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

Detection of platelet activation in vivo is useful to identify patients at risk of thrombotic diseases. Platelet factor 4 (PF4) and ß-thromboglobulin (ß-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 ß-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 ß-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.

Soluble CLEC-2 is generated independently of ADAM10 and is increased in plasma in acute coronary syndrome: comparison with soluble GPVI.

Soluble forms of platelet membrane proteins are released upon platelet activation. We previously reported that soluble C-type lectin-like receptor 2 (sCLEC-2) is released as a shed fragment (Shed CLEC-2) or as a whole molecule associated with platelet microparticles (MP-CLEC-2). In contrast, soluble glycoprotein VI (sGPVI) is released as a shed fragment (Shed GPVI), but not as a microparticle-associated form (MP-GPVI). However, mechanism of sCLEC-2 generation or plasma sCLEC-2 has not been fully elucidated. Experiments using metalloproteinase inhibitors/stimulators revealed that ADAM10/17 induce GPVI shedding, but not CLEC-2 shedding, and that shed CLEC-2 was partially generated by MMP-2. Although MP-GPVI was not generated, it was generated in the presence of the ADAM10 inhibitor. Moreover, antibodies against the cytoplasmic or extracellular domain of GPVI revealed the presence of the GPVI cytoplasmic domain, but not the extracellular domain, in the microparticles. These findings suggest that most of the GPVI on microparticles are induced to shed by ADAM10; MP-GPVI is thus undetected. Plasma sCLEC-2 level was 1/32 of plasma sGPVI level in normal subjects, but both soluble proteins significantly increased in plasma of patients with acute coronary syndrome. Thus, sCLEC-2 and sGPVI are released by different mechanisms and released in vivo upon platelet activation.

Murine monoclonal antibody to platelet factor 4/heparin complexes as a potential reference standard for platelet activation assays in heparin-induced thrombocytopenia.

Quality control of the platelet activation assays to diagnose heparin-induced thrombocytopenia (HIT), (14)C-serotonin release assay (SRA) and platelet aggregation test (PAT) has yet to be established due to lack of reference standards and the difficulty of obtaining significant amounts of HIT antibodies from patients with HIT. We prepared a murine monoclonal antibody to human platelet factor 4 (hPF4)/heparin complexes (HIT-MoAb) and investigated the platelet activating action of HIT-MoAb by using SRA and PAT. The HIT-MoAb activated human platelets at low heparin concentration and the platelet activations were inhibited at high heparin concentration in both SRA and PAT. The HIT-MoAb produced a concentration-dependent effect. Moreover, the platelet activation at low heparin concentration was inhibited by anti-FcγRIIa antibody. These results indicated that HIT-MoAb has characteristics similar to human HIT antibodies regarding heparin-dependent platelet activation. Therefore, it is suggested that HIT-MoAb has the potential to be a positive control or reference standard in platelet activation assays.