INTRODUCTION

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HUS is a clinical syndrome characterized by acute renal failure, thrombocytopenia, and a microangiopathic hemolytic anemia (70). Although HUS can occur at any age, HUS is particularly common among young children, usually in association with a prodromal diarrheal illness (10). In addition to being the most common cause of acute renal failure in children, HUS is also a cause of chronic renal insufficiency and chronic renal failure in 15 and 5% of affected children, respectively (reference 10 and references therein).

Although multiple etiologies have been implicated in the pathogenesis of HUS (70), there is increasing evidence that most cases of community-acquired or idiopathic HUS may follow infection by certain strains of enterohemorrhagic Escherichia coli or VTEC (3, 10, 21). A commensal organism in the digestive tract of some cattle (42), VTEC can contaminate beef products during commercial meat processing. As a result, there have been several large outbreaks of VTEC-associated illnesses after ingestion of undercooked contaminated beef (22). In one recent outbreak in the Pacific Northwest, more than 700 cases of E. coli-related illnesses, including 55 cases of HUS and 4 deaths, were reported (22). Although the mortality rate from VTEC infection in the latter outbreak was less than 1%, mortality rates as high as 35% have been reported (21). Overall, E. coli is the fourth most common food-related illness in the United States, with an estimated incidence of 10,000 to 20,000 cases per year (3). Costs of enterohemorrhagic E. coli-associated illness, due to medical treatment and lost productivity, are reported to range from 216 and 580 million dollars annually (3). As a result of the threat and cost to public health, there is increasing interest by government agencies and food industry officials in the pathogenesis, treatment, and prevention of VTEC-associated illness.

Previous studies over the last two decades have shown that the causative agent of both E. coli-associated gastroenteritis and HUS is a bacterial toxin (73) known as verotoxin or Shiga-like toxin. Like Stx from Shigella dysenteriae, verotoxin is a multimeric toxin consisting of five identical B subunits (MW, 6,500) and a single, enzymatically active A subunit (MW, 32,000) (73). During infection, Stx and verotoxin bind to cell surface receptors via the toxin B subunit (49), followed by endocytosis of the toxin (81) and proteolytic cleavage of the A subunit. The latter then binds and inactivates rRNA, leading to inhibition of protein synthesis and apoptosis (48, 73).

The receptors for Stx and most verotoxins (Stx1 and Stx2) were previously identified as cell surface GSLs which display a terminal Gal1-4Gal or galabiosyl epitope (15). To date, three GSLs which can potentially serve as a receptor for Stx (Stx1 and Stx2) have been identified (15). These GSLs include galabiosylceramide, a gala family ceramide disaccharide, Gb₃ or the P^k antigen, and the P₁ antigen, a neolacto series pentaosylceramide (Table 1). Stx2e, the causative agent of pig edema disease, recognizes the P blood group antigen (20). Tissue-specific differences in the distribution and relative expression of galabiosylceramide, Gb₃, and P₁ antigens are hypothesized to play a role in the clinical spectrum of illness seen with S. dysenteriae and VTEC-related infections (49, 55, 100). The cells and tissues previously shown to bind verotoxins and Stx via cell surface GSLs include human erythrocytes (8, 49), endothelium (54, 62, 74, 91), and kidney (11).

Stx may also potentially bind human platelets (58). Multiple studies have provided laboratory evidence of widespread platelet activation in patients with HUS (4, 25, 36) including histologic evidence of platelet microthrombi occluding small renal vessels (70, 78). In vitro, bacterial supernatants containing verotoxin were shown to potentiate platelet aggregation (80). Furthermore, Gb₃ was reported to be a major platelet GSL antigen (28, 57, 87). To investigate whether platelets can bind Stx, we examined Stx binding to platelets by two-color immunofluorescence flow cytometry. We also examined Stx binding to platelet neutral GSLs isolated from pooled platelet concentrates and from individual platelet donors.

(Part of this work has been reported as a preliminary communication [28a].)

	MATERIALS AND METHODS

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Abbreviations. BSA, bovine serum albumin; C, chloroform; CDH, lactosylceramide (Gal1-4Glc1-1Cer); Cer, ceramide; FITC, fluorescein isothiocyanate; CV, coefficient of variation; DPA, diphenylamine; Fuc, fucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Gb₃, globotriaosylceramide or P^k blood group antigen (Gal1-4Gal1-4Glc1-1Cer); Gb₄, globotetraosyceramide or P blood group antigen (GalNAc1-3Gal1-4Gal1-4Glc1-1Cer); Glc, glucose; GlcNAc, N-acetylglucosamine; GSL(s), glycosphingolipid(s); HPA, Helix pomatia lectin; HPLC, high-performance liquid chromatography; HPTLC, high-performance thin-layer chromatography; HUS, hemolytic-uremic syndrome; IgM, immunoglobulin M; Le, Lewis; Le^a, Lewis^a (Gal1-3[Fuc1-4]GlcNAc-R); Le^b, Lewis^b (Fuc1-2Gal1-3[Fuc1-4]GlcNAc-R); lyso-Gb₃, lysoglobotriaosylceramide or globotriaosylsphingosine; M, methanol; MAb, monoclonal antibody; MW, molecular weight; PBS, phosphate-buffered saline; PE, phycoerythrin; R, carbohydrate residue; R_f, relative mobility; SSEA-3, stage-specific embryonic antigen (Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer); Stx, Shiga holotoxin; Stx-B, Shiga toxin B subunits; VTEC, verotoxin-producing Escherichia coli.

