TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage

Abstract

Key Points• TAILS proteomics identified 2938 human platelet proteins, pervasive proteolytic processing, and precise proteolytic cleavage sites in stored platelets.• During storage, metalloproteinases were predominantly involved in proteolytic processing, while other proteinases were mainly involved in degradation.Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 unique peptides, we characterized N-terminal methionine excision, co-and posttranslational Na acetylation, protein maturation, and proteolytic processing of proteins in human platelets. We also identified for the first time 10 proteins previously classified by the Human Proteome Organization as "missing" in the human proteome. Most N termini (77%) were internal neo-N termini (105 were novel potential alternative translation start sites, and 2180 represented stable proteolytic products), thus highlighting a prominent yet previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metalloproteinase and other proteinase substrates and their respective cleavage sites suggests novel mechanisms of the effect of proteases on protein activity and platelet function during storage. All data sets and metadata are available through ProteomeXchange with the data set identifier PXD000906. (Blood. 2014;124(26):e49-e60)

Inhibiting GPIbα Shedding Preserves Post-Transfusion Recovery and Hemostatic Function of Platelets After Prolonged Storage

“…A characteristic of platelet storage lesion is ectodomain shedding of platelet surface receptor glycoprotein (GP)Ibα, as accumulation of glycocalicin, the product of GPIbα shedding, during platelet storage is reported in many studies 5–7 . As a major part of the GPIb-IX complex, GPIbα is the platelet receptor for von Willebrand factor (VWF) and other ligands present in circulation.…”

“…As a major part of the GPIb-IX complex, GPIbα is the platelet receptor for von Willebrand factor (VWF) and other ligands present in circulation. ADAM17, a widely expressed metalloprotease, cleaves GPIbα at the Gly 464 -Val 465 peptide bond and releases glycocalicin to the plasma 5, 8 . Recent reports showed that inhibiting ADAM17 activity using a broad-spectrum metalloprotease inhibitor GM6001 or p38 MAPK inhibitors during storage improved the post-transfusion recovery of stored murine platelets 6, 9 .…”

“…However, ADAM17 and other metalloproteases have broad substrate specificities. They cleave GPIbα, TNF-α and other protein substrates in platelets 5, 8 . Thus, studies using the inhibitors of ADAM17 activity could not rule out the possibility that shedding of a platelet receptor other than GPIbα mediates platelet clearance.…”

Simple, scalable, and ultrasensitive tip-based identification of protease substrates

“…Upon proteolytic cleavage as part of the common bottom-up proteomics strategy, this labelling allows quantifying N-terminal peptides but also distinguishing them from internal peptides with free Ntermini generated during in vitro digestion. Both methods have been used in numerous studies providing novel insights into proteases and their substrates (1,(8)(9)(10). Nevertheless, likely due to challenges in technical and (particularly in the past) data analysis aspects, the number of labs worldwide applying Nterminomics methods to screen for protease substrates is still limited.…”

Caracterização proteômica comparativa da agregação plaquetária induzida pela trombina e pela PA-BJ, uma serinoproteinase do veneno da Bothrops jararaca

“…Em trabalho recente, Prudova et al, (2014) identificaram mais de 2000 produtos proteolíticos estáveis em concentrado de plaquetas armazenado por 9 dias, sendo as metaloproteinases foram identificadas como responsáveis primárias por esta proteólise. Desta forma, os produtos de degradação identificados em nosso trabalho também podem ter sido gerados por metaloproteinases presentes no sobrenadante plaquetário.…”