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Ex vivo modelling of the formation of inflammatory platelet-leucocyte aggregates and their adhesion on endothelial cells, an early event in sepsis.

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posted on 2019-07-03, 09:29 authored by Azzah Alharbi, Jonathan P. Thompson, Nicholas P. Brindle, Cordula M. Stover
Septicaemia is an acute inflammatory reaction in the bloodstream to the presence of pathogen-associated molecular patterns. Whole blood stimulation assays capture endotoxin-induced formation of aggregates between platelets and leucocytes using flow cytometry. We wanted to assess extent of spontaneous aggregate formation in whole blood stimulation assays and compare the effects of endotoxin and heat-killed, clinically relevant, bacterial pathogens on aggregate formation and then on adhesion of aggregates to TNFα-stimulated endothelial cells. We found that endotoxin (from Escherichia coli or Salmonella enteritidis) was not a suitable stimulus to provoke platelet-leucocyte aggregates in vitro, as it did not further increase the extent of aggregates formed spontaneously in stasis of hirudin-anticoagulated blood. Specifically, whole blood samples stimulated with or without LPS produced aggregates with a mean surface area of 140.97 and 117.68 μm2, respectively. By contrast, incubation of whole blood with heat-killed Klebsiella pneumoniae or Staphylococcus aureus produced significantly enhanced and complex cellular aggregates (with a mean surface area of 470.61 and 518.39 μm2, respectively) which adhered more frequently to TNFα (and free fatty acid)-stimulated endothelial cells. These were reliably captured by scanning electron microscopy. Adhesion of cellular aggregates could be blocked by incubation of endothelial cells with a commercial P-selectin antibody and an angiopoietin-2 ligand trap. In conclusion, we have developed an in vitro method that models the acute inflammatory reaction in whole blood in the presence of sepsis-relevant bacterial pathogen surfaces.


AA received funding from King Abdulaziz University, Jeddah 21589, Saudi Arabia. This manuscript contains part of her PhD thesis. Natalie Allcock and Anna Straatman-Iwanowska from University of Leicester’s Electron Microscope unit are gratefully acknowledged for their expert guidance. We thank Professor Martyn Mahaut-Smith for helpful discussions. Omeed Al-Dalawi is acknowledged for purification of R3. For this part we gratefully acknowledge funding from BHF (PG/15/65/3170). The FACS cell sorter used for this work was funded by a grant from the MRC (G0802524).



Clinical and Experimental Medicine, 2018

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/Organisation/COLLEGE OF LIFE SCIENCES/School of Medicine/Department of Infection, Immunity and Inflammation


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Clinical and Experimental Medicine


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Parts of this work were presented at BJA Research Forum Winter Meeting, 23.-24. 11. 2017, held in Leicester.



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