Autophagy is a self-eating process, which consists of several phases: induction, autophagosome formation, autolysosome formation, and cargo breakdown followed by recycling of degradation products. Autophagosome formation is the central event in autophagy process, and microtubule-associated protein 1 light chain 3 (LC3) is required for the expansion of autophagic membranes and the fusion of autophagosomes with lysosomes. LC3 is cleaved to produce LC3I and converted to LC3II, which is associated with the autophagosome membrane, and thus serves as a good autophagy marker. Autophagy is primarily a non-selective pathway, but a more selective form of autophagy (i.e., mitophagy) is becoming an apparently important phenomenon. The autophagy-mediated protein degradation removes aggregated or dysfunctional proteins to maintain the quality of intracellular proteins. During starvation, lipids are hydrolyzed to release fatty acids and glycogen is broken down to produce glucose for energy production. In addition, anucleate platelets play a crucial role in arterial thrombosis. Collagen is present in the vascular subendothelium and acts as an endogenous platelet activator via binding to glycoprotein (GP) VI and integrin α2β1, which mediates distinct signals in platelet activation. Moreover, -aminobutyric acid (GABA) is the major inhibitory neurotransmitter, we demonstrated platelets contain high level of GABA and it acts as a specific inhibitor of GP VI, thus GABA may play a negative regulator during platelet activation. Aldose reductase (AR), a NADPH-dependent enzyme, converts glucose to sorbitol, leading to oxidative stress in various tissues. Our previous MOST project discovered that transfected AR siRNA inhibits platelet aggregation stimulated by convulxin (GP VI agonist), but not by aggretin (integrin 2agonist). TOF-SIMS analysis revealed both AR protein and sorbitol were increased after convulxin stimulation. The next generation sequencing study showed platelets indeed contain AR mRNA. Based on these results, we conclude that GP VI-mediated increase of AR activity and protein expression are likely via different steps in mRNA regulation. Recently, we found that the NF-B is also present in platelets, and it can translocate from cytosol into mitochondria, and binding to mitochondrial DNA (mtDNA). However, the question remains to know whether NF-B is functionally present in a novel way, unrelated to transcriptional regulation. Furthermore, we also found that GABA or epalrestat significantly inhibited IKKβ phosphorylation, neither the sorbitol level nor AR protein expression was diminished by andrographolide in convulxin-activated platelets, indicating AR seems to be an upstream regulator of NF-B activation. Thus, platelets may be an ideal target for the study of non-genomic functions of NF-B in anucleate cells. Autophagy is important in nucleate cells, however, its role in anucleate cells is less recognized. A study suggested autophagy is occurred in platelets and seems to be related with platelet activation. Our preliminary results revealed that the ratio of autophagy marker proteins LC3II/LC3I and de-phosphorylation of p62 were increased after convulxin but not aggretin stimulation in platelets; nevertheless GABA, epalrestat or andrographolide markedly reversed both reactions. Deconvolution Microscope also observed the autophagy machinery in platelets. Therefore, we hypothesize that autophagy machinery may be induced in GP VI-dependent AR and/or NF-B activation in platelets, and endogenous GABA may play an inhibitory role in platelet autophagy. However, the detailed mechanisms of GABA in these reactions, and the non-genomic functions of NF-κB in the GP VI-mediated autophagy (mitophagy) are still unclear. We therefore proposed this three year project to systematically examine these issues. Specific Aim 1: To investigate the GP VI-dependent platelet autophagy, and the possible inhibitory role of GABA on these events. Specific Aim 2: To explore the energy metabolism in platelet autophagy and its functional role in thrombosis and hemostasis by human platelets and Atg5-deficient platelets from Atg5-/- mice. Specific Aim 3: To determine the mechanisms of NF-B regulates mtDNA contributing to platelet autophagy. Specific Aim 4: To elucidate the GP VI-dependent AR-triggered platelet autophagy from humans and Ar-/- mice in vitro and in vivo studies. Specific Aim 5: To examine the downstream regulatory role of AR-activated NF-B-induced autophagy (mitophagy) in platelets from humans and NF-B-/- mice. This project can provide a novel approach targeting the relationship between platelet activation and autophagy. Improvement of the platelet function by targeting autophagy machinery may provide promising choices for the treatment of arterial thrombosis. More prominently, development of specific autophagy inhibitors of platelets may become an important strategy for anti-thrombotic therapy.
|Effective start/end date||8/1/17 → 7/31/18|
- aldose reductase
- anucleate cell
- glycoprotein VI