A comparative proteomic and glycoproteomic study of platelets from patients with diabetes and non-diabetic healthy individuals

Abstract

Introduction: Diabetes mellitus is considered one of the four major non-communicable disease with the global prevalence of this disease nearly doubling over the past 40 years, making it one of the leading causes of morbidity and contributing to mortality. Diabetes is characterized by elevated blood glucose levels due to insulin deficiency, resistance or a combination of both. The disease is associated with several complications, and some of these complications are attributed to glycation of proteins which impairs their function and stability. Diabetic foot ulcers (DFUs) are one of the most catastrophic and costly complications of diabetes. These foot ulcers do not naturally progress through the phases of the wound healing process and are therefore classified as chronic wounds. There is limited treatment availability for diabetic foot ulcers, and the current treatment strategies have been met with high rates of recurrence and failure. Therefore, there is need for more extensive research to be conducted to improve therapy and minimize the chance of developing complications. Blood platelets play a central role in initiation of wound healing and on account of this, the study was directed towards comparing platelet proteins from diabetic patients and non-diabetic healthy individuals to investigate the possible differences in protein expression and glycation. The aim was to characterize these proteins to further understand the role platelets play in impaired wound healing in DFUs and to provide a possible basis in developing targeted therapies. Methods: All blood samples were tested for HbA1c levels which is an indicator of long term blood glucose levels. This was used as a screening tool to confirm participant status. After the screening test, non-stimulated platelets were isolated from whole blood of diabetic patients and non-diabetic healthy individuals, washed and the total protein complement separated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The gels were visualized using Stain-free™ imaging and then separately stained with Acqua (colloidal Coomassie blue), silver, Oriole™ or Periodic Acid-Schiff (PAS) stains for comparison. Following this, high performance liquid chromatography (HPLC) coupled to fluorescence detection and western blotting were done to check for possible formation of advanced glycation end products (AGEs). In-gel and in-solution trypsin digests of selected samples exhibiting protein band differences between the two groups were performed, followed by peptide sequencing using liquid chromatography tandem mass spectrometry (LC-MS/MS). Results and discussion: Gel electrophoresis results showed similarities in the general pattern of the protein mass fingerprint with subtle band differences identified between the two groups. Results from the PAS stain implied that there was no glycation of platelet proteins in diabetic patients, which led to the proposition that complex advanced AGEs may be forming. This was tested using HPLC with fluorescence detection of the trypsinized samples and the appearance of extra peaks from diabetic patient samples on the chromatograms obtained after HPLC analysis indicated that this was a possibility. Western blotting to confirm the formation of AGEs, showed similarities in the formation of AGEs between the two groups suggesting that there was no difference in AGE formation between diabetic patients and non-diabetic healthy individuals. A few differences in platelet protein abundance were seen between the two groups when downstream LC-MS/MS analysis of the samples was done, which showed the superiority of the analysis technique over SDS-PAGE. Conclusion: The study showed that there were no significant differences in glycation of proteins between the two groups which can possibly eliminate glycation as a potential cause of delayed healing of DFUs. However, LC-MS/MS analysis of samples identified proteins which had differences in abundance between the diabetic patients and non-diabetic healthy individuals, some of which are key proteins in the wound healing process. Therefore, based on these results, a proposition can be made that differences in the abundance of these proteins could be contributing to delayed wound healing of DFUs. Due to this, use of autologous platelet rich plasma from diabetic patients to promote healing of DFUs would result in little treatment enhancement as these platelets show proteomic differences that could provide an excess of undesirable proteins at the wound site. Thus, the study can be supplemented with other studies to make a more substantiated conclusion and possibly develop therapies targeted at these proteins.