Comparative Proteome Analysis To Study Novel Markers of the Blood Platelet Lesion.

Platelet transfusion is a very common live-saving medical procedure for patients with platelet-deficient diseases like leukemia. In contrast to other blood components, the availability of platelets is restricted since they have a limited shelf-life of 5 days for transfusion purposes. This is due to storage-related deterioration in product quality resulting in the clearance from circulation. To overcome this problem, it is important to understand the molecular mechanisms leading to blood platelet lesion during storage. We were using two different proteomic approaches combined with functional biochemistry to investigate time-dependent changes in the blood platelet proteome. One type of analysis consisted of the separation of the platelet proteome at two different time points of storage, day 1 and day 8, using 2-dimensional (2D) gel electrophoresis for qualitative and DIGE technology for quantitative analysis. The identification of proteins changing in spot intensity was carried out by liquid chromatography and tandem mass spectrometry. The second method was based on stable isotope labeling with ITRAQ/ICAT reagents in combination with protease treatment, equivalent mixing, separation of the resulting peptides and quantitative analysis by mass spectrometry. Taken together, for the 2D/DIGE approach we analyzed 977 spots corresponding to 103 different proteins and for the ITRAQ approach 1428 peptides corresponding to 355 proteins, resulting in 37 proteins significantly changing both quantitatively due to protein synthesis or degradation and qualitatively due to post-translational modification and enzymatic activity. The high degree of correlation between the two approaches validates the experimental set-up and confirmed the requirement for complementary tools to enhance proteome coverage. Among others, increased amounts of integrins and other proteins known to form receptor signaling complexes with these integrins as well as proteins observed in platelet activation were detected. This proves, for the first time, that there is an apparent link between blood platelet storage lesion and cell signaling.