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

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.Dissertation (MSc)--University of Pretoria, 2018.PharmacologyMScUnrestricte

Wounds : an overlooked burden (Part 4) – Burn wounds : a searing situation

Burn injuries are a leading cause of deformities and death globally, and in developing countries, the recurrence of burn incidents increases the financial burden on health systems. Appropriate management of burn wounds with timeous referral of patients to burn wound experts must be prioritised to facilitate recovery and prevent complications. This article describes common characteristics of burn wounds based on their aetiology and severity to help the treating clinician determine the appropriate treatment protocol to be applied. Treatment strategies according to burn wound aetiology have been summarised into downloadable reference tables for use in everyday practice.http://www.sagp.co.zaam2022Pharmacolog

Treatment of acute wounds and injuries : cuts, bites, bruises and sprains

Acute wounds and injuries are a common daily occurrence, and their appropriate care is critical to healing. In South Africa, community pharmacists are often the first to encounter acute wounds and injuries such as cuts, bites, bruises and sprains, due to the accessibility and cost constraints associated with primary healthcare facilities. Since pharmacists assist with first aid, refer the patient for further management if necessary, and provide the appropriate dressings during the later healing phase, it is imperative for pharmacists to stay informed on developments in wound care. This review aims to highlight some of the more common acute wounds and injuries encountered by pharmacists, such as cuts, bites, bruises and sprains, and the associated therapeutic strategies.The National Research Foundation – Blue Skies Grant.http://www.sapj.co.za/index.phdm2022Pharmacolog

Wounds : an overlooked burden (Part 2) – Wound treatment : a daunting decision

Various types of wounds are encountered daily in medical practice, and due to the numerous treatment options available on the market, making the most appropriate choice of treatment may be a rather daunting decision. The wound treatment should not inconvenience the patient to the point that the wound care becomes compromised. It is for this reason that a proper assessment of the wound status be conducted in order to select an appropriate treatment intervention or dressing type, as this will directly impact the way in which the wound heals. This article is based on the expertise of three active wound care practitioners and aims to provide guidance to clinicians when providing primary wound care. The downloadable tables can be printed and used as reference materials in daily practice.https://journals.co.za/journal/mp.sagphttps://journals.co.za/journal/mp.sagpam2022Pharmacolog

Wounds : an overlooked burden (Part 3) - Chronic wounds : a conundrum of complications

Wound healing is a complex process which may be hindered by a range of interfering factors, especially in patients with underlying pathologies. This results in the formation of chronic, non-healing wounds which subject the patient to significant discomfort while placing strain on the resources of any medical system. Primary healthcare practitioners are often the first to encounter chronic wounds of various aetiologies. This article provides guidelines on the assessment, wound bed preparation and treatment of various types of chronic wounds, which have been summarised into downloadable reference tables for use in everyday practice.National Research Foundation – Blue Skies Granthttp://www.sagpj.co.za/index.php/SAGPJPharmacolog

Stimulating students' critical thinking skills in pharmacology using case report generation

It is not a debatable issue that the COVID‑19 pandemic created several challenges in the education sector, and institutions across the world had to employ various interventions to ensure continuation of teaching and learning. At the University of Pretoria, one of South Africa’s largest contact universities, exclusive online learning was implemented for the remainder of the first semester of 2020. Although this was helpful to facilitate learning during strict lockdown, it also came with its own problems. For third-year undergraduate students in the disciplines of nursing, dietetics, physiotherapy and medical sciences, pre-existing issues such as failure to apply critical thinking skills in pharmacology were inflated during this period. Critical thinking in education is defined as a learning process where students analyse, evaluate, interpret or synthesise information, and apply creative thought to solve a problem. However, for the majority of the abovementioned student cohort, which comprised 252 students, learning scarcely went beyond memorisation and recall of information and facts. A potential cause of this could have been diminished learning skills owing to pressure and anxiety associated with the pandemic as reported in the literature, or a lack of active interaction among the students, where prior to COVID‑19, problem-solving was often conducted as a team effort during contact sessions. A combination of these causes is also likely.The Office of the Deputy-Dean: Teaching and Learning, Faculty of Health Sciences, University of Pretoria.http://www.ajhpe.org.zaam2021Pharmacolog