Distinct spatio-temporal Ca2+ signaling elicited by integrin α2β1 and glycoprotein VI under flow

We studied how integrin alpha 2 beta 1 and glycoprotein VI (GPVI) contribute to collagen-induced platelet activation under flow conditions by evaluating stable adhesion and intracellular Ca2+ concentration ([Ca2+](i)) of FLUO 3-AM-labeled platelets perfused over acid-soluble type I or microfibrillar type VI collagen. Adhering platelets displayed 2 kinds of [Ca2+](i) oscillations. Rapid alpha-like peaks were unaffected by the membrane-impermeable Ca2+ chelator ethyleneglycoltetraacetic acid but abolished by membrane-permeable BAPTA-AM. Longer-lasting gamma-like peaks were always preceded by at least one alpha-like peak and abolished by intracellular or extracellular Ca2+ chelation. Inhibition of phosphatidylinositol 3-kinase or phospholipase C and modulation of cyclic nucleotides, but not blockage of adenosine diphosphate receptors, prevented both Ca2+ responses. Human or mouse platelets lacking GPVI function exhibited alpha-like but not gamma-like Ca2+ peaks, whereas those lacking alpha 2 beta 1 showed markedly reduced to absent alpha-like and no gamma-like Ca2+ peaks. Specific alpha 2 beta 1 ligation induced alpha-like but not gamma-like peaks. Thus, alpha 2 beta 1 may generate Ca2+ signals that are reinforced by GPVI and required for subsequent longer-lasting Ca2+ oscillation mediated by GPVI through transmembrane ion flux. Our results delineate a GPVI-independent signaling role of alpha 2 beta 1 in response to collagen stimulation. (Blood. 2009;114:2793-2801)