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Identification of the mechanisms important in enhanced platelet activation in diabetes

Summary of Project

Type 2 diabetes mellitus (DM) is a disorder growing in incidence, currently affecting more than 170 million individuals world wide. Type 2 DM increases risk of cardiovascular disease 2-4 fold, leading to 80% of fatalities in DM patients. Platelet hyperactivation and endothelial dysfunction underpin the development of cardiovascular disease in DM.

Endothelial dysfunction occurs through reduced nitric oxide (NO) bioavailability. Under healthy conditions NO acts to keep the endothelium and platelets in a quiescent state. Reduced NO bioavailability causes elevated cellular adhesion molecule expression on ECs, making them ‘sticky’ for platelets, and is a hallmark of DM. The molecular mechanisms causing EC dysfunction are not fully elucidated, but are believed to be caused by a combination of hyperglycaemia, insulin resistance and oxidative stress. The disruption of this nonthrombogenic barrier allows increased platelet adhesion to ECs, the first step in platelet activation. Platelets in diabetic patients are documented to circulate in a semi-activated state, and are characterised by dysregulation of several signalling pathways. Platelet stimulation causes the affinity of integrin receptors for their ligands to be upregulated and induces platelet degranulation. Secreted products act to recruit further platelets into the growing aggregate, as well as having strong inflammatory effects on the endothelium. Thus, platelet interactions with the intact endothelium, which is inhibited in healthy cells but stimulated by endothelial cell dysfunction in diabetes, is the major contributor to pathological thrombus formation.

The relative contributions of the dysregulated endothelium and hyperactivated platelets to the initiation of thrombus formation remains unclear. In addition, the molecular mechanisms behind increased platelet adhesion to endothelial cells seen in diabetics remain to be elucidated. The factors contributing to the balance between increased proaggregatory signalling and loss of inhibitory regulation of platelet-EC interactions remains to be established.

Project aims

  1. Determine to what extent platelet-EC interactions are elevated under diabetic conditions. The ability of healthy and diabetic endothelium to support the adhesion, spreading and aggregation of healthy and diabetic platelets will be investigated. These experiments will determine if diabetic endothelium, diabetic platelets or both are important for enhanced platelet-endothelial interactions. Platelet-EC interactions will be investigated using a combination of quantitative assays, fluorescent microscopy and physiologically relevant flow conditions.
  2. Characterise the mechanisms behind upregulated adhesion. Expression and activation of the receptors important in platelet-EC interactions will be measured. The levels of P-selectin and activated integrin αIIbβ3 on platelets and ICAM-1 and integrin αVβ3 on ECs will be monitored by flow cytometry/microscopy with antibody binding before and after co-incubating platelets and EC’s. Preliminary data in Dr Graham’s lab has already shown EC from diabetes patients have elevated expression of adhesion molecule expression. These experiments are designed to indicate how cell- cell interactions enhance the expression of adhesion molecules. The importance of these receptors for platelet-EC interactions under diabetic conditions will be confirmed by measuring adhesion in the presence of blocking antibodies and peptides against these receptors. The activation levels of normal and diabetic platelets and EC’s will also be determined, both before and after co-incubation, by western blotting. The levels of phosphotyrosine, JNK phosphorylation and p38 phosphorylation will be examined; pathways important in platelet and EC activation. The relevance of these pathways to mediate platelet-EC interactions will be investigated using specific inhibitors against these signalling molecules prior to measuring adhesion.
  3. The ability of exogenous NO to inhibit platelet-EC interactions, signalling and receptor activation will be investigated under diabetic and normal conditions. This will determine if this inhibitory pathway is blunted under diabetic conditions. To confirm if this pathway is activated to the same levels in diabetes and healthy ECs/platelets, the phosphorylation of the protein VASP ( a marker of NO signalling) will be measured. In addition we will examine if endogenous NO machinery in ECs is disrupted in diabetes. The protein and mRNA levels of the enzyme responsible for producing NO, eNOS, will be examined in diabetic and normal endothelium, both before and after co-incubation with platelets. To determine if endogenous NO signalling has different effects under normal and diabetic conditions, we will measure adhesion in the presence of the NOS inhibitor L-NAME. This will indicate if endogenous inhibition is disrupted under diabetes.

Entry requirements

At least 2.i Honours degree or equivalent.

Supervisors

Title and name:
Dr Wayne Roberts
Position:
Lecturer in Medical Sciences
Email address:
Telephone number :
Work+44 (0) 1274 232131
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Title and name:
Dr Anne Graham
Position:
Co-programme lead Healthcare Science and Associate Dean (Research and Knowledge Transfer)
Email address:
Telephone number :
Work+44 (0) 1274 233570
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