Tissue preparation. Outdated (72- to 96-h-old), whole-blood-derived, nonapheresis platelet concentrates (1) from volunteer blood donors were purchased from Siouxland Red Cross (Sioux City, Iowa). Outdated, single-donor, apheresis platelet concentrates, erythrocytes (blood group AB+, P₁+, Le^b+ and B+, P₁, Le^a+, Le^b), lymphocyte, monoblast, and granulocyte concentrates were obtained from the DeGowin Blood Center (Department of Pathology, University of Iowa, Iowa City) as previously described (28, 30). From 13 apheresis platelet donors, an erythrocyte sample was also collected for P₁ typing and erythrocyte GSL analysis. Human kidney, aorta, intestine, heart, lung, liver, brain, and cauda equina were obtained from the hospital autopsy service (Robert Schelper and Van Savell, Department of Pathology). Human synovium was obtained from patients undergoing prosthetic joint replacement and were a kind gift from Stanley Naides, John Callahan, and C. Peradones (Departments of Internal Medicine and Orthopedic Surgery, University of Iowa). Human bronchial epithelial cells were obtained at bronchoscopy from paid volunteers and were supplied as isolated neutral GSLs from Doug Hornick, Department of Internal Medicine. Rabbit erythrocyte GSLs were a gift from Jim Greer (Department of Pathology). Signed consent was obtained from apheresis platelet donors at the time of donation. All tissue procurement was in accordance with the institutional human investigation review committee.

GSL isolation. The total neutral GSL fraction of each tissue or cell type was isolated by the procedure of Ledeen and Yu (59) for all tissues and cells, except platelets, erythrocytes, granulocytes, monoblasts, lymphocytes, and plasma, which were isolated by a modified procedure (30, 57). For single-donor apheresis platelets, single-donor whole-blood-derived platelets and individual erythrocyte samples (2 ml of packed erythrocytes), washed and lyophilized cell pellets were extracted in 100 ml of C-M (1:1 [vol/vol]) for 48 h. The total lipid extract was dried, resuspended in 200 ml of C-M-water (30:60:8 [vol/vol]), and applied to a DEAE-Sephadex column (A25, 10-ml bed volume; Sigma, St. Louis, Mo.). Neutral lipids were eluted with 200 ml of C-M-water (30:60:8) [vol/vol]), evaporated in vacuo, saponified with 35 ml of 0.3 N methanolic sodium hydroxide, and dialyzed against distilled water (MW cutoff, 14,000; Spectra-Por, Houston, Tex.). The dialysis retentate was lyophilized dry, resuspended in 45 ml of C and applied to a silicic acid column (40-µm-diameter, 10-ml bed volume; J. T. Baker, Phillipsburg, N.J.). The column was sequentially washed with 100 ml of C and 50 ml of ethyl acetate to remove cholesterols and steroids, respectively. Neutral GSLs were then isolated by batch elution with 50 ml each of acetone-methanol (9:1 and 7:3 [vol/vol]). The last two fractions were pooled, dried, and resuspended in C-M (1:1 [vol/vol]) prior to use.

Preparation of lyso-Gb₃. Lyso-Gb₃ was prepared from Gb₃ (Sigma) as described by Schwarzmann and Sandhoff (84). Briefly, 500 µg of Gb₃ was dissolved in 0.8 M potassium hydroxide in dry methanol and incubated for 20 h at 100°C. The solution was cooled to 20°C, neutralized with acetic acid, dried under nitrogen, and dialyzed against distilled water for 6 h at room temperature. The dialysate was lyophilized dry, resuspended in C-M (1:1 [vol/vol]) and examined by HPTLC as described below.

HPTLC. HPTLC was performed by published procedures (57) using aluminum-backed HPTLC plates (E. Merck, Darmstadt, Germany). Neutral GSLs were spotted onto HPTLC plates and then developed in solvent A (C-M-water, 65:25:4 [vol/vol]) or solvent B (C-M-0.2% aqueous CaCl₂, 55:45:10 [vol/vol]). GSLs were detected by spraying with DPA reagent (Sigma) (59) or by immunostaining as described below. GSL bands were characterized by intensity (percent total staining density) and R_f by scanning densitometry at 370 nm (Schimadzu Instruments, Columbia, Md.). Error in mobility measurements was less than 0.01 unless otherwise stated. The concentrations of neutral GSL spotted per HPTLC lane was 80 to 100 µg for pooled platelets, erythrocytes, lymphocytes, granulocytes, intestine, heart, lung, liver, kidney, synovium, brain, cauda equina, and aorta. For single-donor erythrocyte- or whole-blood-derived, nonapheresis platelet GSL samples (5.5 × 10¹⁰ platelets total) (1), 1/5 of each sample was spotted per HPTLC lane. For single-donor, apheresis platelet concentrates (3.0 × 10¹¹ platelets total) (1), the total neutral GSL was resuspended in 500 µl of C-M (1:1 [vol/vol]) and between 1/100 and 1/50 of each sample was spotted per HPTLC lane. The amount of GSL spotted was normalized relative to the amount of lactosylceramide in each sample, as determined by scanning densitometry (mean area in square millimeters) of DPA-stained HPTLC plates.

