Towards a novel anti-inflammatory, vasculo-protective agent: inhibition of P-selectin and disruption of platelet-leukocyte interaction by Staphylococcus aureus extracellular fibrinogen binding protein

Lead supervisor: Dr Stefan Bagby, Department of Biology & Biochemistry
Second supervisor: Dr Giordano Pula, Department of Pharmacy & Pharmacology

The inflammatory response requires leukocytes to migrate to an infection or wound site by “crawling” along interior walls of blood vessels. Protein-protein interaction between PSGL-1 on leukocytes and P-selectin on platelets is essential for this process. As such, P-selectin–PSGL-1-mediated platelet–leukocyte interaction contributes to pathological inflammation in several disease models, including atherosclerosis, ischaemia-reperfusion injury, and stroke. P-selectin inhibition is thus an attractive target for the development of therapeutic agents that limit the adverse effects of excessive inflammation. Agents developed to date that disrupt P-selectin–PSGL-1 interaction, including antibodies, small molecules and glycopeptide mimics, have significant drawbacks.

The proposed project builds on our findings that Staphylococcus aureus protein Efb binds with nM affinity to P-selectin and disrupts P-selectin–PSGL-1 and platelet–leukocyte interaction. The project involves four phases: A. Cell-free assays to define the minimum Efb fragment that replicates P-selectin inhibition by full length Efb. B. Structural, biophysical and biochemical analyses to elucidate how Efb interacts with P-selectin, the first such study of a bacterial protein binding to P-selectin, potentially revealing a novel mode of P-selectin recognition. C. Phase B-guided alterations to Efb peptides towards increased affinity and specificity for P-selectin. D. Whole blood assays to verify peptide activity in resting and vascular flow conditions. Outcomes could lead to in vivo testing of Efb-based peptides for prevention and treatment of serious cardiovascular disorders like atherosclerosis.

The project will involve training in a wide range of both molecular and cellular level techniques, most of which are fundamental and routinely used in academia and industry, and some of which are somewhat more specialised. Phases A and B will involve training in molecular cloning, recombinant protein expression using both prokaryotic (E. coli) and eukaryotic (CHO cells) systems, and protein purification. Protein characterisation methods will include analytical ultracentrifugation and size exclusion chromatography-multiangle light scattering. Phases A and C will include the acquisition of skills in peptide design and synthesis, and all four phases will involve training in peptide handling. Biophysical and biochemical analysis of protein-protein/peptide interaction in Phases B and C will include training in thermal shift assay and pull downs, one or both of surface plasmon resonance and isothermal titration calorimetry, and site directed mutagenesis. The project will additionally include training in one, two or three major structural biology techniques (X-ray crystallography, NMR spectroscopy, and small angle X-ray scattering). Phase D1 will involve training in whole blood, platelet rich plasma (PRP) and washed platelet preparation, immunoblotting with quantification by densitometry analysis, and flow cytometry for detection of platelet–leukocyte complexes. Phase D2 will involve monocyte and neutrophil cell culture in the presence or absence of endothelial cells. In addition, the student will be trained in the use of a BioFlux 200 flow system and the assessment of platelet adhesion/thrombus formation under physiological flow conditions.