Measurement of soluble C-type lectin-like receptor 2 in human plasma
Measurement of soluble C-type lectin-like receptor 2 in human plasma
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DOI：
10.3109/09537104.2015.1021319
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发表时间：
2015-01-01
期刊：
影响因子：
3.3
通讯作者：
Ozaki, Yukio
中科院分区：
文献类型：
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作者：
Kazama, Fuminori;Nakamura, Junya;Ozaki, Yukio
体内血小板活化的检测有助于识别处于血栓性疾病风险的患者。血小板因子 4 (PF4) 和 β-血栓球蛋白 (β-TG) 用于此目的；然而，在采样过程中发生最小程度的血小板激活时，它们很容易被释放。血小板激活后会释放出几种可溶形式的血小板膜蛋白；然而，可溶形式的 C 型凝集素样受体 2 (sCLEC-2) 尚未得到充分研究。使用抗 CLEC-2 抗体进行的蛋白质印迹显示，用胶原模拟物刺激的洗涤的人血小板中释放出 sCLEC-2。为了检测血浆中的 sCLEC-2，我们使用 F(ab')2 抗 CLEC-2 单克隆抗体建立了夹心酶联免疫吸附测定 (ELISA)。尽管 PF4 和 β-TG 测定需要血浆与柠檬酸盐、腺苷、茶碱和腺苷 (CTAD) 混合，但抗凝剂（EDTA、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 测定的血液采样和样品制备需要特殊技术，但日常临床实验室测试中使用的标准血液采集程序已证明足以进行 sCLEC-2 分析。在这项研究中，我们发现血小板激活后释放两种形式的 sCLEC-2：脱落片段和微粒结合的全长蛋白，这两种形式都可以通过 sCLEC-2 ELISA 检测到。 10名健康个体血浆中sCLEC-2的平均浓度为97+/-55pg/ml，而25名糖尿病(DM)患者血浆中sCLEC-2的平均浓度为149+/-260pg/ml。 DM患者中sCLEC-2浓度增加的趋势可能反映了患者体内血小板活化，表明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。
Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and beta-thromboglobulin (beta-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 beta-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 beta-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.