Measurement of soluble C-type lectin-like receptor 2 in human plasma

Abstract

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.

• Biomarker
• CLEC-2
• platelet activation
• platelets
• thrombosis

Acknowledgements

We thank Hideyuki Tanaka, Chiaki Komatsu, Hisaichiro Nakazawa, and Toshiaki Shirai for technical assistance.

Declaration of interest

We received support in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology (20569628, M.O.) and the Japan Society for the Promotion of Science (JSPS) through the Funding Program for Next Generation World-leading Researchers (NEXT Program) (LS052, K.S.-I.).

Junya Nakamura and Mitsuru Oosawa are employees of Mitsubishi Chemical Medicine Corporation. Yukio Ozaki, Katsue Suzuki-Inoue, Junya Nakamura, and Mitsuru Oosawa have pending patent applications from the last two years related to this report (Japanese patent application No. 2012-215900).

Notes

†Some of these results were reported in an abstract for the XXIV Congress of the International Society on Thrombosis and Haemostasis in July 2013.