Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines

Background: Glycoprotein (GP)VI is the major signaling receptor for collagen on platelets and signals via the associated FcR-gamma-chain, which has an immunoreceptor tyrosine-containing activation motif (ITAM). Objective: To determine why GPVI-FcR gamma signals poorly, or not at all, in response to collagen in hematopoietic cell lines, despite robust responses to the GPVI-reactive snake venom toxin convulxin. Methods and results: Using a nuclear factor of activated T-cells (NFAT) transcriptional reporter assay, a sensitive readout for sustained ITAM signaling, we demonstrate collagen-induced GPVI-FcR gamma signaling in hematopoietic cell lines. This is accompanied by relatively weak but sustained protein tyrosine phosphorylation, in contrast to the stronger but transient response to convulxin. Sustained signaling by collagen is also observed in platelets and is necessary for the maintenance of spreading on collagen. Finally, in cell lines, the inhibitory collagen receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), which is not expressed on platelets but is present on most hematopoietic cells, inhibits GPVI responses to collagen but not convulxin. Conclusion: The inability of previous studies to readily detect GPVI collagen signaling in cell lines is probably because of the weak but sustained nature of the signal and the presence of the inhibitory collagen receptor LAIR-1. In platelets, we propose that GPVI-FcR gamma has evolved to transmit sustained signals in order to maintain spreading over several hours, as well as facilitating rapid activation through release of feedback agonists and integrin activation. The establishment of a cell line NFAT assay will facilitate the molecular dissection of GPVI signaling and the identification of GPVI antagonists in drug discovery.