血栓调节蛋白 α通过凝血酶存在下的羧肽酶 B2 发挥抗炎和抗纤维蛋白溶解作用。

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin.

机构信息

Laboratory for Pharmacology, Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, Shizuoka, Japan.

出版信息

Thromb Res. 2016 Nov;147:72-79. doi: 10.1016/j.thromres.2016.09.011. Epub 2016 Sep 17.

DOI:10.1016/j.thromres.2016.09.011

PMID:27693845

Abstract

BACKGROUND

Thrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro.

METHODS

CPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay.

RESULTS

CPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor.

CONCLUSIONS

TM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro.

摘要

背景

血栓调节蛋白（TM）α是一种重组人可溶性 TM，可增强凝血酶对前羧肽酶 B2（pro-CPB2）的激活。激活的 pro-CPB2（CPB2）发挥抗炎和抗纤维蛋白溶解作用。因此，TMα 也可能通过 CPB2 发挥抗炎和抗纤维蛋白溶解作用。然而，TMα 的这些作用尚未阐明。在本研究中，我们研究了 TMα 通过 CPB2 对补体成分 C5a 失活（作为抗炎作用）和凝血酶诱导的纤维蛋白溶解时间延长（作为抗纤维蛋白溶解作用）的体外影响。

方法

通过比色法测定 CPB2 活性和组织因子诱导的凝血酶生成。通过 C 末端切割 C5a 和抑制 C5a 诱导的人中性粒细胞迁移来评估 C5a 失活。通过组织型纤溶酶原激活剂诱导的纤维蛋白溶解试验测定纤维蛋白溶解时间延长。

结果

TMα 和凝血酶以浓度依赖的方式增加人血浆中的 CPB2 活性。TMα 抑制组织因子诱导的凝血酶生成，并同时增强人血浆中 pro-CPB2 的激活。用 TMα、凝血酶和 pro-CPB2 处理的 C5a 的质谱在 156m/z 处减少，表明 TMα 增强了 C5a 向 C 末端切割的 C5a 的加工，即 C5a 的无活性形式。用 TMα、凝血酶和 pro-CPB2 处理 C5a 后，C5a 诱导的人中性粒细胞迁移减少。TMα 延长人血浆中的纤维蛋白溶解时间，并且该作用通过添加 CPB2 抑制剂完全消除。