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. Disclosures: No relevant conflicts of interest to declare.

Spike-Mediated and Graded Inhibitory Synaptic Transmission Between Leech Interneurons: Evidence for Shared Release Sites

Inhibitory synaptic transmission between leech heart interneurons consist of two components: graded, gated by Ca2+ entering by low-threshold [low-voltage–activated (LVA)] Ca channels and spike-mediated, gated by Ca2+ entering by high-threshold [high-voltage–activated (HVA)] Ca channels. Changes in presynaptic background Ca2+ produced by Ca2+ influx through LVA channels modulate spike-mediated transmission, suggesting LVA channels have access to release sites controlled by HVA channels. Here we explore whether spike-mediated and graded transmission can use the same release sites and thus how Ca2+ influx by HVA and LVA Ca channels might interact to evoke neurotransmitter release. We recorded pre- and postsynaptic currents from voltage-clamped heart interneurons bathed in 0 mM Na+/5 mM Ca2+ saline. Using different stimulating paradigms and inorganic Ca channel blockers, we show that strong graded synaptic transmission can occlude high-threshold/spike-mediated synaptic transmission when evoked simultaneously. Suppression of LVA Ca currents diminishes graded release and concomitantly increases the ability of Ca2+ entering by HVA channels to release transmitter. Uncaging of Ca chelator corroborates that graded release occludes spike-mediated transmission. Our results indicate that both graded and spike-mediated synaptic transmission depend on the same readily releasable pool of synaptic vesicles. Thus Ca2+, entering cells through different Ca channels (LVA and HVA), acts to gate release of the same synaptic vesicles. The data argue for a closer location of HVA Ca channels to release sites than LVA Ca channels. The results are summarized in a conceptual model of a heart interneuron release site.