CLEC-2 expression is maintained on activated platelets and on platelet microparticles

The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hemimmunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP) VI and Fc gamma RIIa. Activation of either of the ITAMreceptors induces shedding of GPVI and proteolysis of the ITAM domain in Fc gamma RIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of similar to 2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and Fc gamma RIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41(+) microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.