Toxins and immunologic reagents. Stx (from S. dysenteriae), isolated Stx-B, and rabbit anti-Stx polyclonal antibody were a kind gift from Arthur Donohue-Rolfe, Tufts University, Boston, Mass. (32). MAb 38.13 (anti-Gb₃ or P^k, rat IgM) (72), PE-labeled anti-platelet glycoprotein IIb/IIIa (MAb P2, CD41; mouse IgG) and a biotinylated anti-rat IgM were purchased from Immunotech, Inc., Westbrook, Maine. Monoclonal antibodies Pk002 (anti-P^k and P₁, mouse IgM) and P001 (anti-globo: anti-P^k, P, P₁>Forssman, SSEA-3; mouse IgM) were purchased from Accurate Chemical and Scientific Corp., San Diego, Calif. (14). Monoclonal anti-blood group P or Gb₄ (MAb MC631, mouse IgM) was purchased as a hybridoma supernatant from the Developmental Studies Hybridoma Bank maintained by the Department of Biology at the University of Iowa, Iowa City, under contract no. N01-HD-2-3144 (51, 52). Monoclonal anti-Forssman (MAb M1/22.25.8; TIB-121, mouse IgM) was purchased from the American Type Culture Collection (Rockville, Md.) and used as a hybridoma supernatant (96). Gal-13 (mouse IgM), an anti-Gal1-3Gal1-4GlcNAc-R MAb (38), was a kind gift from Uri Galili, University of California at San Francisco, San Francisco. SC802, a recombinant P-fimbriated E. coli strain was metabolically labeled with [³⁵S]methionine by the method of Bock et al. (9) and was provided by Steven Clegg and Doug Hornik (Departments of Microbiology and Internal Medicine, University of Iowa). Monoclonal anti-blood group A (Birma-1, mouse IgM) (64), anti-blood group Le^a (LM112/161, mouse IgM) (37), and anti-Le^b (LM119/181) (40) were purchased from Gamma Biologicals, Houston, Tex. Biotinylated anti-mouse IgM, anti-rabbit IgG, avidin-linked alkaline phosphatase (ABC kit) and alkaline phosphatase substrate (SK-5000) were purchased from Vector Laboratories (Burlingame, Calif.).

HPTLC-immunostaining. HPTLC-immunostaining was performed as previously described (18). Briefly, air-dried, solvent-developed HPTLC plates were dipped in a hexane solution of 0.04% poly(isobutyl)methacrylate (Polysciences, Warrington, Pa.) for 60 s and then air dried. The plates were blocked with buffer A (40 mM Tris-HCl, 150 mM NaCl, 1% BSA, 0.1% sodium azide [pH 7.8]) for 45 min. For Stx overlays, blocked plates were incubated with Stx (final concentration, 50 ng/ml) or isolated Stx-B (final concentration, 0.5 µg/ml) in buffer A for 2 h at room temperature. A toxin-free plate was also included as a negative control. Plates were washed with PBS (pH 7.4) and then incubated with rabbit anti-Stx polyclonal antibody diluted in PBS-1% BSA (pH 7.4) (final concentration, 1:5,000) for 1 h. The plates were washed again with PBS, incubated with biotinylated anti-rabbit IgG in PBS-1% BSA (1 h), washed, and then allowed to react with an avidin-linked alkaline phosphatase (Vector) for 30 min. Bound antibody was detected with an alkaline phosphatase substrate (Vector) in cold (4°C) 100 mM Tris-HCl, pH 9.5.

Glycosidase digestion. Platelet neutral GSLs were digested with -galactosidase from green coffee beans (EC 3.2.1.22) by the method of Ariga et al. (5). Briefly, approximately 50 µg of total platelet neutral GSL or 15 µg of Gb₃ (Sigma) was suspended in 100 µl of sodium citrate buffer (100 mM, pH 5.0) containing 10% sodium taurodeoxycholate and 20 µl of -galactosidase (50 U/1.0 ml of 3.2 M ammonium acetate; Boehringer Mannheim, Indianapolis, Ind.). The mixture was incubated for 24 h at 37°C. The reaction was terminated by the addition of 100 µl of C-M (1:1 [vol/vol]) and dried under nitrogen. For analysis, samples were resuspended in C-M (1:1 [vol/vol]), spotted onto HPTLC plates, and examined by DPA and HPTLC-immunostaining with Stx. An enzyme-free, buffer control was also included.

