The Novel Roles of Aldose Reductase (AR) in Collagen GPVI- and Thrombin PAR4-Mediated Platelet Activation and Apoptosis---Regulatory Machinery of Arf-AR Signaling in Anucleate Cells

Project: A - Government Institutionb - National Science and Technology Council

Project Details


Aldose reductase (AR), a NADPH-dependent enzyme, converts glucose to sorbitol associated with increasing oxidative stress. Sorbitol accumulates inside the tissues, as it cannot permeate to cell membranes, leading to ionic imbalance and protein insolubilization, consequently causes chronic diabetic complications. Recently, AR was strongly suggested to regulate cell proliferation, apoptosis and atherothrombosis in normal glucose condition. Platelets, anucleate cells, play a crucial role in hemostatic processes and arterial thrombosis. Collagen and thrombin induce platelet activation via binding to integrin α2β1 and glycoprotein (GP)VI, and protease-activated receptors (e.g., PAR1 and PAR4), respectively, and these receptors mediate distinct signals in platelet activation. Furthermore, the Ras superfamily (e.g., ADP-ribosylation factor, Arfs) appears to link platelet activation. Among the Arfs family, Arf6, a membrane bound enzyme is reported to play a key element in early platelet activation; nevertheless, its detailed role in platelet activation remains obscure. In addition, it is esteemed that megakaryocytes invest platelets with a varied repertoire of mRNAs, which are competent for translation. Quiescent platelets display minimal translational activity and their activation could lead to the rapid translation of existing mRNAs. Despite it has long been assumed that splicing events are negligent in platelets, surprisingly, a recent study found that platelets still contain functional spliceosomal components in their cytoplasm. Furthermore, caspase-dependent and -independent apoptosis exist in nucleated cells. In caspase-dependent apoptosis both the extrinsic death receptor (DR) and the intrinsic mitochondrial pathways may be involved, whereas apoptogenic factors may involve in caspase-independent pathway. Platelets can express several members of the caspases; however, the mechanisms of the apoptotic machinery in platelets are not yet clearly understood. Therefore, we hypothesize that AR plays a crucial role in platelet activation and apoptosis in normal glucose condition. Our preliminary results indicated that epalrestat, a selective AR inhibitor, markedly inhibited platelet aggregation stimulated by collagen and convulxin, an agonist of GPVI, but not by aggretin, an agonist of integrin 2 (Fig.1). We also found that epalrestat clearly abolished platelet aggregation stimulated by thrombin and PAR4-AP, but not by PAR1-AP (Fig.1). These results suggest that GPVI and PAR4 might play crucial roles in AR activation in human platelets. Moreover, AR protein expressions were increased after convulxin and PAR4-AP stimulation (Fig.2A), which were reversed in the presence of epalrestat (Fig.2B), cycloheximide (Fig.3B) or transfected AR siRNA (Fig.3C). The RT-PCR and next generation sequencing (NGS) studies revealed that platelets indeed contain AR mRNA (Figs.3A, 4). The intracellular level of sorbitol was markedly increased after convulxin stimulation (resting, 2.6 ± 0.2 vs. convulxin-activated, 3.3 ± 0.1 g/ml; n=3) by LC-MS/MS (Fig.5A) and Time-of-Flight Secondary Ion Mass Spectrum (TOP-SIMS) analysis (Figs.5B-C). Based on these results, we conclude that GPVI- or PAR4-mediated increase of AR activity and AR protein expression are likely to be via different steps in mRNA regulation. In addition, convulxin markedly stimulated both Arf6 and caspase 3 activation; epalrestat clearly inhibited caspase 3 activation, but not Arf6 (Fig.6), indicating that Arf6 seems to be an upstream regulator of AR activation and AR activation might be involved in platelet apoptosis. Our preliminary results revealed that AR might play a vital role in platelet activation; however, the molecular mechanisms of AR in these processes and regulatory machinery, particularly in increasing AR protein expression and the translational machinery of AR mRNA in platelets, remain unclear. Therefore, we will systematically examine these issues through this proposed project (Fig.7). Aim 1: To elucidate the activity of AR and AR-triggered signal events in GPVI- or PAR4-mediated platelet activation under normal glucose condition. Aim 2: To determine the differences in upstream regulators of AR (e.g., Arf family and/or unknown proteins) that control AR activation in GPVI- or PAR4-mediated platelet activation. Aim 3: To study the translational machinery of GPVI- or PAR4-mediated increases in AR protein expression. Aim 4: To determine the possible apoptotic roles of AR using platelets from humans and Ar-knockout mice. This project can provide a novel therapeutic approach targeting AR activation to prevent atherothrombosis.
Effective start/end date8/1/147/31/15


  • aldose reductase
  • anucleate cell
  • platelet
  • apoptosis
  • glycoprotein VI
  • protease-activated receptor 4


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