Platelet microparticles (pMPs) are small membrane-coated vesicles that are released from the plasma membrane upon platelet activation. In the joint fluid of patients with
rheumatoid arthritis, pMP can interact with and activate fibroblast-like synoviocytes (FLS), which are important effector cells that mediate both immune activation and joint destruction. The signaling process by which engagement of
glycoprotein VI (GPVI), a
surface glycoprotein receptor for
collagen which is expressed on platelets, triggers pMP generation is poorly understood, but has been suggested to involve
Spleen Tyrosine Kinase (SYK), best known as an upstream activator of
Bruton's Tyrosine Kinase (BTK) in B cells. In this study, we showed that activation of human platelets triggered by
convulxin or
collagen, specific
ligands for GPVI receptor, or alternatively by antibody-mediated cross-linking of another platelet
receptor, C type lectin-like receptor 2 (CLEC2), resulted in phosphorylation of BTK and downstream effector,
phospholipase Cγ2 (PLCγ2). A potent and selective BTK inhibitor,
RN486, inhibited GPVI- or CLEC2-mediated PLCγ2 phosphorylation and pMP production in a dose-dependent manner. BTK is also an essential effector of B cell receptor (BCR)-induced B cell signaling. Consistent with the biology, the IC50s of BTK inhibitors with varying potencies in a BCR-dependent B cell activation marker assay correlated with those in the GPVI-mediated PLCγ2 phosphorylation. In a co-culture system consisting of human primary synovial FLS and activated human platelets,
convulxin stimulation resulted in elevated production of pro-inflammatory
cytokines,
IL-6 and
IL-8, an effect which was dose-dependently blocked by
RN486. The effects are specific as
RN486 abrogated platelet aggregation induced by GPVI
ligands but not by other platelet surface receptor agonists. Taken together, our data further support the potential therapeutic utility of BTK inhibitors in RA
therapy, by inhibiting GPVI-mediated platelet activation and thus subsequent amplification of
inflammation driven by pMP-induced FLS
cytokines production.