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.

A quantitative analysis of H-2 linked effects on hepatic ganglioside composition.

We have conducted a quantitative analysis of the gangliosides extracted from brain, spleen, thymus, and liver tissue of 8-week-old male mice from H-2 congenic mouse strains on the B10 background, using high performance thin-layer chromatography (HPTLC). An analysis of variance of replicate samples of liver from strains B10, B10.A, and B10.G revealed that the time of sample and colony of origin were not sources of significant variance but that for N-glycolylated gangliosides GM2, GM1, and GD1a, the differences detected between strains were significant. Particularly important were the differences for GM1: the values of 0.0% for B10, 19.0% for B10.A, and 36.0% for B10.G were each significantly different from the others (P less than 0.0005). Further studies with liver tissue from B10/A H-2 recombinant strains also revealed three significantly different levels of GD1a: less than or equal to 4.0% [B10, B10.A (3R), B10.A (5R), B10.A (18R)], 11.0% (B10.A), and 33% [B10.A (1R), B10.A (2R), B10.A (4R)]. Our findings support prior studies which indicate that a gene linked to the H-2 complex affects hepatic GM2 galactosyltransferase activity. However, they also indicate that the current model, which classifies all strains as possessing either an allele for "high" enzyme activity or a single alternative allele for "low" enzyme activity, is probably oversimplified, since at least three levels of enzyme activity appear to exist as stable phenotypic markers. Moreover, the current model cannot readily account for the three different levels of GD1a observed with B10/A H-2 recombinants. Alternative models are proposed, including the novel suggestion that a distinct H-2 linked gene may affect hepatic GM1 sialyltransferase activity. These findings demonstrate that further study of H-2 linked genes affecting the activities of glycosyltransferases is indicated.

The effect of storage on the ganglioside content of human platelets.

Gangliosides are glycolipids which contain sialic acid and are found in the membranes of mammalian cells. By analogy with recent studies of other cells, it is possible that gangliosides play a role in the membrane functions and in vivo survival of platelets. In order to determine if ganglioside destruction plays a role in the storage-induced loss of platelet viability and function (storage lesion), the ganglioside content of platelets was measured after 24 and 96 hours of storage. Samples were taken from platelet concentrates that were stored either on a flat-bed shaker (n = 6) or on a circular rotator (n = 6). Total ganglioside content was determined colorimetrically from the total lipid extracts of purified platelet pellets using the Svennerholm resorcinol method. Ganglioside GM3 content was determined by Folch partitioning, high performance thin-layer chromatography, and densitometric scanning. Ganglioside content, measured as microgram of lipid-bound sialic acid per 10(10) platelets, was significantly decreased (p less than 0.005) between 24 and 96 hours of storage, whether measured as total or GM3 ganglioside. The mean values +/- SEM at 24 and 96 hours of storage were 9.4 +/- 0.6 and 6.7 +/- 0.6, respectively (n = 12 for each). These data indicate that storage causes irreversible loss of membrane ganglioside, which may be detrimental to the function and in vivo survival of platelets.

Effect of phenytoin on [3H]norepinephrine release from synaptosomes.

Phenytoin reduces depolarization-linked [3H]norepinephrine release from rat brain synaptosomes. When choline chloride was substituted for NaCl the phenytoin effect was attenuated but still significant. This is consistent with the theory that phenytoin reduces both Na and Ca influx during depolarization.

Acetylcholine release from synaptosomes and phenytoin action.

The release of labeled acetylcholine from synaptosomes loaded with methyl-[3H]choline has been measured in Krebs-Ringer-Bicarbonate (KRB) media containing either 5.6 or 56 mM KCl. Experiments have been performed in media containing either 1.0 mM Ca or 0 Ca with 1 mM EGTA. Phenytoin, 2 X 10(-4) M, reduced the depolarization-dependent release of acetylcholine in media containing 1.0 mM Ca and 56 mM KCl. It also significantly increased the release of acetylcholine from undepolarized samples in 5.6 mM KCl irrespective of the Ca concentration. The drug did not affect release from synaptosomes depolarized in Ca-free media. These results confirm the hypothesis that phenytoin has a dual effect on transmitter release.