Shiga Toxin Binds Human Platelets via Globotriaosylceramide (Pk Antigen) and a Novel Platelet Glycosphingolipid

Abstract

Hemolytic-uremic syndrome is a clinical syndrome characterized by acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia that often follows infection by Shiga toxin-or verotoxin-producing strains of Escherichia coli. Because thrombocytopenia and platelet activation are hallmark features of hemolytic-uremic syndrome, we examined the ability of Shiga toxin to bind platelets by flow cytometry and highperformance thin-layer chromatography (HPTLC) of isolated platelet glycosphingolipids. By HPTLC, Shiga toxin was shown to bind globotriaosylceramide (Gb 3) and a minor platelet glycolipid with an R f of 0.03, band 0.03. In a survey of 20 human tissues, band 0.03 was identified only in platelets. In individuals, band 0.03 was expressed by 20% of donors and was specifically associated with increased platelet Gb 3 expression. Based on glycosidase digestion and epitope mapping, band 0.03 was hypothesized to represent a novel glycosphingolipid, IV 3-␤-Gal␣1-4galactosylglobotetraosylceramide. Based on incidence, structure, and association with increased Gb 3 expression, band 0.03 may represent the antithetical Luke blood group antigen. By flow cytometry, Shiga toxin bound human platelets, although the amount of Shiga toxin bound varied in donors. Differences in Shiga toxin binding to platelet membranes did not reflect differences in platelet Gb 3 expression. In contrast, there was a loose association between Shiga toxin binding and decreasing forward scatter, suggesting that Shiga toxin and verotoxins bind more efficiently to smaller, older platelets. In summary, Shiga and Shiga-like toxins may bind platelets via specific glycosphingolipid receptors. Such binding may contribute to the thrombocytopenia, platelet activation, and microthrombus formation observed in hemolytic-uremic syndrome.

Neutrophil Extracellular Traps Induced by Shiga Toxin and Lipopolysaccharide-Treated Platelets Exacerbate Endothelial Cell Damage

“…This central finding prompted us to investigate the mechanism by which Stx2 was potentiating the Plts-mediated stimulation of netosis. Previous reports have described the presence of Gb 3 and also other Stx receptors on Plts and Stx binding to Plts (Tao et al, 1973;Cooling et al, 1998). In our experiments, we found an increase in the Gb 3 receptor on Plts after treatment that was parallel with an increased Stx2 binding.…”

Cell density-induced changes in lipid composition and intracellular trafficking

“…In the human body, the expression of Gb3 is restricted to only several tissues. Normally, the highest levels of Gb3 are found in the kidney epithelium and endothelium [68][69][70], in microvascular endothelial cells [71,72] and in platelets [73]. In the carbohydrate defined P histo-blood group system, Gb3 constitutes the rare P k antigen present on the erythrocytes [74].…”

Many cell types are Shiga toxin targets

“…Stx leads to an increase in Gb3 synthesis, an increase in the also has direct effects on polymorphonuclear cells [13], expressed functional Gb3 receptor and increased cell and there is evidence that erythrocytes and platelets have death after exposure to Stx [7]. receptors for Stx [14]. After intestinal absorption, Stx There is increasing evidence that endothelial cells are must pass through the liver before reaching the systemic not the only targets for Stx in the kidney, and that several circulation.…”