Evidence for two distinct epitopes within collagen for activation of murine platelets

It has recently been shown that the monoclonal antibody JAQ1 to murine glyc oprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody c rosslinking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (N ieswandt, B,, Bergmeier, W,, Schulte, V,, Rackebrandt, K,, Gessner, J, E,, and Zirngibl, H, (2000) J. Biol, Chem, 275, 23998-24002), In the present st udy, we have shown that crosslinking of GPVI by JAQ1 results in tyrosine ph osphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) gamma -chain, Syk, LAT, SLP-76, and phospho lipase C gamma2. In contrast, platelet aggregation and tyrosine phosphoryla tion of these proteins were inhibited when mouse platelets were preincubate d with JAQ1 in the absence of cross-linking and were subsequently stimulate d with a collagen-related peptide (CRP) that is specific for GPVI and low c oncentrations of collagen. However, at higher concentrations of collagen, b ut not CRP, aggregation of platelets and tyrosine phosphorylation of the ab ove proteins (except for the adapter LAT) is re-established despite the pre sence of JAQ1, These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelet s, is occupied in the presence of high concentrations of collagen. Although this could be a second site on GPVI that is activated by a novel motif wit hin the collagen molecule, the absence of LAT phosphorylation in response t o collagen in the presence of JAQ1 suggests that this is more likely to be caused by activation of a second receptor that is also coupled to the FcR g amma -chain. The possibility that this response is mediated by a receptor t hat is not coupled to FcR gamma -chain is excluded on the grounds that aggr egation is absent in platelets from FcR gamma -chain-deficient mice.