Fluorescein labeling of Stx-B. For flow cytometry, Stx-B were labeled as previously described (8). Briefly, 100 µg of lyophilized Stx-B was resuspended in 100 µl of sodium bicarbonate buffer (0.5 M, pH 9.5) containing 2 mg of FITC (isomer I; Sigma) per ml and incubated for 2 h at 25°C in the dark. Unbound FITC was removed by filter centrifugation (Centricon 30; Amicon, Beverly, Mass.). FITC-labeled Stx-B were resuspended in 1 ml of PBS-1% BSA (pH 7.4), aliquoted, and stored at 20°C until use. The toxin-free filtrate was diluted 1/5 with PBS-1% BSA and saved as a negative control.

Flow cytometry. Whole-blood-derived, single-donor platelet concentrates from 16 blood group A and two blood group O donors were leukodepleted by differential centrifugation (350 × g, 15 min), and the resulting platelet-rich supernatant was counted on a Coulter counter (model Z; Coulter, Hialeah, Fla.). For flow cytometry, one million platelets were incubated with 5 µl of PE-labeled anti-platelet glycoprotein IIb/IIIa (MAb P2; CD41), 20 µl of FITC-labeled Stx-B (100 µg/ml stock), and PBS buffer (PBS with 1% BSA, 0.2% EDTA, and 0.1% sodium azide [pH 7.4]) in a final volume of 100 µl for 2 h at 4°C. A toxin-free FITC filtrate was also included as a negative control. For some samples, platelets were stained in parallel with MAb P2 and 5 µl of FITC-labeled HPA (10 µg/ml working dilution; Sigma). Platelets were washed twice with PBS buffer (1,000 × g, 10 min) and resuspended in 400 µl of PBS-1% paraformaldehyde. All samples were performed in duplicate and analyzed on a Becton Dickinson 440 flow cytometer equipped with an argon laser. A minimum of 10,000 events were measured per sample. FITC and PE spectral overlaps were corrected with electronic compensation. Data was collected on a VAX station 3200 computer equipped with DESK software (kindly supplied by Wayne Moore, Stanford University). For histograms, platelets were gated on forward scatter, orthogonal scatter, and MAb P2 positivity and the percent platelets positive for Stx-B or HPA was determined. Results were recorded as the mean (n = 2) percent platelets positive for CD41 and Stx-B. Final graphic output was performed with Canvas software for the MacIntosh computer.

Serology. In apheresis platelet donors, an erythrocyte sample (5 ml) was collected for erythrocyte phenotyping. To type donors for the P₁ and P₂ phenotypes, a drop of 2% erythrocyte suspension was incubated with 2 drops of human polyclonal anti-P₁ antisera (Gamma Biologics) for 15 min at 4°C according to the manufacturer's instructions. Donors with erythrocytes which agglutinated with anti-P₁ were considered P₁ positive. Donors with erythrocytes which failed to agglutinate were considered to be of the P₂ phenotype. The latter was confirmed by isolating the erythrocyte neutral GSLs from P₂ donors and demonstrating the presence of both P^k and P antigens (67).

Statistics. Platelet GSL expression was compared by using a two-tailed Student t test, Mann-Whitney U-test, Spearman rank sum, and Pearson product-moment correlation (17). A P of <0.05 was considered statistically significant.

	RESULTS

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Stx binds Gb₃ and a novel GSL in human platelets. Gb₃ or the P^k blood group antigen, the physiologic receptor for Stx on human umbilical vein endothelium (62, 74, 75, 92), rabbit intestinal mucosa (49), Vero (61), HeLa (49), MDCK (82), and Daudi cells (26), was previously reported to be present on human platelets (28, 57, 87). To confirm the latter, we isolated the total neutral GSL fraction from pooled, type-specific platelet concentrates from more than 100 whole-blood donors. Following isolation, the total platelet neutral GSL fraction was separated by HPTLC and chemically stained with DPA reagent, which nonspecifically reacts with the oligosaccharide moiety on GSLs (59).

View larger version (68K): [in this window] [in a new window]   FIG. 1.   Stx binding to platelet neutral GSLs. The total neutral GSL fraction of human platelets was spotted onto HPTLC plates (80 µg per HPTLC lane). GSLs were visualized chemically with DPA (lane 1) or by HPTLC-immunostaining with Stx (lane 2), Stx-B (lane 3), or toxin-free, buffer control (lane 4). Major DPA and Stx-positive GSL bands were characterized by their mobility relative to that of the solvent front. Rfs are shown to the right of the gel. GSLs were separated in solvent A. OR, origin.

Band 0.03 is a platelet-specific glycolipid. To determine whether Stx band 0.03 was specific for human platelets or was expressed by other human tissues, we screened the neutral GSL fraction from 20 human tissues for GSLs capable of binding Stx. Tissues of interest included hematopoietic cells as well as several tissues potentially affected in HUS and thrombotic thrombocytopenia purpura. On HPTLC-immunostaining, Stx recognized four GSL species with R_fs of 0.40, 0.25, 0.06, and 0.03 (Table 3). Stx band 0.25 was identified in the vast majority (17 of 20) of tissues screened, whereas Stx bands 0.40, 0.06, and 0.03 showed marked tissue restriction: Stx band 0.40 was identified only in kidney and small bowel mucosa, Stx band 0.06 was specifically expressed in P₁ (but not P₂) erythrocytes, and (Stx) band 0.03 was identified only in the GSL fraction of human platelets. Band 0.03, therefore, appeared to represent a potentially platelet-specific GSL.

