Collagen mediates changes in intracellular calcium in primary mouse megakaryocytes through syk-dependent and -independent pathways

We have characterized changes in [Ca2+](i) in primary mouse megakaryocytes in response to fibrillar collagen and in response to cross-linking of the c ollagen receptor, the integrin alpha(2)beta(1). The response to collagen wa s markedly different from that seen to a triple helical collagen-related pe ptide (CRP), which signals via the tyrosine kinases p59(fyn) and p72(syk). This peptide binds to the collagen receptor glycoprotein VI (GPVI), but not to the integrin alpha(2)beta(1). 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 (similar to 30%) inhibited by the src-family kinase inhibitor PP1 (10 mu mol/L) or by microinjection of the tandem SH2 domains of p72(syk), Collagen also caused an increase in [Ca2+](i) in megakaryocytes deficient i n either p59(fyn) or p72(syk), although the response was reduced by approxi mately 40% in both cases: Cross-linking of the alpha(2) integrin increased [Ca2+](i) in these cells exclusively via Ca2+ influx. This response was red uced by approximately 50% after PP1 pretreatment, but was significantly inc reased in fyn-deficient megakaryocytes. Collagen therefore increases [Ca2+] (i) in mouse megakaryocytes via multiple receptors, including GPVI, which c auses Ca2+ mobilization, and alpha(2)beta(1) which stimulates a substantial influx of extracellular Ca2+. (C) 1999 by The American Society of Hematolo gy.