Human platelets express gangliosides with LKE activity and ABH blood group activity.

BACKGROUND: Platelets express several neutral glycosphingolipids with ABH and P blood group activity that may play a role in infectious, autoimmune, and alloimmune thrombocytopenia. In RBCs, sialylated glycosphingolipids or gangliosides with blood group activity have also been reported. To determine whether similar antigens are expressed by platelets, the total platelet ganglioside fraction was isolated and screened for blood-group-active glycosphingolipids. STUDY DESIGN AND METHODS: Platelet gangliosides were isolated by organic extraction, base hydrolysis, anion exchange, silicic acid, and high-performance liquid chromatography. Gangliosides were identified and characterized by high-performance thin-layer chromatography-immunostaining with blood group-specific MoAbs and glycosidase digestion. RESULTS: Group A, but not group O, platelets express five gangliosides with group A activity. Of five A MoAbs and lectins examined, only MoAbs Birma-1 and MHO4 recognized all five sialyl A bands. The sialyl A bands were sensitive to endoglycoceramidase and neuraminidase. One sialyl A band may represent a branched ganglioside with sialyl-I and group A activity. Platelets also express an LKE-active ganglioside consistent with sialyl-galactosylgloboside. CONCLUSION: In addition to sialyl-iI and sialyl-Le(x) gangliosides, group A platelets express gangliosides with LKE activity and group A activity. Like RBCs, group A-active gangliosides may act as alloantigens and autoantigens to naturally occurring isohemagglutinins.

Evidence of a phospholipid binding species within human fibrinogen preparations.

Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent Kd=5 microM, Bmax=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3-6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143-210 in the alpha chain, and positions 59-77 and 101-139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.

Shiga toxin binds human platelets via globotriaosylceramide (Pk antigen) and a novel platelet glycosphingolipid.

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 an Rf 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-beta-Galalpha1-4galactosylglobotetraosylceramide. 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.

The specific hospital significantly affects red cell and component transfusion practice in coronary artery bypass graft surgery: a study of five hospitals.

BACKGROUND: Interhospital differences in blood transfusion practice during coronary artery bypass graft (CABG) surgery have been noted, but the underlying issues have not been identified. STUDY DESIGN AND METHODS: Records of 3217 consecutive CABG cases in five university teaching hospitals in 1992 and 1993 were stratified by hospital, type of revascularization conduit, patients' sex, and other factors. Statistical methods were used to compare patient characteristics, transfusion outcomes, and hospital outcomes. RESULTS: Forward two-step logistic regression using patient likelihood of red cell transfusion factors in the first step and the specific hospital in the second step revealed a significant effect of hospital on the delta odds ratios for red cell transfusion. This finding was confirmed by analyses of a highly stratified subset of cases, males in diagnosis-related group 107 (primary cases of coronary bypass without coronary catheterization) who underwent revascularization with venous and internal mammary artery grafts, revealing variations among hospitals from 109 to 457 units of red cells transfused per hundred cases. Corresponding variations in transfusions of all blood components were from 324 to 1019 units by hospital. Variation in red cell transfusion practice among surgeons in the same hospital was not responsible for these interhospital differences. CONCLUSION: The effect of the specific hospital on transfusion practice is attributed to institutional differences that, through reasons of training or hierarchy, become ingrained in hospitals.

Determinants of red cell, platelet, plasma, and cryoprecipitate transfusions during coronary artery bypass graft surgery: the Collaborative Hospital Transfusion Study.

BACKGROUND: Very little is known about the determinants of blood transfusions in patients undergoing coronary artery bypass graft surgery. STUDY DESIGN AND METHODS: To identify factors that influenced the transfusion of red cells, platelets, plasma, and cryoprecipitate, statistical methods were used to study 2476 consecutive diagnosis-related group 106 and 107 patients in five teaching hospitals who underwent coronary artery bypass surgery between January 1, 1992, and June 30, 1993. RESULTS: The likelihood of red cell transfusion was significantly associated with 10 preoperative factors: 1) admission hematocrit, 2) the patient's age, 3) the patient's gender, 4) previous coronary artery bypass surgery, 5) active tobacco use, 6) catheterization during the same admission, 7) coagulation defects, 8) insulin-dependent diabetes with renal or circulatory manifestations, 9) first treatment of new episode of transmural myocardial infarction, and 10) severe clinical complications. Platelet and/or plasma transfusions were strongly associated with the dose of red cells transfused. Transfusion requirements and other in-hospital outcomes were associated with patient characteristics, surgical procedure (reoperation vs. primary procedure), and the conduits used for revascularization (venous graft only, venous and internal mammary artery graft, or internal mammary artery graft only). Blood resource use and donor exposures were evaluated with respect to the risk to patients of contracting hepatitis C virus and human immunodeficiency virus infections. CONCLUSION: The classification of coronary artery bypass graft patients on the basis of attributes known preoperatively and by conduits used yields subsets of patients with distinctly different transfusion requirements and in-hospital outcomes.