Band 0.03 is a novel Stx-binding GSL, distinct from galabiosylceramide, Gb₃, and P₁ antigens. The ability of Stx bands 0.40, 0.25, 0.06, and 0.03 to bind Stx suggested that these GSLs, by definition, possessed a terminal Gal1-4Gal-R or galabiosyl epitope. In order to identify and compare these GSLs, the neutral GSL fractions from platelets (Stx bands 0.25 and 0.03), kidney (Stx bands 0.40 and 0.25), and P₁ erythrocytes (Stx bands 0.25 and 0.06) were immunostained with several anti-globo MAbs and lectins with overlapping epitopes (Table 4). Additional controls included rabbit erythrocytes and commercially available Gb₃, Gb₄, and Forssman antigen GSL standards.

View larger version (70K): [in this window] [in a new window]   FIG. 2.   GSL specificity of Stx, MAb Pk002, and MAb 38.13. The total neutral GSL fraction from pooled human platelets (lane 1) and a P1+ erythrocyte control (lane 2) were spotted onto HPTLC plates and immunostained with Stx (ST), MAb Pk002 (PK002) (anti-Pk and anti-P1), or MAb 38.13 (anti-Pk only). Numbers to the left are the Rfs of the MAb or Stx-positive GSL bands. The apparent binding of Stx to Gb4 and MAb 38.13 to CDH and GlcCer (near top of plate) represents nonspecific binding. GSLs were separated in solvent A.

Band 0.03 is not lyso-Gb₃. Although it appeared that band 0.03 was unrelated to Gb₃, we could not exclude the possibility that band 0.03 was a lyso-Gb₃: Lyso-Gb₃ has a much slower mobility by HPTLC (R_f 0.04) than Gb₃ has (R_f, 0.25) due to the loss of an N-acyl fatty acid moiety. To determine if band 0.03 was lyso-Gb₃, lyso-Gb₃ was prepared from Gb₃ as previously described (84) and then immunostained with MAb 38.13. Unlike band 0.03, Gb₃ and lyso-Gb₃ were recognized by MAb 38.13 (data not shown).

Glycosidase digestion of band 0.03. To help confirm that band 0.03 was a Gal1-4Gal-R-Ceramide, the total neutral platelet GSL fraction was incubated with -galactosidase, an exoglycosidase specific for terminal -linked galactose residues (5, 31). As shown in Fig. 3, digestion of Gb₃ with -galactosidase (lane 2) resulted in a 77% conversion of Gb₃ to CDH on DPA (Fig. 3A), with a concomitant loss of Stx binding by HPTLC-immunostaining (Fig. 3B) compared to the buffer control (lane 1). Similarly, digestion of platelet neutral GSL with -galactosidase resulted in a complete loss of Stx binding to band 0.03 (Fig. 3B, lane 4).

View larger version (60K): [in this window] [in a new window]   FIG. 3.   Sensitivity of band 0.03 to -galactosidase. The total platelet neutral GSL fraction (lanes 3 and 4) and a Gb3 standard (lanes 1 and 2) were incubated with buffer only (lanes 1 and 3) or -galactosidase (lanes 2 and 4). For analysis, GSLs were separated on HPTLC plates (solvent A) and visualized with DPA (A) or by HPTLC-immunostaining with Stx (B).

Band 0.03 is a hexa- to octaosylceramide. Because band 0.03 appeared to be at least a hexosylceramide based on its mobility relative to that of the P₁ antigen, we compared the mobility of band 0.03 in two different solvents relative to other blood group-active GSL antigens present in the neutral GSL extract of platelets (Table 2) (29, 46). Based on R_f alone, band 0.03 appeared to be a hexa- to octaosylceramide. Because of the generally greater mobility of fucosylated GSLs (12), the lower mobility of band 0.03 relative to blood group A (A-6) and Lewis GSLs (Le^b-6) in solvent B may overestimate the size of the oligosaccharide moiety on band 0.03.

Gb₃ and band 0.03 expression by individual platelet donors. In an effort to determine whether Gb₃ and/or band 0.03 expression differed in individuals, we examined the neutral GSL extracts isolated from 25 individual, single-donor apheresis platelet donors. As shown in Table 5, the donors ranged from 19 to 56 years of age and included nearly equal numbers of male (n = 14) and female (n = 11) donors. With the exception of blood groups O and B, the distribution of ABO types among our sample of platelet donors was similar to the expected distribution of ABO phenotypes among Caucasian donors (2). As before, the total platelet neutral GSL fraction from each individual donor apheresis platelet concentrate was isolated and examined by scanning densitometry of DPA-stained HPTLC plates (Table 5).

