Analysis of syndecan-4 core protein as a receptor for antithrombin III

近年来，急性呼吸窘迫综合征（ARDS）的治疗效果有所改善。然而，ARDS的预后仍然较差。特别是合并败血症的ARDS预后极差。因此，我们应尽快建立治疗ARDS的方法。抗凝血酶III （AT）由于其抗凝作用，有时应用于治疗弥散性血管内凝血。AT结合凝血酶和其他凝血剂并使其失活。另一方面，meta分析显示AT治疗严重脓毒症的有效性。该结果提示AT具有抗炎作用。我们证明，AT很可能减少中性粒细胞中f -肌动蛋白的形成，从而减少肺中中性粒细胞的积累，这反过来又会抑制大鼠肺中氧自由基的产生。我们还证明了用AT和潜伏AT预处理中性粒细胞可减弱F-actin的形成，并降低其可变形性。这些数据提示在中性粒细胞的细胞膜上一定存在新的AT受体。2006年，我们首先检测了syndecan-4的表达，并从分离的人中性粒细胞中去除硫酸肝素。然而，对于这类实验，我们需要大量的中性粒细胞。2007年，我们发现人类肿瘤细胞系HTI080表达syndecan-4。因此，我们使用HT1080代替分离的人中性粒细胞。用肝素酶或氯酸钠从培养的肿瘤细胞株HT1080细胞表面去除硫酸肝素后，除肝素结合位点外，AT仍与AT结合。然后，我们用质谱法分离并分析了与AT结合的蛋白质。即使使用肝素酶或氯酸钠从细胞表面去除硫酸肝素，也能检测到Syndecan-4，一种糖胺聚糖。为了进一步证明结果，我们应用了IP-western，并证明即使在使用肝素酶或氯酸钠去除硫酸肝素后，AT也能与syndecan-4结合。这些数据表明syndecan-4是AT的受体。少

Recently, the outcome of the treatment for Acute Respiratory Distress Syndrome (ARDS) has been improved. However, the prognosis of ARDS is still poor. Especially, the prognosis of ARDS associated with sepsis is extremely poor. Therefore, we should establish the method to cure ARDS as soon as possible. Antithrombin III (AT) sometimes applies for the treatment of disseminated intravascular coagulation, because of its anti-coagulant effect. AT binds and inactivates thrombin and other coagulants. On the other hand, the meta-analysis demonstrated that the effectiveness of AT for severe sepsis. This results suggests that AT has an anti-inflammatory effect. We demonstrated that AT most likely reduces F-actin formation in neutrophil thereby reducing neutrophil accumulation in the lung, which would in turn inhibit oxygen radical production in the rat lung. We also demonstrated that pretreatment of neutrophil with both AT and latent-AT attenuate F-actin formation and decreasing of deformability … More due to fMLP treatment. These data suggest that there must be new receptor for AT on the cell membrane of neutrophil. In 2006, at first we examined expression of syndecan-4 and removed heparan sulfate from isolated human neutrophil. However, for these kinds of experiment, we need a large amount of neutrophil. In 2007, we found that human tumor cell line HTI080 express syndecan-4. Therefore, we used HT1080 instead of isolated human neutrophil. After removing heparan sulfate from cell surface of the cultured tumor cell line HT1080 using heparinase or sodium chlorate, AT was still bound to AT except for heparin binding site. Then, we isolated and analyzed the proteins bound to AT using mass spectrometry. Syndecan-4, one of the glycosaminoglycan, was detected even when heparin sulfate was removed from the cell surface using heparinase or sodium chlorate. To further proof the results, we applied IP-western and demonstrated that AT bound to syndecan-4 even after removing heparin sulfate using heparinase or sodium chlorate. These data suggest syndecan-4 is a receptor for AT. Less