Multiple glycosphingolipids determine the tissue tropism of parvovirus B19.

Infection with human parvovirus B19, the etiologic agent of fifth disease, is associated with numerous hematologic and nonhematologic complications. Recently, the receptor for parvovirus B19 was reported to be globoside (Gb4), a neutral glycosphingolipid (GSL) of red cell membranes. To ascertain if tissue Gb4 expression correlates with B19-associated disease, neutral GSLs from 16 human tissues were isolated and analyzed using high-performance thin-layer chromatography and immunostaining with anti-Gb4 monoclonal antibodies or B19 empty capsids. Gb4 was identified as a major neutral GSL in 11 tissues, especially in those of mesodermal origin. In addition to recognizing Gb4, B19 capsid bound to several tissue-specific GSLs, including two complex globo series GSLs (SSEA-3, SSEA-4) and paragloboside (neolactotetraglycosylceramide), as was demonstrated in red cell, granulocyte, kidney, liver, and bowel tissue. There was good correlation between tissue-neutral GSL expression, B19 capsid binding, and the tissue tropism observed clinically in B19 parvovirus-associated disease.

Detection in human blood platelets of sialyl Lewis X gangliosides, potential ligands for CD62 and other selectins.

Activated platelets are known to express P-selectin, a lectin-like adhesion receptor (CD62), through which they bind to sialyl Lewis X (sLex) ligands displayed on the membranes of leukocytes. To determine whether direct platelet-platelet interactions via P-selectin/sLex interactions are also possible, we have examined the ganglioside extract of human blood platelets for the presence of sLex ligands. Using the sensitive method of high-performance thin-layer chromatography (HPTLC)-immunostaining with the monoclonal antibody (mAb) CSLEX or with sialidase followed by mAbs MC480 or PM81, eight sLex bands were demonstrated at Rf 0.01, 0.03, 0.05, 0.06, 0.08, 0.10, 0.14 and 0.21 in the solvent 45:55:10 chloroform-methanol-aqueous 0.02% CaCl2. The sensitivity of all eight bands to sialidase or endoglycoceramidase confirmed that they were gangliosides. Comparison of the HPTLC mobilities and densities of platelet bands with those from five other human tissues (granulocytes, monoblasts, kidney, aortic endothelium and erythrocytes) in three different solvents revealed three major bands associated with platelets: 3 (Rf0.03), 6 (0.08) and 14 (0.21). Platelet bands were demonstrated not to have resulted from granulocyte contamination. Partial purification of platelet sLex gangliosides by high-performance liquid chromatography and their reaction with 14 oligosaccharide-specific mAbs (FH4, FH5, LM112-161, LM119-181, A5, 1B2, BR55-2, BE2, ES4, MC631, MH04, SH34, P001 and MC813-70) revealed that band 6 is a multifucosylated neolacto ganglioside and band 14 is a branched, disialo neolacto fucoganglioside. Platelet band 3 combined the features of both bands 6 and 14, and reacted differently than granulocyte band 3. These partial structures resemble gangliosides associated with adhesion in other cell systems. It is concluded that platelets express tissue-specific sLex gangliosides (sLex ligands). Thus, it is possible that platelet-platelet binding may be mediated at least partially through P-selectin/sLex interactions, especially after platelet activation.

Concurrent comparison of the safety of paid cytapheresis and volunteer whole-blood donors.

BACKGROUND: Historically, paid blood donors were found to transmit hepatitis at higher rates than volunteers. In those older studies, paid donors frequently were recruited from prisons or slum areas--a finding consistent with the belief that monetary payment in itself did not necessarily lead to the high-risk status of commercial blood. Instead, it was the population base from which the donors were recruited that was important. STUDY DESIGN AND METHODS: Today, cytapheresis donors are in great demand. Because payment is one incentive that might entice donors to undertake the increased commitment of repeated cytapheresis donation, the results were studied of infectious disease history and laboratory testing performed concurrently in 917 volunteer whole-blood donors and 1240 paid cytapheresis donors, who were enrolled in distinct programs at the DeGowin Blood Center from October 7, 1987, through November 30, 1990. RESULTS: When first, repeat, and overall donations made by these donors were evaluated separately, paid cytapheresis donors were found to exhibit no increase in infectious disease history or test results beyond those of volunteer whole-blood donors. CONCLUSION: Thus, paid cytapheresis donors, when managed within a formal program, should not necessarily be presumed to be more dangerous than volunteers, from an infectious disease aspect. However, definitive proof of safety (comparison of transfusion-transmitted infection rates in two groups of patients receiving blood components exclusively from either paid cytapheresis or volunteer donors) was not pursued by long-term follow-up studies.

