human factor Ⅶ from Cohn fraction Ⅲ precipitate involves dissolving fraction Ⅲ,absorbing factor Ⅶ onto barium citrate and eluting,ammonium sulfate fractionation and DEAE-Sepharose Fast Flow ion exchange chromatography,and Sephadex G-100 gel filtration chromatography.Results 10.1mg purified FⅦ was obtained from 400g Cohn fraction Ⅲ precipitate.The purified FⅦ has a specific clotting activity of 1775.8U/mg and the overall yield of FⅦ specific clotting activity is 17.6% of the starting material.The purity of FⅦ was judged by SDS-PAGE and there was only one protein band on the gel.Conclusion The procedure of purifying Ⅶ from Cohn fraction Ⅲprecipitate is established with satisfactory purity.