-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the CNS and it also appears in peripheral tissues and blood. Three main types of GABA receptors are identified: A, B, and C. GABAA receptor is the most prominent type, which is a ligand-gated chloride ion channel. Platelets play a central role in thromboembolic diseases and have been shown to possess a GABA uptake system. Despite some benzodiazepine analogues are reported to inhibit platelet aggregation through different mechanisms, there is relatively rare information on the study of GABA in platelet function. Recently, GABA (4 M) was reported to potentiate platelet aggregation, whereas our preliminary study revealed that GABA obviously inhibited platelet aggregation and [Ca+2]i in activated platelets at relative lower concentrations (0.5-1 M) (Figs.1&2). Since the concentrations of GABA were approximated at 98.6±33.9 ng/ml (~0.7-1.3 M) in human plasma, the pharmacological concentration of GABA (0.5~1 μM) employed to inhibit platelet aggregation are reasonable. Electron microscopic observation and LC-MS/MS analysis of our preliminary study revealed that GABA is abundantly distributed in the cytoplasm of resting platelets (Fig.3) and it levels in platelets is about 1.03 ng/106 cells (Fig.4), respectively. These results clearly indicate that human platelets contain high levels of GABA, which, upon stimulation, it is readily released from platelets and further contributes to be circulating GABA, and it may be taken as an endogenous antithrombotic agent. Recent our study also found that the transcription factor, NF-B, is present in platelets (anucleated cells) (Fig.5), it can translocate from the cytosol into mitochondria (Fig.6), and binding to the mitochondrial DNA (mtDNA). However, the question remains as to whether or not this transcription factor is functionally present in a novel way, unrelated to transcriptional regulation. Moreover, apoptotic (i.e., mitochondrial dysfunction, caspase activation, etc) and autophagic (i.e., LC3II etc) events have also been found in platelets. Our study have found that GABA (0.5 and 1 μM) markedly inhibited both NF-B and caspase 3 activation (Figs.5&7) in activated platelets. Thus, platelets may be an ideal target for the study of the non-genomic functions (i.e., regulation of platelet activation, apoptosis, and autophagy) of NF-B in anucleated cells. Our preliminary results also demonstrated that GABA plays an important role in regulation of platelet activation and apoptosis. However, the inhibitory mechanisms of GABA in these reactions, and the non-genomic roles of NF-κB involved in GABA-mediated signal events are still unclear. We therefore aimed for the first time to systematically examine these issues in this project (Fig. 8). Specific Aim 1 (1st year): To further quantify the levels of GABA by LC-MS/MS and capillary zone electrophoresis (CZE) and elucidate their specific localization (i.e., granules, dense granules or open canalicular system) in platelets by transmission electron microscope. The signaling events during the GABA-mediated inhibition of platelet activation and whether platelets express GABA-like binding receptors are also determined. We also determine the antithrombotic effect of GABA in vivo. Specific Aim 2 (2nd year): To determine whether inhibition of NF-B (p65/p50) (IKKβ phosphorylation, IκBα degradation, and p65 phosphorylation) contributes to GABA-mediated inhibitory effect in platelet activation, and to clarify the relationship between NF-κB and GABA-mediated signaling events (i.e., cyclic nucleotides, PLCγ2-IP3-PKC cascades, and MAPK, etc). Specific Aim 3 (2nd year): To determine whether translocated NF-B binds to mtDNA by using of isolated platelet mitochondria to conduct EMSA using an oligonucleotides of NF-B binding sequence (5’-AGTTG AGGGG ACTTT CCCAG G-3’) or NFB-like binding sequence (5’-ACATC ATTAC CGGGT TTTCC TCTTG TAAAT-3’) in mtDNA, and by chromatin immunoprecipitation assay, we also verify this binging regulates mtDNA-encoded protein expressions (i.e., cytochrome c oxidase III, NADH dehydrogenase 4), contributing to platelet apoptosis. We also evaluate whether GABA regulates the NF-B translocation or mtDNA-encoded protein expressions in platelets. Specific Aim 4 (3rd year): To investigate the pivotal roles of NF-B regulation in Bax-associated apoptotic signals (i.e., mPT, AIF, Endo G, apoptosome formation, caspase activation, and mtDNA fragmentation, etc) in human platelets, and concurrently to compare the differences of apoptotic events in platelets isolated from NF-B knockout (NF-B-/-) and wild type mice. We also determine whether GABA can reverse some of those reactions. Specific Aim 5 (3rd year): To investigate whether NF-B stimulates the machinery of autophagy (i.e., beclin-1, LC3/Atg8, Atg5, and Atg12 proteins, and autophagic vacuoles formation, etc) in platelets from human and NF-B-/- mice, and GABA inhibits these signaling events of autophagy through inhibition of NF-B activation. Knowledge of these regulatory mechanisms could provide new insights into the role of GABA on regulation of NF-B-mediated platelet activation, apoptosis, and autophagy in anuclear cells, platelets.
|Effective start/end date||8/1/13 → 7/31/14|