Abstract 271: Dynamic Regulation of the GP Ib-IX/14–3–3 Interaction in a 32 Adrenergic Receptor/Adenyl Cyclase Dependent Manner
The platelet receptor for von Willebrand factor (vWf), glycoprotein GPIb-IX, mediates transient platelet adhesion and rolling under high shear conditions. GPIb-IX initiates signals that activate α Iibβ 3 integrin, leading to stable platelet adhesion and aggregation. In this way, GP Ib-IX contributes to platelet pathology at sites of high shear and atherosclerotic plaques. The cytoplasmic tail of GP Ib interacts with the signaling molecule 14−3−3. Using a CHO cell model, we have shown that by sequestering 14 –3–3 this interaction prevents it from interacting with integrin clusters, where it can stimulate outside-in signaling and cell spreading. The goal of this study was to determine whether the association of 14 –3–3 with GP Ib in platelets is regulated in a physiological manner. Washed human platelets were incubated with platelet agonists, GP Ib-IX immunoprecipitated with an antibody against GP Ibα, and the presence of 14 –3–3 detected with 14 –3–3 antibodies. Incubation of platelets with epinephrine or exposure of platelets to high-shear forces decreased co-immunoprecipitation of 14 –3–3. Epinephrine decreases platelet cAMP levels by a Gz-dependent mechanism, with resulting inhibition of protein kinase A (PKA). One of the residues known to be phosphorylated by PKA in platelets is S166 on GPIbβ; in vitro experiments have shown that the phosphorylation of this residue is involved in the interaction of 14 –3–3 with GP Ib-IX. Thus, we examined the possibility that phosphorylation of this residue is regulated by epinephrine. Using an antibody that specifically recognizes the phosphorylated form of S166, we determined that phosphorylation of S166 was decreased in epinephrine-stimulated platelets. In contrast, agents such as forskolin that activate PKA resulted in both increased phosphorylation of S166 and increased association of 14 –3–3 with GP Ib-IX. We suggest that the association of 14 –3–3 with GP Ib-IX is regulated in a dynamic manner by changes in cAMP levels within platelets. In this way variable GP Ib-IX/14 –3–3 interactions may regulate integrin-induced signaling, stable adhesion and platelet aggregation.