Glycosphingolipid inhibition of the adhesion of thrombin-activated platelets to surfaces is potentiated by albumin.

Previous studies have shown that exogenous glycosphingolipids (GSLs) inhibit the adhesion of thrombin-activated platelets (TAP) to polystyrene plates coated with various RGD-ligands (where RGD is the peptide sequence Arg-Gly-Asp), suggesting that GSLs can modulate the platelet integrin receptor glycoprotein IIb-IIIa. However, albumin was always used as a plastic surface-blocking agent in these studies. In order to evaluate the role of albumin in these experiments, we studied the effect of various GSLs and albumin on the interaction between TAP and hydrophobic surfaces in a solid-phase assay using indium-111-labelled platelets and polystyrene plates. TAP (10(8) platelets/ml) adhered to polystyrene (half-saturation time 40 +/- 3 min) with a maximal adhesion density of 56 +/- 1 x 10(3) platelets/mm2. Platelet adhesion was only slightly affected (< 11% inhibition) by immobilized bovine serum albumin, immobilized mixed bovine brain gangliosides (MBG) or fluid-phase MBG. In contrast, fluid-phase MBG was an effective inhibitor of platelet adhesion to polystyrene (> 46% inhibition), but only after albumin was first immobilized to the plate. Covering albumin-coated polystyrene with MBG, followed by washing, was as effective as fluid-phase MBG at inhibiting platelet adhesion, thus indicating that a ganglioside-albumin interaction at the polystyrene surface was responsible for effective inhibition. When purified GSLs were substituted for MBG, it was found that all those tested (GT1b, GD1a, GM1, asialo GM1 and globoside) had similar inhibitory activity.

The 3G11+ antigen, a marker for murine CD4+ TH1 lymphocytes, is a ganglioside.

Two monoclonal antibodies (mAbs), SM3G11 and SM6C10, can be used to discriminate between functionally distinct murine CD4+ T cell subsets. In this study we use high-performance thin-layer chromatography and immunostaining techniques to show that the 3G11 mAb reacts with two bands of a ganglioside fraction from murine spleen and thymus, and rat spleen. The 6C10 antibody shows no evidence of glycolipid reactivity. The 3G11+ bands have a mobility between those of the reference gangliosides GD1a and GD1b from human brain. The 3G11+ reactive bands were eluted in the disialyl fraction of rat spleen gangliosides using DEAE anion-exchange chromatography. Treatment of spleen gangliosides with endoglycoceramidase eliminates 3G11 antibody binding over time, indicating that the antigen contains a Glc beta 1-1'ceramide linkage, characteristic of a glycosphingolipid. Treatment of thymus or spleen gangliosides with sialidase eliminates binding of 3G11, thus indicating that the 3G11 epitope is dependent on the expression of one or more sialic acid residues. Immunostaining studies with a variety of reagents indicate that the 3G11+ gangliosides: (i) probably do not contain either the asialo-GM1 or the GM1 core structures; (ii) are not recognized by mAbs specific for the oligosaccharides of asialo-GM2, GM2, GD2 and GD3 gangliosides; and (iii) are also not recognized by antibodies or reagents that are specific for several structures representative of other major glycosphingolipid classes. Overall, these studies strongly suggest that the 3G11+ gangliosides have structures that have not been previously recognized in murine lymphoid tissue. Structures that could account for the known properties of the 3G11+ molecules are described. Finally, ways in which the selective expression of 3G11+ gangliosides might be linked to functionally distinct T-cell behaviours are discussed.

Structure of an extracellular polysaccharide produced by Erwinia chrysanthemi.

