title: Evidence for two distinct epitopes within collagen for activation of murine platelets.
abstract: It has recently been shown that the monoclonal antibody JAQ1 to murine glycoprotein VI (GPVI) can cause aggregation of mouse platelets upon antibody cross-linking and that collagen-induced platelet aggregation can be inhibited by preincubation of platelets with JAQ1 in the absence of cross-linking (Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner, J. E., and Zirngibl, H. (2000) J. Biol. Chem. 275, 23998-24002). In the present study, we have shown that cross-linking of GPVI by JAQ1 results in tyrosine phosphorylation of the same profile of proteins as that induced by collagen, including the Fc receptor (FcR) gamma-chain, Syk, LAT, SLP-76, and phospholipase C gamma 2. In contrast, platelet aggregation and tyrosine phosphorylation of these proteins were inhibited when mouse platelets were preincubated with JAQ1 in the absence of cross-linking and were subsequently stimulated with a collagen-related peptide (CRP) that is specific for GPVI and low concentrations of collagen. However, at higher concentrations of collagen, but not CRP, aggregation of platelets and tyrosine phosphorylation of the above proteins (except for the adapter LAT) is re-established despite the presence of JAQ1. These observations suggest that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, 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 within the collagen molecule, the absence of LAT phosphorylation in response to 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 gamma-chain. The possibility that this response is mediated by a receptor that is not coupled to FcR gamma-chain is excluded on the grounds that aggregation is absent in platelets from FcR gamma-chain-deficient mice.
authors.affiliation: Department of Molecular Oncology, General Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany.
authors.forename: V
authors.initials: V
authors.lastname: Schulte
authors.forename: D
authors.initials: D
authors.lastname: Snell
authors.forename: W
authors.initials: W
authors.lastname: Bergmeier
authors.forename: H
authors.initials: H
authors.lastname: Zirngibl
authors.forename: S P
authors.initials: SP
authors.lastname: Watson
authors.forename: B
authors.initials: B
authors.lastname: Nieswandt
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D003094:Collagen / Q000037:antagonists & inhibitors / Q000276:immunology* / Q000494:pharmacology*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003435:Crotalid Venoms / Q000494:pharmacology; D000939:Epitopes / Q000276:immunology*; D037181:Lectins, C-Type; D051379:Mice; D008815:Mice, Inbred Strains; D010446:Peptide Fragments / Q000037:antagonists & inhibitors / Q000276:immunology / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000276:immunology / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D003435:Crotalid Venoms; D000939:Epitopes; D037181:Lectins, C-Type; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; C098341:platelet membrane glycoprotein VI; D019000:Phosphotyrosine; C028890:convulxin; D003094:Collagen