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Virogen现货谷胱甘肽抗体101-A助力蛋白质二硫键异构酶研究发表在JCI

更新时间:2025-01-19   点击次数:171次

中文摘要:

启动蛋白组织因子(TF)的激活可能是导致纤维蛋白形成的凝血过程中的关键步骤。诱导TF激活的刺激在很大程度上是不确定的。在这里,我们发现氧化还原酶蛋白二硫键异构酶(PDI)在体内血栓形成过程中直接促进TF依赖性纤维蛋白的产生。在小鼠颈动脉内皮剥脱后,PDI在损伤部位从粘附的血小板和破裂的血管壁细胞中释放出来。通过活体荧光显微镜测定,PDI的抑制降低了不同体内血栓形成小鼠模型中TF引发的纤维蛋白形成。PDI输注增加,在血小板粘附减少的情况下,PDI抑制减少了损伤部位的纤维蛋白生成,表明PDI可以直接启动凝血。在体外,人血小板分泌的PDI有助于激活微囊泡(微粒)上的隐性TF。质谱分析表明,TF的部分细胞外半胱氨酸209是组成型谷胱甘肽化的。混合二硫化物的形成有助于将TF保持在低功能状态。我们提出,减少的PDI通过将混合二硫化物和游离硫醇异构化为分子内二硫化物来激活TF。我们的研究结果表明,二硫化物异构酶可以作为损伤反应信号,触发血管损伤后纤维蛋白形成的激活。

英文摘要:

The activation of initiator protein tissue factor (TF) is likely to be a crucial step in the blood coagulation process, which leads to fibrin formation. The stimuli responsible for inducing TF activation are largely undefined. Here we show that the oxidoreductase protein disulfide isomerase (PDI) directly promotes TF-dependent fibrin production during thrombus formation in vivo. After endothelial denudation of mouse carotid arteries, PDI was released at the injury site from adherent platelets and disrupted vessel wall cells. Inhibition of PDI decreased TF-triggered fibrin formation in different in vivo murine models of thrombus formation, as determined by intravital fluorescence microscopy. PDI infusion increased — and, under conditions of decreased platelet adhesion, PDI inhibition reduced — fibrin generation at the injury site, indicating that PDI can directly initiate blood coagulation. In vitro, human platelet–secreted PDI contributed to the activation of cryptic TF on microvesicles (microparticles). Mass spectrometry analyses indicated that part of the extracellular cysteine 209 of TF was constitutively glutathionylated. Mixed disulfide formation contributed to maintaining TF in a state of low functionality. We propose that reduced PDI activates TF by isomerization of a mixed disulfide and a free thiol to an intramolecular disulfide. Our findings suggest that disulfide isomerases can act as injury response signals that trigger the activation of fibrin formation following vessel injury.


论文信息:

论文题目:

Protein disulfide isomerase acts as an injury response signal that enhances fibrin generation via tissue factor activation

期刊名称:J Clin Invest.

时间期卷:2008;118(3):1110-1122.

在线时间:2008年2月14日

DOI:doi.org/10.1172/JCI32376.

产品信息:

货号:101-A

规格:100ug

品牌:Virogen

产地:美国

名称:Anti-Glutathione mAb 100

授权现货代理:Target Technology(靶点科技)

Virogen品牌谷胱甘肽抗体Anti-Glutathione助力蛋白二硫键异构酶(PDI)研究,美国Virogen货号101-A见刊于JCI:

Virogen现货谷胱甘肽抗体101-A助力蛋白质二硫键异构酶研究发表在JCI


Virogen现货谷胱甘肽抗体101-A助力蛋白质二硫键异构酶研究发表在JCI的材料和方法

Virogen现货谷胱甘肽抗体101-A助力蛋白质二硫键异构酶研究发表在JCI


Virogen现货谷胱甘肽抗体101-A助力蛋白质二硫键异构酶研究发表在JCI的材料和方法

Immunoprecipitation. For the detection of the constitutive glutathionylation of TF, solubilized monocytes were centrifuged 3 times at 3,000 g for 2 minutes at 4°C, and the supernatant was incubated with the monoclonal anti-GSH antibody (Virogen; or control IgG) overnight at 4°C. In other experiments, intact monocytes were incubated with anti-GSH antibody for 60 minutes at room temperature. Then, protein A Sepharose (Sigma-Aldrich; 50 μl) was added and the mixture incubated for 60 minutes at 4°C. Immunoprecipitated complexes were collected by centrifugation at 3,000 g for 2 minutes at 4°C. After washing 3 times with PBS, the pellets were incubated in SDS sample buffer (without reducing reagents) for 60 minutes at 20°C. After centrifugation, the supernatant was subjected to SDS-PAGE (7.5%), followed by electroblotting onto the nitrocellulose membrane. The membranes were then exposed to a monoclonal anti-TF antibody (or control IgG), followed by a horseradish peroxidase–conjugated anti-mouse IgG. The experiments were repeated 3 times.

TF glutathionylation. For the labeling of GSH with biotin, equimolar amounts (10 mM) of NHS-biotin (Pierce Biotechnology) and GSH dissolved in PBS were incubated at room temperature. The reaction was terminated after 1 hour, the residual biotin being quenched with ethanolamine (50 mM). To enable the protein incorporation of the labeled glutathione, the cells (or sTF) were incubated for 30–60 minutes with biotin-GSH (5 mM). Subsequently, the cells were washed and solubilized with 1% Triton X-100 in PBS. sTF was dialyzed using centricon filters. Then streptavidin beads (Sigma) were added to the biotinylated proteins of the cells (or sTF) and the mixture was incubated overnight. After sedimentation of the beads, they were washed twice with PBS containing 0.2% Triton X-100. The proteins were eluted in SDS sample buffer, separated by SDS-PAGE, transferred to nitrocellulose membranes, and the monoclonal anti-TF antibody was employed to detect the glutathionylated protein.



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