Platelet band 0.03 is related to platelet CDH and Gb₃ expression. To determine whether there was any relationship between band 0.03 expression and other globo series GSLs, we compared the mean expression of CDH, Gb₃, and Gb₄ in band 0.03-positive and -negative platelet donors. Differences between band 0.03-positive and -negative donors were considered statistically significant if the P was <0.05 by the Student t test or the Mann-Whitney U-test. As shown in Table 5, the percent Gb₃ and the Gb₃/CDH ratio were significantly higher in band 0.03-positive donors. In contrast, there was no association between band 0.03 and the percent Gb₄ or the Gb₃/Gb₄ ratio. The increased percent Gb₃ and Gb₃/CDH ratio suggest a possible increased conversion of CDH to Gb₃ in band 0.03 donors.

Platelet band 0.03 expression is not related to donor P₁ or P₂ phenotype. The frequency of band 0.03 (20%) in our sample of 25 platelet donors suggests that band 0.03 may be related to a P₂ (P₁-negative) erythrocyte donor phenotype. Among Caucasian blood donors, the P₂ phenotype is observed in approximately 21% of donors (2). Furthermore, studies of GSLs isolated from P₁ and P₂ donor erythrocytes have shown an increase in the mean percent Gb₃ and the Gb₃/CDH ratio in P₂ erythrocytes (35). Of 13 donors for which an erythrocyte sample was available, 11 (85%) donors were P₁ positive and 2 (15%) were P₁ negative or P₂ (Table 5).

Stx binding to platelets is unrelated to the relative fraction of Gb₃. To determine whether individual differences in Gb₃ and band 0.03 expression affected the ability of Stx to bind circulating platelets, we compared platelet GSL expression to Stx binding in 18 blood donors by HPTLC-immunostaining and two-color immunofluorescence flow cytometry. For flow cytometry, whole-blood-derived platelet concentrates from 16 type A and 2 type O blood donors were incubated with FITC-labeled Stx-B and a PE-labeled anti-platelet glycoprotein IIb/IIIa MAb (CD41; MAb P2). For a negative control, platelets were incubated with a toxin-free FITC filtrate. Platelets were gated on CD41 positivity, forward scatter, and orthogonal scatter. Results were reported as the percent platelets positive for anti-CD41 and Stx-B (Table 6). For GSL analysis, the remaining platelet concentrate was washed, lyophilized, and isolated as described before. Isolated GSLs were analyzed by scanning densitometry of DPA-stained HPTLC plates.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   Stx and HPA binding to platelets is related to platelet size. The mean (FITC) fluorescence intensity of HPA and Stx binding relative to platelet forward scatter (x axis) was plotted for platelets from five donors. As shown, there was an increase in Stx and decrease in HPA binding with decreasing forward scatter. Mean fluorescence intensity represents the mean of two independent samples per lectin (HPA and Stx) per donor (standard error of mean = 2.4 [17]).

	DISCUSSION

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As previously reported (57, 87), Gb₃ is a major neutral GSL of human blood platelets and can be shown to bind Stx by HPTLC-immunostaining. Platelets also express a second Stx receptor, band 0.03. The latter appears to represent a potentially novel Stx-binding GSL, immunologically distinct from either Gb₃, P₁, or galabiosylceramide. Furthermore, band 0.03 may be platelet specific: in a survey of 20 human tissues, band 0.03 is identified only in platelets. In contrast, Gb₃, the putative physiologic receptor for Stx, is ubiquitously expressed in most tissues arising from the embryonic mesoderm (28).

In HUS (10, 19), Stx binding to either Gb₃ or band 0.03 could contribute to microthrombus formation (70, 78), thrombocytopenia, and platelet activation (4, 25, 36), possibly through induction of the sphingomyelin/ceramide signal transduction pathway (43, 99). Evidence supporting Stx-induced platelet activation includes a study by Rose et al. (80) which showed an increase in agonist-induced platelet aggregation after incubation of plasma with verotoxin. Stx binding to platelet membranes may also contribute to thrombocytopenia via splenic removal of toxin-coated platelets.

We also observed a reasonably good correlation between clinical disease and the expression of Stx receptors on other tissues. Both renal failure (11) and toxin-mediated diarrhea (49, 50, 55, 65) could reflect Stx binding to Gb₃ and/or galabiosylceramide (Stx band 0.40) on kidney and small intestinal mucosa. On vascular endothelium, Stx binding to Gb₃ is believed to play a critical role in the pathogenesis of VTEC-associated HUS and thrombotic thrombocytopenic purpura through upregulation of vascular addressins such as E- and P-selectin (68), decreased synthesis of tissue plasminogen activator and inhibitor (90), and endothelial cell death (54, 62, 74, 91, 92) with exposure of the thrombogenic subendothelium. On monocytes, Stx binding can lead to a release of inflammatory cytokines such as tumor necrosis factor alpha (56, 77, 93, 94). Tumor necrosis factor alpha and other inflammatory cytokines may further enhance Stx-mediated cell injury via cytokine-induced increases in Gb₃ synthesis (54, 75, 77, 91-94). On erythrocytes, Stx binding to Gb₃ and P₁ antigen may act synergistically with RTX toxin to promote erythrocyte hemolysis (6-8). Likewise, Stx binding to Gb₃ on other tissues (liver, heart, and lung) could contribute to the etiology of thrombotic thrombocytopenia purpura (76, 97) and multisystem organ failure sometimes seen with VTEC infections (10).

