Dana Virginia Devine

Platelets are an integral part of transfusion medicine. In Canada, over 80% of the platelet units for transfusion are produced via the buffy coat method from whole blood and stored in plasma. The storage condition is at 22°C under constant agitation for a maximum of 7 days. These are known as room temperature stored platelets (RPs). There are several disadvantages associated with RPs. First, RPs experience a significant decrease in platelet quality during storage known as the platelet storage lesion. Second, RPs have increased risks of microorganism growth. Last, RPs have a very limited shelf life which contributes to product wastage.Platelets stored at 4°C or cold-stored platelets (CPs) have been gaining renewed interest due to their potential of overcoming the disadvantages of RPs. Preliminary studies have shown that CPs have preserved mitochondrial functions, superior hemostatic functions and stable pH compared to RPs. Additionally, CPs have been hypothesized to be a superior product especially for bleeding patients. In this thesis, we compared the in vitro storage characteristics of CPs versus RPs over 14 days of storage. We showed that CPs have better aggregation response to physiological agonists and formed stronger clots compared to RPs. We also examined the underlying biological changes of CPs and RPs during storage by performing an untargeted metabolomic study. We showed that CPs exhibited significantly less metabolomic stress during storage compared to RPs as exemplified by the stable reduced glutathione pools in the CPs but not RPs. Additionally, we explored the potential benefits of CPs in bleeding scenarios by using an in vitro trauma transfusion model. From this experiment, we showed that donor whole blood mixed with CPs is more resistant to hyperfibrinolysis that is commonly observed in trauma. Finally, we explored the importance of oxidative stress in platelet storage by inhibiting the activity of the glucose 6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway. We found that RPs with G6PD inhibitors seem to have higher P-selectin expression and lower maximum clot firmness, indicating worse quality.These results indicate that CPs should be further considered as a transfusion product specifically for bleeding patients.

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Red blood cell (RBC) transfusion is a cornerstone therapeutic intervention in contemporary medicine. Stored RBCs undergo multiple biochemical and biophysical changes, collectively referred to as the RBC storage lesions. One of the most prominent alterations is hemolysis, the rupturing of RBC and the subsequent release of hemoglobin-rich cytosolic content. Hemolysis may compromise the red cell concentrate (RCC) therapeutic efficacy with a decrease in viable and functional cells and an increase in bioactive molecules that may disrupt vascular and immune homeostasis. Thus, hemolysis is used as a key surrogate indicator of RCC storage quality; however, this parameter is evaluated at expiry and all RCCs are transfused without knowing their levels of hemolysis. With aims to optimize product quality for transfusion recipients and to improve donor and inventory management efficiency, the identification of predictive markers for storage hemolysis warrants further investigation. While the molecular mechanism(s) of hemolysis remain elusive, a few factors such as donor characteristics and manufacturing processes are known to influence its development. From a unique donor population composed of repeat donors exhibiting high hemolysis, membrane-associated proteins involved in antioxidant pathways - peroxiredoxin-2, catalase, and 20s proteasome - were identified as potential quality markers using isobaric tag for relative and absolute quantitation. Additionally, in a novel comparative study between RCC subjected to gamma-irradiation and pathogen inactivation (PI), the level of these candidate protein markers displayed robust negative linear relationship with storage hemolysis. These candidate protein markers hint at the role of oxidative damage in product quality deterioration and hemolysis development. While deoxygenation treatment successfully rescued elevated hemolysis in PI-treated RCCs, the application of these protein markers did not yield similar relationship to hemolysis development. This observation suggests that PI-induced damage is heavily reliant on the presence of oxygen and that the identified protein quality markers may not be specific to oxidative damage alone. Taken together, the findings presented here propose three potential candidate protein markers for product quality and further support that cumulative oxidative damage contributes to storage hemolysis development. However, the results suggest that there may be additional underlying metabolic and/or molecular mechanisms that are important for hemolysis development.

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Transfusions of platelet concentrates (PCs) are given to maintain primary hemostasis in patients with various thrombocytopenic disorders. There is poor correlation between in vivo PC transfusion outcome and in vitro tests, which typically do not test the functional effectiveness of platelets, but rather measure platelet characteristics. Thus a PC quality assay that would accurately predict transfusion efficacy should test the efficacy of platelet activation and clot formation in a manner that more closely models these same processes in the bloodstream.The first aim of this thesis is to determine whether Thromoelastography (TEG)/rotational Thromboelastometry (ROTEM) technologies involving global hemostatic analyzers could be used to assess the quality of PCs under a variety of conditions. Due to their procoagulant properties, platelet microvesicles’ (PMVs’) contribution to the clot signature was assessed. The second aim was to investigate the effect of pathogen inactivation technology (PI) using riboflavin/UV light (Mirasol) on the hemostatic potential of PCs and plasma in transfusion trauma packages composed of reconstituted whole blood (WB). The packages were composed of red blood cells (RBC), plasma, and platelet, in a ratio of 1:1:1.As there is an increasing interest by practitioners in returning to the use of WB (2-7 days old) in the civilian setting for the treatment of massively hemorrhaging patients, our third aim was to determine whether ROTEM could be used to assess the impact of PI-treated WB in a trauma model. Due to the reduction in the activity of multiple plasma coagulation proteins following PI-treatment, supplementation of fibrinogen to correct the negative impact was assessed. Hemostatic analysis showed no significant change in maximum clot formation during the storage of PCs up to Day 10. Hemostatic measurement was sufficiently sensitive to dissect platelet and PMV contributions to clot formation and to detect PCs stored under poor conditions. This study suggests a potential solution to the apparent reduction in the hemostatic capability of blood products as caused by treatments with Mirasol; the use of fibrinogen supplementation appears to largely correct the Mirasol defect.

