title: Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression.
abstract: Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10 μg/ml, 1 h) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100 μmol/l, 4 h) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.
authors.affiliation: aCritical Illness Research, Lawson Health Research Institute, London, Ontario bDepartment of Physiology and Pharmacology cDepartment of Medical Biophysics dDepartment of Anesthesia and Perioperative Medicine, Critical Care Western, University of Western Ontario, London, Canada.
authors.forename: Dan
authors.initials: D
authors.lastname: Secor
authors.forename: Scott
authors.initials: S
authors.lastname: Swarbreck
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Ellis
authors.forename: Michael D
authors.initials: MD
authors.lastname: Sharpe
authors.forename: Qingping
authors.initials: Q
authors.lastname: Feng
authors.forename: Karel
authors.initials: K
authors.lastname: Tyml
mesh_terms: D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D019007:P-Selectin / Q000378:metabolism*; D010973:Platelet Adhesiveness; D018805:Sepsis / Q000378:metabolism*
chemical_list: D019007:P-Selectin; D001205:Ascorbic Acid