Like previous investigators (87), we initially examined GSLs isolated from a large pool of platelet donors. However, a number of small studies have suggested normal biologic variability in GSL expression in individuals. In a small study of two apheresis platelet donors, Holgersson et al. (46) demonstrated clear, donor-specific differences in platelet globo-GSL expression. Likewise, Fletcher et al. (35) noted differences in erythrocyte Gb₃ and CDH expression which were related to the donor P₁ or P₂ phenotype. In human kidney (11), Gb₃ levels can reportedly vary from 0.254 to 0.896 nmol of Gb₃/mg of kidney cortex. Likewise, Obrig and colleagues (62, 75) observed marked differences in Stx-induced endothelial cytotoxicity among different endothelial cell lines, possibly reflecting a difference in Gb₃ expression by different donors.

To determine whether individuals may differ in the number and/or type of Stx receptors expressed on platelet membranes, we isolated and examined the GSLs from 25 platelet donors. In agreement with Holgersson's (46) findings, there is significant heterogeneity in the relative expression of the three major platelet neutral GSLs in individual donors, particularly for Gb₃. Band 0.03 expression also varied in donors and may represent a low-frequency platelet alloantigen. Although there may be a relationship between donor P₁ or P₂ phenotype and Gb₃ expression on platelets, there is no correlation between a P₂ phenotype and band 0.03 expression. There is, however, a significant correlation between band 0.03 expression and increased percent Gb₃ and Gb₃/CDH ratios. The latter may suggest a possible association between Gb₃ and band 0.03 synthesis.

In erythrocytes (8) and vascular endothelium (91, 92), differences in Stx receptors have been shown to correlate with Stx binding and cytotoxicity. To determine whether the same were true for platelets, we compared platelet Gb₃ content with the binding of Stx to intact platelet membranes by flow cytometry and HPTLC. As expected, there are notable differences in both platelet Gb₃ content and Stx binding between donors. In individual donors, however, there is no correlation between Gb₃ expression and the ability of Stx to bind platelets (percent Stx-positive platelets).

Interestingly, there is a loose association between Stx binding and platelet size. Specifically, mean fluorescence intensity (FITC-Stx-B) tends to increase with decreasing forward scatter. Since forward scatter is related to cell size (85), these results suggest that Stx binds with greater efficiency to smaller and older platelets (79), possibly reflecting a decrease in the platelet glycocalyx as platelets age. Evidence for the latter includes parallel experiments in which type A platelets were labeled with HPA, a lectin specific for the blood group A antigen (86). In contrast to Stx, there is a parallel decrease in forward scatter and HPA fluorescence. Because a large portion of blood group A antigen is expressed on platelet glycoproteins (46, 83), this decrease in HPA positivity would support a decrease in platelet glycoproteins with decreasing platelet size (and increasing platelet age). Similar findings have been reported for aging erythrocytes (34).

This age-associated decrease in platelet glycoproteins may be important in the pathophysiology of Stx infections. Due to their size and proximity to the cell membrane, short-chain GSLs are frequently cryptic antigens on the surfaces of many cells (41, 95). As a consequence, many GSL antigens must be exposed through enzymatic modification of cell membranes by proteases or neuraminidase (41). This was specifically demonstrated for Gb₃ in ARH77 cells (95) and MDCK cells (82). In the latter study, treatment of MDCK cells with either pronase or neuraminidase resulted in a nearly twofold increase in Stx binding. Unmasking of GSL receptors on platelets and other tissues may also occur in vivo. At least two studies have reported the identification of cysteine proteases in the sera of patients with HUS and thrombotic thrombocytopenia purpura (33, 69).

Although we were able to demonstrate the presence of two Stx receptors on platelets, only Stx band 0.25 or Gb₃ was positively identified. Gb₃ was identified by both physical and immunologic methods including HPLC, HPTLC, HPTLC-immunostaining, and one- and two-dimensional nuclear magnetic resonance of the isolated GSL (29). In contrast, the low incidence of band 0.03 (20% of donors), coupled with the fact that band 0.03 was a minor platelet GSL, even in band 0.03-positive donors, made isolation of band 0.03 for biophysical studies difficult. As a consequence, the structure of band 0.03 was deduced by glycosidase digestion and epitope mapping with a series of anti-globo MAbs and lectins with differing specificities.

