The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

Carla Freitas,Maria-Cristina Assis,Camila Castro Figueiredo,Maria-Cristina Plotkowski,Veronica Maria Morandi,Alessandra Mattos Saliba,Mirian Pereira

doi:10.1590/s0074-02762012000600004

Abstract

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Highlights

Pseudomonas aeruginosa is a major agent of sepsis, a clinical syndrome characterised by the infection-triggered activation of the inflammatory and coagulation systems and by endothelial dysfunction (Esmon 2005)
ExoU enhanced the release of von Willebrand factor (vWF) and vWF-bearing microparticles from microvascular endothelial cells - As shown in Fig. 1, the concentrations of vWF in both the cell culture supernatant (Fig. 1A) and subendothelial matrix (Fig. 1B) of endothelial cells infected with the ExoU-producing bacterial strain were significantly higher than those in control cultures or in cultures infected with the exoU-deficient mutant
The vWF expression indices for microparticles shed after PA103 infection were significantly higher than those for microparticles shed by control cells or by cells infected with the PA103ΔexoU mutant (Fig. 2C, E)

Summary

Introduction

Pseudomonas aeruginosa is a major agent of sepsis, a clinical syndrome characterised by the infection-triggered activation of the inflammatory and coagulation systems and by endothelial dysfunction (Esmon 2005). We investigated whether ExoU would promote the release of vWF from endothelial cells, thereby contributing to enhanced platelet adhesion/aggregation and potentially to the generation of prothrombotic conditions.

Results

Conclusion