Platelet proteomics in transfusion medicine: a reality with a challenging but promising future.

Dear Sir, Platelet proteomics is a young field that took off at the beginning of this century and soon yielded relevant results. The basic concept for platelet proteomic researchers is that, because platelets do not have a nucleus, proteomics is an ideal tool to approach their biochemistry. This concept proved to be true and thus, during the last ten years proteomics allowed the discovery of new platelet receptors and signaling proteins, some of which were shown to play a significant role in platelet activation. Indeed, some of these proteins are being studied as potential anti-thrombotic drug targets1. The initial success was taken with cautious optimism by platelet researchers, some of whom took the challenge of applying platelet proteomics to more clinical orientated studies. The aim was to take advantage of the technology in a clinical environment in order to find novel biomarkers and drug targets for those pathologies where unwanted platelet activation plays a relevant role. One of the fields where platelet clinical proteomics has shown more productive results is transfusion medicine. The storage of platelets is of critical importance in today’s clinical practice. Platelet concentrates (PLCs) are obtained, either by apheresis or pooled buffy coats, and administered routinely as life-saving procedures during surgery or chemotherapy to patients with low numbers of platelets or those who have their function impaired2,3. The problem with PLCs is their short shelf life when stored at 22 °C, which may lead to a deterioration of platelet functionality, a condition known as platelet storage lesion. Even though improvements in storage materials have lengthened the storage time of PCs, it is still restricted to 5 days for the risk of bacterial infection3. In contrast to other blood products, which can be stored for longer periods of time using colder temperatures (e.g. erythrocyte concentrates and plasma), platelets can’t be preserved in cold conditions because they undergo intense modifications in shape and functionality. These limitations create a constant shortage of PLCs in blood transfusion services. Platelet proteomics aims to help to understand the altered morphological, biochemical and functional features that constitute the phenomenon of storage lesion. By filling this gap of knowledge, proteomics will improve the understanding of the loss of function during storage and help to design new strategies and methods to enhance our ability to maintain platelets in optimal conditions during longer periods of time. During the last 5 years a combination of two-dimensional gel electrophoresis (2-DE)- and mass spectrometry (MS)-based proteomic approaches have been used to profile alterations in platelet proteins during storage. Back in 2007, Thiele and coworkers used two-dimensional differential in-gel electrophoresis (2D-DIGE) technology and MS to assess the effects of storage on the global proteome profile of therapeutic PCs and identify proteins that could be used as sensitive markers for storage related changes2. Their results showed that the platelet proteome remains quite stable during the first 5–9 days of storage, in fact, 97% of the cytosolic platelet proteins didn’t suffer any alterations at day 9. Major alterations of the platelet proteome occurred between day 9 and 15 of storage and these changes are most likely caused by degradation2. Proteins that were detected to change in the remaining 3% of cytosolic platelet proteins after 9 days of storage included septin-2, gelsolin and β-actin. Interestingly septin 2 and gelsolin are affected during apoptosis, indicating that apoptosis in PCs may have an impact on platelet storage. Moreover, those altered proteins could also be used as sensitive markers of platelet deterioration during storage. This study - as the authors recognize - suffers from one the most important limitations of 2-DE-based proteomics, the under-representation of membrane proteins due to their high hydrophobicity. To overcome this limitation, and comprehensively analyze changes suffered by PLCs during storage, Thon and co-workers performed a multi-technique study of the platelet proteome in PLCs over a 7-day storage period3. The authors combined protein-centric (2-DE/DIGE) and peptide-centric techniques (isotope-coded affinity tagging, ICAT; and isotope tagging for relative and absolute quantitation, iTRAQ) and could identify a total of 503 differentially expressed proteins. More precisely, 93 proteins were identified by 2-DE /DIGE, 355 by iTRAQ, and 139 by ICAT. Comparative analysis of 2-DE/DIGE, iTRAQ, and ICAT indicated that only five proteins were common to all three proteomic approaches employed3. There was some overlap among studies, especially between those with the same orientation (peptide- or protein-centric). They could also identify the altered proteins detected by Thiele and collaborators2 (septin-2, gelsolin, β-actin), which further consolidate them as possible markers for changes happening in PLCs. Other protein changes, like the ones seen in talin, 14-3-3, tubulin and thrombospondin, were also in excellent agreement between the two studies. Nevertheless, there were also some disagreements, which might be due to differences in instrumentation, variations in protocols from laboratory-to-laboratory, undiscovered changes in post-translational modifications, or the lack of specific protein detection due to low abundance.4 Platelets stored for transfusion produce prothrombotic and pro-inflammatory mediators implicated in adverse transfusion reactions. Correspondingly, these mediators are central players in pathological conditions including cardiovascular disease, the major cause of death in diabetics. In view of this, in 2009 Springer and coworkers performed a MS-based study where they analyzed platelet proteome changes in PLCs from diabetic patients and healthy donors over a 5-day storage period5. They identified 122 proteins that were either up- or down-regulated in type-2 diabetics relative to non-diabetic controls and 117 proteins whose abundances changed during the storage period. They could replicate some of the findings by Thiele and colleagues2 seeing that septin and actin were increased with storage. The study by Springer and colleagues was the first to characterize the proteome of platelets from diabetics before and after storage for transfusion. Their findings allowed the authors to formulate new hypotheses and experimentation to improve clinical outcomes by targeting “high risk platelets” that render platelet transfusion less effective or even unsafe. In conclusion, the application of platelet proteomics to address clinically relevant issues in the transfusion medicine field is already a promising reality. Obviously, there are many challenges ahead that should be addressed before we can have standardized methods that can be routinely introduced in the clinical practice. The proteomic method/s of choice should be robust, reproducible, and relatively rapid. Multi-center studies could bring light into these issues but there is no doubt the future of this young field is as promising as it was its beginning.