Identification of O‐GlcNAcylated Proteins in Human Platelets

Abstract

Platelets play a central role in haemostasis and thrombosis. Here, we focus on a newly-discovered, post-translational modification in platelets: a single N-acetylglucosamine attached to serines and/or threonines (O-GlcNAc). This modification is added by O-GlcNAc transferase (OGT) and removed by O-GlcNAcase. Both enzymes are present in platelets. Western blotting of SDS-PAGE gels with O-GlcNAc-specific antibodies (RL2 and CTD110.6) shows that platelets contain 15-20 major O-GlcNAcylated proteins. Upon stimulation by agonists (e.g. thrombin, TRAP, collagen, U46619, and A23187), the extent of modification displays protein- and agonist-specific changes. Treatment of platelets with three O-GlcNAcase inhibitors causes an accumulation of O-GlcNAc on specific proteins suggesting that O-GlcNAcylation dynamically cycles in resting platelets. To understand the effects of O-GlcNAc in platelets, we have undertaken a proteomic approach to identify modified proteins. We performed a series of western blots of 2-dimensional (2D) gels with O-GlcNAc-specific antibodies and identified positive spots by mass spectrometry (MS) analysis. To date 24 potentially modified proteins have been identified and their modification is in the process of being confirmed. Also, we are using wheat germ agglutinin affinity chromatography to purify additional O-GlcNAcylated proteins from platelet cytosol. These proteins will be identified by MS analysis. (Supported by NIH grant HL56652)