AbstractWe have characterized changes in [Ca2+]iin primary mouse megakaryocytes in response to fibrillar collagen and in response to cross-linking of the collagen receptor, the integrin α2β1. The response to collagen was markedly different from that seen to a triple helical collagen-related peptide (CRP), which signals via the tyrosine kinases p59fyn and p72syk. This peptide binds to the collagen receptor glycoprotein VI (GPVI), but not to the integrin α2β1. Collagen elicited a sustained increase in [Ca2+]i composed primarily of influx of extracellular Ca2+ with some Ca2+release from internal stores. In contrast to CRP, this response was only partially (∼30%) inhibited by the src-family kinase inhibitor PP1 (10 μmol/L) or by microinjection of the tandem SH2 domains of p72syk. Collagen also caused an increase in [Ca2+]i in megakaryocytes deficient in either p59fyn or p72syk, although the response was reduced by approximately 40% in both cases: Cross-linking of the α2 integrin increased [Ca2+]iin these cells exclusively via Ca2+ influx. This response was reduced by approximately 50% after PP1 pretreatment, but was significantly increased in fyn-deficient megakaryocytes. Collagen therefore increases [Ca2+]i in mouse megakaryocytes via multiple receptors, including GPVI, which causes Ca2+ mobilization, and α2β1, which stimulates a substantial influx of extracellular Ca2+.