Erwinia chrysanthemi pv zeae strain SR260, a phytopathogen of corn, produced from lactose an acidic extracellular polysaccharide which was purified and found to consist of L-rhamnose, D-mannose, D-glucose, and D-glucuronic acid in the ratio of 3:1:1:1. A combination of chemical (carboxyl-group reduction, methylation analysis, periodate oxidation, Smith degradation, and lithium-ethylenediamine degradation) and physical (1 and 2D NMR spectroscopy) methods revealed that the polysaccharide is composed of a hexasaccharide repeating unit 1: [formula: see text]

Feasibility and success of a single-donor red cell program for pediatric elective surgery patients.

Although the risks of allogeneic blood transfusions are small, it is wise to limit donor exposure whenever possible. A program has been developed in which one donor provided all red cell (RBC) units for each patient awaiting elective surgery. Patients were mostly children who were ineligible for autologous blood donation. Seventy-three patients and 115 donors (mostly parents) entered the program. Of the 115 donors, 90 (78%) were eligible to participate and 25 (22%) were ineligible; 21 were ineligible because of RBC incompatibility. For each of the 73 patients, one eligible donor was selected to donate all RBC units. Preoperative RBC orders were 1 to 2 units for 41 patients and > or = 3 units for 32 patients. Of the 73 donors, 58 (79%) gave all RBC units ordered; 15 (21%) failed to complete all donations, but only 1 because of anemia (hematocrit < 33% [0.33]). Of 73 patients entered, 46 (63%) underwent transfusion, and 27 (37%) did not. Of 46 patients transfused, 38 (83%) received only single-donor RBCs. Thus, the RBC needs of nearly all pediatric elective surgery patients were provided by a single donor for each patient. Single-donor blood programs should be considered for elective surgery patients who are ineligible for autologous blood donation and who would otherwise be exposed to multiple donors.

The role of membrane lipid in the platelet storage lesion.

Because of their hemostatic and structural importance and their chemical and physical lability, membrane lipids are likely to be involved in the development of the platelet storage lesion. Chemical analysis using the new method of high-performance liquid chromatography with laser light scattering detection (HPLC-LLS) reveals platelet lipid to be composed of more than 22 individual components, the most abundant of which are phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (C), sphingomyelin (SM), phosphatidylserine (PS), and phosphatidylinositol (PI). Surprisingly, an asymmetric distribution of these lipids is maintained in the resting platelet with PS concentrated in the inner leaflet of the plasma membrane. The exposure of PS may be important in platelet activation because of its powerful procoagulant effect. Studies of the effect of blood bank storage on platelet lipid composition have repeatedly shown a steady loss of all components, which may be temperature dependent. Studies of platelet factor 3 activity and flow cytometry of stored platelets have revealed the lipid is lost through the process of microvesiculation. Coupled to this storage induced depletion of platelet lipid is a loss of more than half of the potential capacity of lipid-dependent platelet functions by day 5. The most likely underlying mechanism for this loss of lipid mass and functional capacity is lipid peroxidation, a process that could be blocked with antioxidants. Lipid peroxidation may also interfere with other membrane constituents such as glycoprotein IIb/IIIa and the aminophospholipid-specific translocase. Thus, lipid peroxidation should be a major focus in studies aimed at preventing or reversing the platelet storage lesion.

Donor reactions during DDAVP-stimulated plasmapheresis.

Donor exposure can be strikingly reduced for patients with classical hemophilia A and von Willebrand's disease when large volumes of potent cryoprecipitated AHF are prepared from donors following DDAVP (1-deamino-8-D-arginine vasopressin) stimulation and automated plasmapheresis--a procedure called "plasma exchange donation." Although this procedure has been reported to be relatively safe for donors, data are limited. Accordingly, we studied 20 donors during 48 procedures using DDAVP (0.3 micrograms/kg IV) followed by 2-3 L plasma collection. Replacement fluid for each initial plasma exchange donation was plasma protein fraction; autologous cryoprecipitate-poor plasma was used for subsequent procedures. Mild reactions, particularly facial flushing, were noted in all 48 procedures. No procedure was discontinued, but four were modified due to either an increased pulse rate (> or = 20/min from baseline) or a fall in systolic or diastolic blood pressure (> or = 20 mm Hg from baseline). No donor was deferred or withdrew from the program. Based on our modest experience, DDAVP stimulated plasma exchange donation appears to be a safe and effective method for collecting large quantities of plasma from which potent cryoprecipitated AHF can be prepared.

Two-dimensional 1H-, 13C-, and 31P-nuclear magnetic resonance and molecular-mechanics investigation of D-fructose 2,6-bisphosphate.

