Kazama Fuminori, Nakamura Junya, Osada Makoto, Inoue Osamu, Oosawa Mitsuru, Tamura Shogo, Tsukiji Nagaharu, Aida Kaoru, Kawaguchi Akio, Takizawa Soichi, Kaneshige Masahiro, Tanaka Shoichiro, Suzuki-Inoue Katsue, Ozaki Yukio
a Department of Clinical and Laboratory Medicine, Faculty of Medicine , University of Yamanashi , Chuo , Yamanashi , Japan .
b Department of Antibody Group, Narita R&D Department, Research and Development Division , LSI Medicine Corporation , Takomachi, Katori-gun , Chiba , Japan .
Platelets. 2015;26(8):711-9. doi: 10.3109/09537104.2015.1021319. Epub 2015 Apr 9.
Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and β-thromboglobulin (β-TG) are used for this purpose; however, they are easily released upon the minimal platelet activation that occurs during sampling. Soluble forms of several platelet membrane proteins are released upon platelet activation; however, the soluble form of C-type lectin-like receptor 2 (sCLEC-2) has not yet been fully investigated. Western blotting with an anti-CLEC-2 antibody showed that sCLEC-2 was released from washed human platelets stimulated with collagen mimetics. To detect sCLEC-2 in plasma, we established a sandwich enzyme-linked immunosorbent assay (ELISA) using F(ab')2 anti-CLEC-2 monoclonal antibodies. Although plasma mixed with citrate, adenosine, theophylline and adenosine (CTAD) is needed for the PF4 and β-TG assays, effects of anti-coagulants (EDTA, citrate and CTAD) on the sCLEC-2 ELISA were negligible. Moreover, while special techniques are required for blood sampling and sample preparation for PF4 and β-TG assay, the standard blood collections procedures used in daily clinical laboratory tests have shown to suffice for sCLEC-2 analysis. In this study, we found that two forms of sCLEC-2 are released after platelet activation: a shed fragment and a microparticle-bound full-length protein, both of which are detected by the sCLEC-2 ELISA. The average concentration of sCLEC-2 in the plasma of 10 healthy individuals was 97 ± 55 pg/ml, whereas that in the plasma of 25 patients with diabetes mellitus (DM) was 149 ± 260 pg/ml. A trend towards an increase in sCLEC-2 concentration in the DM patients may reflect in vivo platelet activation in the patients, suggesting that sCLEC-2 may have clinical significance as a biomarker of in vivo platelet activation.
体内血小板活化的检测有助于识别有血栓性疾病风险的患者。血小板因子4（PF4）和β-血小板球蛋白（β-TG）用于此目的；然而，在采样过程中发生的最小程度的血小板活化时，它们就很容易释放出来。几种血小板膜蛋白的可溶性形式在血小板活化时会释放出来；然而，C型凝集素样受体2（sCLEC-2）的可溶性形式尚未得到充分研究。用抗CLEC-2抗体进行的蛋白质印迹分析表明，sCLEC-2是从用胶原模拟物刺激的洗涤过的人血小板中释放出来的。为了检测血浆中的sCLEC-2，我们使用F(ab')2抗CLEC-2单克隆抗体建立了一种夹心酶联免疫吸附测定（ELISA）。虽然PF4和β-TG检测需要与柠檬酸盐、腺苷、茶碱和腺苷（CTAD）混合的血浆，但抗凝剂（乙二胺四乙酸、柠檬酸盐和CTAD）对sCLEC-2 ELISA的影响可以忽略不计。此外，虽然PF4和β-TG检测需要特殊的血液采样和样品制备技术，但日常临床实验室检测中使用的标准血液采集程序已证明足以进行sCLEC-2分析。在本研究中，我们发现血小板活化后会释放出两种形式的sCLEC-2：一种脱落片段和一种与微粒结合的全长蛋白，这两种形式都能被sCLEC-2 ELISA检测到。10名健康个体血浆中sCLEC-2的平均浓度为97±55 pg/ml，而25名糖尿病（DM）患者血浆中的平均浓度为149±260 pg/ml。DM患者中sCLEC-2浓度有升高趋势，这可能反映了患者体内的血小板活化，提示sCLEC-2作为体内血小板活化的生物标志物可能具有临床意义。
