Shiga Toxin Binds Human Platelets via Globotriaosylceramide (P^kAntigen) 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 high-performance thin-layer chromatography (HPTLC) of isolated platelet glycosphingolipids. By HPTLC, Shiga toxin was shown to bind globotriaosylceramide (Gb3) and a minor platelet glycolipid with anRf 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 Gb3 expression. Based on glycosidase digestion and epitope mapping, band 0.03 was hypothesized to represent a novel glycosphingolipid, IV3-β-Galα1-4galactosylglobotetraosylceramide. Based on incidence, structure, and association with increased Gb3 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 Gb3 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.

Blood Groups in Infection and Host Susceptibility

“…NOR red cells express P k -Gb4 derivatives that possess a Gb4 core and a terminal ␣1-4Gal epitope (342) ( Table 7). A related globo-GSL can be observed in 10% of platelet donors (band 0.03; Gal␣1-4 -Gb5) and is associated with increased Gb3 synthesis (350,351). The Forssman antigen is a common globo-GSL in animals (for example, chickens and sheep) but is absent from humans (352).…”

“…The P 1 antigen is red cell specific and is labile during storage at 4°C (341,350). In contrast, Gb3/P k and Gb4/P antigens are widely expressed on tissues, particularly those arising from the embryonic mesoderm (368,369).…”

“…S. dysenteriae always expresses Stx1, whereas EHEC can express either Stx1 or Stx2 (429). In general, Stx recognizes GSLs bearing a terminal galabiose or Gal␣1-4Gal disaccharide (350,436). These GSLs include P 1 , Gb3, galabiosylceramide (Gal 2 Cer), and an extended globo-GSL, "band 0.03."…”

“…The latter is a trace GSL, related to MSGG/LKE, found on some platelet donors (350). Stx1 and Stx2 also display weak binding to Gb4 by thin-layer chromatography, enzyme-linked immunosorbent assays (ELISAs), and surface plasmon resonance (350,437,438). Gb3 is considered the primary physiological receptor for Stx1 and human Stx2 strains (Stx2, Stx2c, and Stx2d).…”

“…Clinically, there is a good correlation between Gb3 expression and disease. The gut epithelium expresses Gb3, although it is a minor GSL (111,350,368). Stx is toxic to colonic epithelial cell lines and can induce colonic secretion in animal models (378,387,433).…”