Two-dimensional nuclear magnetic resonance studies have been carried out to assign unequivocally all the proton, carbon, and phosphorus resonances of D-fructofuranose 2,6-bisphosphate (1) and to verify its structure using a 400-MHz spectrometer. Several unexpected chemical-shift values and coupling constants were obtained. Molecular mechanics calculations (Sybyl) carried out to minimize the conformational energy of 1 yield phi C-1,P-2 = + 84, phi C-3,P-2 = - 155, and phi C-5,P-6 = + 175. Thus the unusual near-gauche orientations of C-1 and C-3 to P-2 in 1 can explain their small vicinal coupling constants (3JC-1,P-2 = 1.2, and 3JC-3,P-2 = 3.8 Hz), in contrast to the expected larger value seen for 3JC-5,P-6 namely, 6.9 Hz. Treatment of a sample of this compound with sodium borohydride did not affect its nuclear magnetic resonance spectrum, substantiating that O-2 is phosphorylated. Oxidation with sodium periodate yielded an intermediate which decomposed by a beta-elimination mechanism involving the 6-phosphate group. These data establish unequivocally the 1H, 13C, and 31P assignments and explain the observed anomalous shifts. Moreover they indicate that the activator of fructose 6-phosphate 1-kinase is the beta anomer of the 4T3 conformer of D-fructose 2,6-bisphosphate.

Serum volumes should not be reduced when testing neonates for unexpected red cell antibodies.

Serum-to-red cell ratio (volume:volume) significantly affects the sensitivity of antibody detection. Obtaining sufficient serum quantity can be a problem when testing blood from transported neonates not accompanied by maternal samples. Reducing serum volumes used for testing might result in missed antibodies. The authors studied 1,177 sera for unexpected red cell antibodies by comparing one versus two drops of patient serum using a technique with LISS at 37 degrees C through the antiglobulin phase. The serum-to-red cell ratio using two drops was approximately 50: 1. Results of 60% (58/96) of samples containing antibody benefited by the use of increased serum. Eighteen percent; (17/96) had antibodies detected only with two drops that were missed entirely by one, and 43% (41/96) showed stronger positive reactions using two drops versus one. Importantly, antibodies detected only with or enhanced by two drops were clinically significant (anti-D, anti-K, anti-M, anti-c, anti-E). Thus, reducing serum-to-cell ratio is potentially dangerous, and blood banks should insist on adequate sample size to ensure patient safety.

Antibodies against platelet glycosphingolipids: detection in serum by quantitative HPTLC-autoradiography and association with autoimmune and alloimmune processes.

In order to assess the importance of glycosphingolipids (GSL) in the immunology of the platelet, serum antibody binding to immobilized, purified platelet GSLs have been quantitatively measured via high-performance thin-layer chromatography (HPTLC), 125I-radio-immunolabeling, autoradiography, and densitometry. Thirteen neutral GSL bands were detected at Rf.03 through .64 (CHCl3-CH3OH-H2O, 65:25:4) after extraction and chromatography (DEAE-Sephadex and Bio-sil A). Both IgM and IgG serum antibody binding was determined for 50 subjects from four groups: normal blood donors (NBD, n = 18); leukemia patients with nonimmune thrombocytopenia (NIT, n = 10); patients with systemic lupus erythematosus (SLE, n = 10); and patients with chronic autoimmune thrombocytopenia (CATP, n = 12). The ABO typing of these 50 subjects also allowed correlation of serum antibody binding with A blood group antigen expression. These studies reveal: (1) anti-GSL binding at band .06 is associated with blood group A alloantigen expression for both IgG and IgM (P less than .0001) antibodies; (2) binding at bands .17, .27, and/or .46 is associated with general autoimmunity (SLE and CATP) for IgM (P less than .0001); (3) binding at bands .35 and/or .38 is associated with platelet-specific autoimmunity (CATP) for IgG and/or IgM (P less than .005); and (4) binding at bands .03, .20, .23, and/or .43 is frequently observed for sera from all groups. The platelet specificity of bands .35 and .38 was confirmed by comparative studies with human intestinal smooth muscle GSLs. Quantitation of the intensity of CATP-associated anti-GSL binding (86 +/- 88 mm2) is comparable to anti-A alloantigen binding (57 +/- 52 mm2). Two of the GSL bands associated with SLE and CATP appear to be the long-chain fatty-acyl forms of globotriaosyl ceramide (.27) and globotetraosyl ceramide (.17), which are the Pk and P blood group antigens, respectively. These studies indicate that neutral GSLs may be important antigens in both autoimmune and alloimmune processes involving the blood platelet.