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

  • Stuart Wallis

Student thesis: Doctoral ThesisPhD


Platelet–leukocyte complexation, essential for the initiation of inflammation, occurs via interaction between P-selectin on platelets and PSGL-1 on leukocytes. Neutrophils recruit platelets to vascular surfaces to initiate inflammation, but platelets can in turn mediate aberrant recruitment of additional leukocytes to vascular surfaces, thus contributing to thromboinflammatory pathologies. Controlling platelet–leukocyte complexation by disrupting the P-selectin–PSGL-1 interaction could alleviate collateral inflammatory damage of blood vessels. Extracellular fibrinogen binding protein (Efb), an immune evasion protein secreted by Staphylococcus aureus, has been previously shown to inhibit P-selectin-PSGL-1-dependent formation of platelet-leukocyte complexes. An attractive long-term objective, therefore, is to identify an Efb-derived agent that slows down or even prevents the development and progression of major cardiovascular pathologies associated with vascular inflammation and atherosclerosis. This project aimed to develop the smallest functional peptide of Efb that inhibits the complexation of P-selectin and PSGL-1, and hence inhibits platelet-leukocyte interaction.
Seven peptide fragments of Efb and their scrambled versions were obtained and used in flow cytometric platelet interaction trials. The smallest of these peptides that bound to platelets was Efb 68-87, which interacted with platelets with a KD value of 7.4 M. A significant reduction in Efb 68-87-platelet binding in the presence of PSGL-1 indicated competition for the binding site. To understand how Efb 68-87 might affect thrombus formation, in vitro flow adhesion assays were performed. Collagen-coated flow adhesion assays showed no significant difference in thrombus formation on collagen in the presence of Efb 68-87 or scrambled 68-87. Efb 68-87-coated flow-adhesion assays did, however, confirm significant platelet binding by Efb 68-87 at venous flow rates compared to scrambled Efb 68-87. Platelet aggregation trials revealed that Efb 68-87 did not stimulate platelet aggregation or alter platelet aggregation that had been stimulated by thrombin, collagen or U46619. Whole blood cell binding trials confirmed that Efb 68-87 binds to platelets and platelet-leukocyte aggregates in whole blood only. Flow cytometry with P-selectin and PAC-1 markers indicated that Efb 68-87 had little effect on the platelet stimulation response. Further whole blood flow cytometry trials suggested that increasing Efb 68-87 concentration has a negative correlation to platelet-leukocyte aggregate (PLA) levels. Additionally, neutrophil extracellular trap (NET) formation assays demonstrated that Efb 68-87 significantly reduced NET formation.
In conclusion, this project has defined Efb 68-87 as a novel P-selectin-binding peptide capable of inhibiting the platelet-leukocyte interactions necessary for PLA and NET formation in vitro. As PLAs and NETs play a significant role in vascular inflammation, Efb 68-87 represents a promising option for the development of a novel peptide therapeutic to treat thromboinflammatory pathologies.
Date of Award25 May 2022
Original languageEnglish
Awarding Institution
  • University of Bath
SupervisorStefan Bagby (Supervisor), Giordano Pula (Supervisor) & Ian Eggleston (Supervisor)

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