Platelet activation by collagen is mediated by the sequential tyrosine phosphorylation of the Fc receptor g-chain (FcR g-chain), which is part of the collagen receptor glycoprotein VI, the tyrosine kinase Syk and phospholipase C-g 2 (PLC-g 2). In this study tyrosine-phosphorylated proteins that associate with PLC-g 2 after stimulation by a collagen-related peptide (CRP) were characterized using glutathione S-transferase fusion proteins of PLC-g2 Src homology (SH) domains and by immunoprecipitation of endogenous PLC-g 2. The majority of the tyrosinephosphorylated proteins that associate with PLC-g 2 bind to its C-terminal SH2 domain. These were found to include PLC-g 2, Syk, SH2-domain-containing leucocyte protein of 76 kDa (SLP-76), Lyn, linker for activation of T cells (LAT) and the FcR g-chain. Direct association was detected between PLC-g 2 and SLP-76, and between PLC-g2 and LAT upon CRP stimulation of platelets by far-Western blotting. FcR g-chain and Lyn were found to co-immunoprecipitate with PLC-g2 as well as with unidentified 110-kDa and 75-kDa phosphoproteins. The absence of an in vivo association between Syk and PLC-g2 in platelets is in contrast with that for PLC-g1 and Syk in B cells. The in vivo function of PLC-g2 SH2 domains was examined through measurement of Ca 2+ increases in mouse megakaryocytes that had been microinjected with recombinant proteins. This revealed that the C-terminal SH2 domain is involved in the regulation of PLC-g2. These data indicate that the C-terminal SH2 domain of PLC-g2 is important for PLC-g2 regulation through possible interactions with SLP-76, Syk, Lyn, LAT and the FcR g-chain.

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor ␥ chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase C␥2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the Gprotein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.

We 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 2β1. 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 2β1. 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 2 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 2β1, which stimulates a substantial influx of extracellular Ca2+.

SummarySince megakaryocytes are the cellular precursors of platelets we have investigated whether they share responses to platelet agonists, in particular collagen. Although previous studies have reported responses to thrombin in non-human megakaryocytes, through studies of single cell calcium responses and protein tyrosine-phosphorylation we demonstrate for the first time that both isolated human megakaryocytes and CD41/61-positive megakaryocytes derived in culture from CD34+ cells share responses to the platelet agonists collagen, collagen-related peptide and thrombin. The responses to either collagen or CRP were seen only in the most mature megakaryocytes and not in mega-karyocyte-like cell lines, suggesting that the response to collagen is a characteristic developed late during megakaryocyte differentiation. These primary cells offer the opportunity to use many molecular and cellular techniques to study and manipulate signalling events in response to platelet receptor agonists, which cannot be performed in the small, anucleate platelet itself.