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的预后仍然很差。尤其是并发脓毒症的ARDS预后极差。因此，应尽快建立治疗ARDS的方法。由于抗凝血酶III(AT)的抗凝作用，有时用于治疗弥漫性血管内凝血。AT结合并使凝血酶和其他凝血剂失活。另一方面，Meta分析表明，AT治疗严重脓毒症的有效性。这一结果提示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，从而减少中性粒细胞在肺中的聚集，这反过来又会抑制大鼠肺中氧自由基的产生。我们还证明，用AT和潜伏期-AT共同处理中性粒细胞可以减少F-肌动蛋白的形成，并降低变形性…更多要归功于fMLP治疗。提示中性粒细胞细胞膜上可能存在新的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了硫酸乙酰肝素。然而，对于这类实验，我们需要大量的中性粒细胞。2007年，我们发现人肿瘤细胞株HTI080表达Syndecan-4。因此，我们用HT1080代替了分离的人中性粒细胞。用肝素酶或氯酸钠去除培养的HT1080细胞表面的硫酸肝素后，除肝素结合部位外，AT仍与AT结合。然后，我们分离并分析了与AT结合的蛋白质。即使用肝素酶或氯酸钠将硫酸肝素从细胞表面去除，也能检测到糖胺聚糖中的Syndecan-4。为了进一步证明结果，我们应用IP-Western，并证明了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。较少

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