Takahashi Yohei, Htwe Soe Soe, Wang Dengli, Wake Hidenori, Yata Mariko, Tomonobu Nahoko, Kinoshita Rie, Sakaguchi Masakiyo, Nishibori Masahiro
Department of Translational Research & Drug Development, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
Department of Pharmacology, University of Medicine 2, Yangon, Myanmar.
Blood Vessel Thromb Hemost. 2024 Oct 8;2(1):100032. doi: 10.1016/j.bvth.2024.100032. eCollection 2025 Feb.
It has been suggested that a serine proteinase inhibitor, antithrombin (AT), exerts anti-inflammatory effects on different types of cells, independent of thrombin inhibition. In this study, we aimed to identify a specific receptor for AT by a screening method using a transmembrane-tethered AT ligand expressed on HEK293T cells together with the coexpression of candidate receptors, followed by the immunoprecipitation of a complex of AT ligand with a receptor. We identified C-type lectin family 1A (CLEC1A) as a receptor for AT. We confirmed the binding of AT to the extracellular domain of CLEC1A using surface plasmon resonance. Recombinant as well as native AT concentration-dependently induced the rounding of purified human neutrophils in shape, associated with the suppression of spontaneous reactive oxygen species production in vitro, but argatroban did not, indicating the independence of AT effects on thrombin inhibition. Native AT maintained the passage of neutrophils through the artificial microcapillaries. Both AT enhanced the phagocytosis of pHrodo-labeled and prolonged the viability of the neutrophils. The cellular effects of AT were similar to those of histidine-rich glycoprotein, which has the same CLEC1A as a receptor, and were partially inhibited by the addition of anti-CLEC1A antibody to the incubation media. These results suggested that CLEC1A is a novel receptor for AT, and the stimulation of CLEC1A by AT at least in part mediates the important functional changes of human neutrophils.
有人提出，一种丝氨酸蛋白酶抑制剂——抗凝血酶（AT），可对不同类型的细胞发挥抗炎作用，且独立于凝血酶抑制作用。在本研究中，我们旨在通过一种筛选方法来鉴定AT的特异性受体，该方法使用在HEK293T细胞上表达的跨膜连接的AT配体以及候选受体的共表达，随后对AT配体与受体的复合物进行免疫沉淀。我们鉴定出C型凝集素家族1A（CLEC1A）为AT的受体。我们使用表面等离子体共振证实了AT与CLEC1A细胞外结构域的结合。重组AT以及天然AT均浓度依赖性地诱导纯化的人中性粒细胞形态变圆，这与体外抑制自发性活性氧产生相关，但阿加曲班则无此作用，表明AT的作用独立于凝血酶抑制作用。天然AT维持了中性粒细胞通过人工微毛细血管的过程。两种AT均增强了对pHrodo标记物的吞噬作用，并延长了中性粒细胞的存活时间。AT的细胞效应与富含组氨酸的糖蛋白相似，后者具有相同的CLEC1A作为受体，并且向孵育培养基中添加抗CLEC1A抗体可部分抑制这些效应。这些结果表明，CLEC1A是AT的一种新型受体，AT对CLEC1A的刺激至少部分介导了人中性粒细胞的重要功能变化。
