P2Y12 Inhibitors Restore the Ability of GPIb-IX to Inhibit Outside-in Signaling Across alphaIIbbeta3.

Abstract 3006

Poster Board II-982

P2Y12 inhibitors decrease the reactivity of platelets. Since the P2Y12 receptor acts through Gαai-induced signals, which inhibit adenyl cyclase, P2Y12 inhibitors are thought to act through phosphorylation of protein kinase A (PKA) substrates. One PKA substrate is serine166 (S166) of GPIbβ. Using a phospho-specific antibody, Western blots, and FACS of whole blood, we found that ADP and epinephrine, which activate P2Y12 receptors caused a dose-dependent dephosphorylation of S166 while apyrase and a P2Y12 inhibitor prevented this dephosphorylation. Since S166 functions with S609 of GPIbαa to bind 14-3-3, we determined whether i) dephosphorylation was associated with release of 14-3-3 from GPIb, and ii) 14-3-3 regulated αaIIbβ3 function. GPIb bound ∼91 ± 5% of the 14-3-3, as determined by its insolubility in streptolysin O-permeabilized platelets and its solubilization by phospho-S609 and -S166 peptides. Quantitation of 14-3-3 in GPIb immunoprecipitates showed that graded Gαai-induced dephosphorylation of S166 was accompanied by graded release of 14-3-3 from GPIb. Evidence that 14-3-3 participates in αaIIbβ3 signaling came from its co-immunoprecipitation with αaIIbβ3 from sheared platelets. Moreover, when CHO/αaIIbβ3 cell 14-3-3 was sequestered by expression of GPIb, αaIIbβ3-induced activation of RhoGTPases and cell spreading were inhibited; overexpression of 14-3-3 restored these functions. These studies: i) show that 14-3-3 is critical for αaIIbβ3-induced spreading; ii) describe a novel function of GPIb in dampening the transition to firm adhesion through the regulated sequestration of 14-3-3; and iii) suggest that the availability of 14-3-3 for αaIIbβ3-mediated adhesion is determined by the Gai-modulated level of GPIb phosphorylation.