In summary, band 0.03 is positive with Stx, E. coli SC802, and MAb P001, consistent with the presence of a terminal galabiose epitope. In addition, Stx binding to band 0.03 is sensitive to both -galactosidase and glycan ceramidase, also consistent with a R-Glc1-1'Cer GSL terminating in an -linked Gal (31, 60). Band 0.03 is negative with MAbs 38.13, Pk002, and Gal-13, suggesting that band 0.03 is a Gal1-4Gal-R-Cer, where R is an oligosaccharide not beginning with Glc or GlcNAc. Therefore, R may be an oligosaccharide beginning with either a Gal or GalNAc. In support of the latter, band 0.03 is weakly positive with MAb MC631, a MAb which recognizes globoside (P antigen) as well as extended globo-series GSLs such as the SSEA-3, SSEA-4, globo-H, and Forssman antigens (13, 51, 52). Finally, a comparison of band 0.03 with other platelet (Table 2) and erythrocyte (P₁; R_f, 0.06) GSLs suggest that band 0.03 is a hexa- to octaosylceramide. The proposed structure for band 0.03, based on available data, is a ceramide hexosylceramide with a globoside or type 4 chain oligosaccharide core and a terminal galabiosyl epitope (IV³--Gal1-4Gal-Gb₄) (Fig. 5).

View larger version (11K): [in this window] [in a new window]   FIG. 5.   Proposed structure of platelet band 0.03 based on enzyme digestion and epitope mapping with a series of anti-globo series MAbs and lectins.

The incidence, as well as the structure of band 0.03, suggests that band 0.03 may be related to the Luke blood group antigen. A high-frequency antigen on human erythrocytes, the Luke blood group antigen is an extended globo-series ganglioside related to the P blood group family (see below) (67, 89). In studies of volunteer blood donors, Luke antigen can be detected on 98% of donors, with 80% of donors having a strong Luke phenotype and 18% of the donors with a weak Luke phenotype (66, 89). A Luke-negative phenotype is observed in 0.2 to 2% of P₁ and P₂ donors (16, 66, 89). A Luke-negative phenotype is also seen in the rare p (Gb₃, Gb₄, P₁) and P^k (Gb₃+, Gb₄) phenotypes, reflecting the absence of globo-GSL precursors necessary for the synthesis of the Luke blood group antigen (67).

Interestingly, studies of families of Luke-negative donors show a reciprocal relationship between expression of the P^k and Luke antigens (16). Specifically, donors lacking Luke antigen have significantly increased P^k expression, as determined by erythrocyte agglutination. This apparent increase in P^k was suggested to reflect the presence of a P^k-like GSL, related to Luke antigen, on Luke-negative erythrocytes (88). As stated by Mollison et al. (67) "...it has been postulated that the terminal neuraminic acid of the hypothetical Luke structure could be replaced by galactose in such individuals, giving rise to a Gal1-4Gal-globoside structure which could react with anti-P^k." The latter GSL is essentially identical to that postulated for band 0.03. (Luke antigen, NeuAc1-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1'Cer; antithetical Luke antigen, Gal1 - 4Gal1 - 3GalNAc1 - 3Gal1-4Gal1-4Glc1-1'Cer.) The incidence of the band 0.03 phenotype (20%) suggests that band 0.03 may be expressed on platelets of weak Luke and Luke-negative donors.

The discovery of a potentially novel P^k-like GSL, related to the Luke antigen system, may have important implications in the diagnosis, prognosis, and pathogenesis of VTEC-associated HUS. Unlike Gb₃ or the P^k antigen, which is ubiquitously expressed by most tissues arising from the embryonic mesoderm (28), the Luke antigen has been identified in only a few tissues and cell types. In addition to erythrocytes (89), Luke antigen has been identified in teratocarcinoma (51, 52), endothelium, smooth muscle (39), dorsal root ganglion (45), platelets (29), and human kidney (53). In Luke-negative or weak Luke individuals, therefore, band 0.03 or an "antithetical" Luke antigen may serve as an additional and, due to its size, more accessible receptor for Stx in vascular endothelium, erythrocytes, platelets, and kidney.

Luke-negative and weak Luke antigen donors could also be at increased risk from Stx-mediated disease due to a constitutive increase in Gb₃ expression on target cell membranes (16, 27). Previously, Newburg et al. (71) demonstrated an association between VTEC-associated HUS and Gb₃ levels in human erythrocytes. In endothelial cells, cytokine-stimulated increases in Gb₃ synthesis and expression are associated with increased Stx cytotoxicity (54, 62, 75, 82, 91-93). Luke-negative and weak Luke individuals, therefore, may have an inherited increased sensitivity and susceptibility to Stx due to increased membrane Gb₃ expression, analogous to the effects of inflammatory cytokines. If true, a negative or weak Luke phenotype may represent a previously unrecognized risk factor in Shigella and VTEC infections.

In summary, we have shown that Stx can bind human platelets. Since the Gal1-4Gal disaccharide linkage essential for Stx binding is expressed only on GSLs (98), Stx presumably bind platelets via GSL receptors on the platelet cell membrane. Platelets possess two GSL receptors for Stx, Gb₃, and a novel GSL, band 0.03. Preliminary data suggest that the latter may represent the antithetical Luke antigen and, as such, may serve as a fourth GSL receptor for Stx on platelets, kidney, endothelium, and erythrocytes. Binding of Stx to platelets may play a role in the etiology of thrombocytopenia in S. dysenteriae (58) and VTEC infections (10) through either platelet activation or splenic removal of toxin-coated platelets.