Transfusion medicine and proteomics: an alliance for blood quality and safety.

Abstract

The main goal of clinical transfusion medicine is to provide safe and effective blood products for the treatment of patients affected by a wide variety of malignant and non malignant diseases, by severe trauma, or candidate to general, cardio-vascular, orthopaedic and transplantation surgery. Moreover, pharmaceutical plasma-derived products still represent life-saving therapeutics for millions of patients. Recent advances in the field of high-tech proteomic analyses along with transfusion medicine clinical studies are increasingly showing how these two approaches may represent complementary facets. Cooperation of clinicians and researchers specialized in these two disciplines has the potential for major improvements in the treatment of patients with blood-derived therapeutics1,2,3. A primary goal in this joint agenda could be represented by the increase of our understanding of the changes that occur in each very phase of the preparation and storage of blood products, mainly during the storage of erythrocyte and platelet concentrates. Thus we could both design better storage systems and provide evidence to regulate storage more effectively. While current blood system regulation allows to guarantee a high degree of blood safety in most developed countries, future challenges might be characterized by the deepening of our knowledge about emerging transfusion transmissible infections, the role of cytokines in transfused products and the effects of blood storage lesions, all of which could still represent a significant threat to blood safety. The results of research on the above issues could be eventually translated into clinical routine practice and in advanced regulation, substantiated by renewed clinical and epidemiological approaches as well as the implementation of innovative technologies and tests on which to base the improvement of blood products’ standards. To this end, both clinical and academic efforts are fundamental. On this ground, over the past three years the Italian National Blood Centre (Centro Nazionale Sangue - CNS) has promoted and funded research studies involving laboratories from different universities. In particular, a scientific agreement has been recently established with the Proteomic Laboratory of Tuscia University of Viterbo, directed by Prof. L. Zolla. The final aim of this joint effort is to investigate to which extent proteomic applications are valuable tools for the study of blood products and what is their potential to improve our understanding of some crucial aspects of collection, processing and storage of blood products. In this framework, red blood cells (RBCs) appear to be uniquely positioned for these kinds of scientific breakthroughs. Thoughtful manipulation of RBC storage conditions coupled with observation of the proteomic consequences has a promising future. RBC storage lesions can be most generally resumed as the sum of all of the bad things that happen to RBCs during storage. In the long run, understanding erythrocyte storage lesions will presumably offer the best chance for guiding the development of better RBC storage and informing blood banking regulation4. It is well known that storage lesions include metabolic effects such as the breakdown of metabolic sugars to create lactate and protons. The protons produced by glycolysis in turn decrease pH leading to the loss of 2,3-diphosphoglycerate. Cold storage slows glycolysis, but also reduces sodium and potassium pumping, thus leading to increasingly elevated supernatant potassium concentrations with likely increased risks of adverse reactions when blood is rapidly transfused. Moreover, during storage proteins and lipids are oxidized and plasticizers from the storage bags might intercollate into the red cell membranes. Furthermore, most evident changes regard RBC morphological alterations accumulating over storage, as they undergo shape change and membrane loss. Ultimately, a number of RBCs break down during storage and a larger number fail to survive when they are returned to the circulation. The relationships of these storage lesions to the function and fate of transfused RBCs is largely unknown and increasingly attracting the attention of the scientific community. Rapid advances in the proteomics of RBCs and the resulting improved understanding of their changes during storage could suggest a path to cope with possible limitations of currently widespread RBC collection and storage systems; this might reveal pivotal for regulating safety and improving effectiveness of blood products. Proteomic techniques allow enumeration of RBC proteins, monitoring their oxidative decoration and damage, and following the occurrence of shed micro- and nano-vesicles. Thanks to proteomics, we now know that a RBC contains about 1,578 different cytosolic proteins5 and about 340 associated with the membrane6. While being devoid of a nucleus and thus lacking any new protein synthesis activity, the RBC protein complement to the genome is all but stable, either in vivo and in vitro. These considerations enhance the need to update this inventory of RBC structural and metabolic proteins investigating in parallel RBCs approaching the end of their lifespan (120 days in vivo) and RBCs for transfusion, which are usually stored for 42 days. As proteins in biological systems do not work on isolation - rather they form a web of complicated interactions - the knowledge of how the proteome changes over time will ultimately be coupled with the understanding of how proteins do interact either among each other or with membrane lipids and metabolic sugars. This information will hopefully allow the construction of formal mathematical models of the life of a RBC. Unfortunately, we are just at the beginning of that process. Future research efforts in RBC biopreservation science should be enriched with emerging principles, techniques and knowledge regarding the RBC interactome7, the formation of lipid rafts and other functionally important multi-protein complexes, the repair/destroy mechanisms, the response to oxidative stress, the post-translational processing, the protein sorting into the vesicles and the membrane as a place of execution of the senescence-related apoptosis-like events. This advanced, sophisticated approach would definitely contribute to the final understanding of the mechanisms that define life and death of RBCs in vivo, in storage plastic bags and in the circulation upon transfusion. This innovative and integrated molecular and in silico strategy might represent a further step towards a successful optimization of the quality and safety of RBC preservation and consequently of transfused RBCs and their clinical outcomes. CNS and University of Tuscia are working on the hypothesis to improve RBC storage. In particular, main emphasis is being put on the concept that better storage is more important than longer storage. So far, suggested strategies have mainly relied on correcting storage lesions by the use and continuous improvement of specific additive solutions, which tackle biochemical alterations but not oxidationinduced protein fragmentation and aggregation events. It is currently under investigation an alternative approach, which rather aims at preventing the occurrence of oxidative-stress-induced irreversible lesions targeting proteins and thus improving erythrocyte damage-free survival and viability over storage. The RBC has many defences against oxidative injury and owns evolved ways to survive despite oxidative attack. As the damage to individual proteins is clearly irreversible in the enucleated RBC system, RBCs do have tools to avoid the consequences of this ongoing damage. Since the underlying cause of irreparable denaturation of proteins following fragmentation and aggregation catalysed by free radicals is the prolonged oxidative stress to which RBCs are exposed during storage, Zolla’s group have recently suggested a storage protocol aimed at tackling the problem at its source. The pilot project proposes storing blood directly in an atmosphere of inert gas. The clinical outcome of this protocol has already been tested with respect to classical standards (haemolysis and red blood cell survival at 24 hours posttransfusion) with encouraging results; slowing in the decreases of 2,3-DPG and ATP was also observed8. In support, by using classical proteomic methods the total protein profile of RBCs stored in the absence of oxygen was compared with that of control units stored at 4°C under normal atmospheric oxygen pressure9. No signs of fragmentation or aggregation were found in RBCs stored under an atmosphere of inert gas in the medium term (during the first 2 weeks); detrimental effects began to be seen, albeit to a reduced extent, towards the end of the storage period (42 days). In other terms, from a molecular point of view, RBC concentrates stored according to this alternative protocol were potentially safer than those stored under conventional conditions. To show that better storage conditions are actually gained, biomarkers of oxidative damage/aging status of RBCs and/or changes in the protein composition of RBC membrane related to the storage period should be identified as RBC damage markers, in order to develop new technological approaches and feasible routine tests for blood component quality control and quality assurance. In this respect, storage under anaerobic conditions demonstrated a suppression of the recruitment of a series of biomarker-like proteins to the membrane which are inevitably generated by reactive oxygen species under conventional storage conditions9. These results are drawn from preliminary studies and further investigations, both molecular and clinical, are needed. In this perspective a considerable amount of research on blood proteomics is currently underway paving new paths that involve transfusion medicine and proteomics in a fascinating alliance.