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.

Functional significance of the platelet immune receptors GPVI and CLEC-2

“…Treatment with the CLEC-2 mAb INU1 achieves sustained depletion of CLEC-2 from mouse platelets in vivo (60) due to internalization and intracellular degradation (61), and severe but transient thrombocytopenia. CLEC-2, however, is not shed from activated platelets, and only very low levels of the CLEC-2 ectodomain are present in blood (62). CLEC-2-positive microvesicles are elevated in the plasma of patients with rheumatoid arthritis (23).…”

Podoplanin-positive periarteriolar stromal cells promote megakaryocyte growth and proplatelet formation in mice by CLEC-2

“…It has been known that CLEC-2 is released from activated platelet as a soluble form by shedding. 33 Therefore, we also investigated the possibility that soluble CLEC-2 is involved in a megakaryocyte expansion associated with BM FRC-like cells. We compared the megakaryocyte expansion in coculture with BM FRC-like cells in the presence or absence of GM6001 and GM6001.…”

Plasma C‐type lectin‐like receptor 2 as a predictor of death and vascular events in patients with acute ischemic stroke

“…Atherosclerotic lesion formation is the main cause of atherosclerotic diseases. In addition, Kazama et al [21] showed a trend towards an increase in soluble pCLEC-2 concentration in DM patients, who are a high-risk population for stroke. However, the mechanisms underlying the association between pCLEC-2 and death or recurrence of vascular diseases are still unclear and more studies, especially animal experiments, are needed to explore them.…”