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Pathogen inactivation (PI) techniques are designed to increase the safety of blood products by damaging RNA and DNA of pathogens. Even though platelets are anucleate, they synthesize proteins using RNA and the ribosomal machinery derived from megakaryocytes. The role of protein synthesis in platelets, however, is still poorly understood. PI-treated platelets show signs of accelerated storage lesion, but the effect of PI on platelet mRNA and subsequent protein synthesis remains unclear. In this dissertation we investigated to what extend platelet mRNA is affected by PI using Mirasol as a representative PI. In chapter 3 we demonstrated that the Mirasol treatment affected platelet mRNA negatively in a target specific manner, but prolonged the mRNA half-life. The long mRNA half-life suggested the presence of a mechanism, protecting platelet mRNA from degradation. We investigated the role of p38 as a potential regulator of platelet mRNA and if stress granule (SG) formation could be involved in protecting platelet mRNA. The kinase p38 is a key regulator of a wide range of platelet function. In nucleated cells, UV-induced stress activates the p38 and ultimately leads to increased mRNA stability through the regulation of RNA binding proteins. UV illumination is a key feature of all PI technique, and p38 is activated in Mirasol-treated platelets. In chapter 4 we demonstrated that inhibition of p38 in Mirasol-treated platelets increased the mRNA half-life, but not through the RNA binding proteins HuR or TTP. SG formation is a protective response in nucleated cells to temporarily store mRNA and consists of mRNAs and specific SG proteins. Chapter 5 is a description of the preliminary experiments performed to test the effect of a known SG inducer (arsenite) and inhibitor (nocodazole) in platelets, especially in the context of the Mirasol treatment. Nocodazole did not impact the platelet mRNA levels, but affected the mean platelet volume and count in Mirasol-treated platelets. Arsenite exposure did not affect platelet total RNA or GAPDH mRNA, but showed a reduction in extent of shape change. Our experiments showed that platelets possessed the potential to form stress granules, but a definitive mechanism was not demonstrated.

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Platelet products have a short shelf life (5 to 7 days) owing in part to the deterioration ofthe quality of platelets stored at 22°C. This creates significant inventory challenges, and blood banks may suffer shortages and high wastage as a result. Proteomics offers a global quantitativeapproach to investigate changes occurring in stored blood products. These data sets can identifyprocesses leading to storage-associated losses of blood component quality such as the plateletstorage lesion (PSL). Changes to the platelet proteome between days 1 and 7 of storage wereanalysed with 3 complementary proteomic approaches with final mass spectrometric (MS)analysis: 2-dimensional (2D) gel electrophoresis/differential gel electrophoresis (DIGE), isobarictagging for relative and absolute quantification (iTRAQ), and isotope-coded affinity tagging(ICAT). Although proteomics analyses identified many storage-associated protein changes, thesevaried significantly by method suggesting that a combination of protein-centric (2D gel orDIGE) and peptide-centric (iTRAQ or ICAT) approaches is necessary to acquire the mostinformative data.Validation of the proteomics results by western blotting, flow cytometry, quantitative real-time polymerase chain reaction (qRT PCR) and ³ٰ⁵S-methionine incorporation confirmed thatplatelets are capable of synthesising biologically relevant proteins ex vivo throughout a 10-daystorage period with particularly long-lived mRNA (half-life of approximately 2.4 days), and hasprovided the first evidence for one of the mechanisms of the PSL. The development of an ³ٰ⁵Smethionineassay has since shown that stored human blood platelets incorporate ³ٰ⁵S-methionine at a rate that is proportional to time and substrate concentration, and is slower for freshly drawn platelets than those stored in pooled buffy coat derived units for 10 days. More interesting still are the observations that the overall ³ٰ⁵S-methionine incorporation rate was higher in pooled buffy coat platelet units versus freshly drawn platelets, that this rate increased upon agonist exposure in both, and that day 8 platelets showed significantly greater total protein translation than on days 2,3,7 and 10 of storage. This may be indicative of translational regulation of the platelet proteome during storage and upon activation. Translational control is a consequence of remarkable cellular specialisation and precise biochemical pathways which, in the case of platelets, may lead to storage-associated losses of blood component quality and must be understood if platelet storage times are to be extended.

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