Different effects of lectins on the ligand binding of the NMDA receptors and sigma sites in rat brain hippocampus synaptic membranes

The effects of the lectins concanavalin A, WGA, ricin, abrin, and the mistletoe lectins from Viscum album MLI, MLII, and MLIII on the binding of ligands of the NMDA and sigma receptors in rat hippocampus synaptic plasma membranes were investigated. Binding of [³H]MK-801, [³H]glutamate, [³H]5,7-DCKA, and [³H]glycine to the membranes was decreased by 40-60% after addition of galactose-specific lectins (mistletoe lectins MLI, MLII, ricin, abrin) at concentrations of 0.01 mg/ml, but was not affected by the glucose- and mannose-specific lectin Con A, an acetylglucosamine-specific lectin WGA, or an acetylgalactosamine-specific lectin MLIII. The binding of [³H]SKF 10047 was decreased only in the presence of MLIII and did not change after addition of the other lectins. It is suggested that lectin-sensitive ligand binding sites of sigma- and NMDA receptors are located separately, and that the carbohydrate side chains of the sigma receptor do not participate in the modulation of the NMDA-receptor.     

Mechanisms of Xenogeneic Baboon Platelet Aggregation and Phagocytosis by Porcine Liver Sinusoidal Endothelial Cells

Background

Baboons receiving xenogeneic livers from wild type and transgenic pigs survive less than 10 days. One of the major issues is the early development of profound thrombocytopenia that results in fatal hemorrhage. Histological examination of xenotransplanted livers has shown baboon platelet activation, phagocytosis and sequestration within the sinusoids. In order to study the mechanisms of platelet consumption in liver xenotransplantation, we have developed an in vitro system to examine the interaction between pig endothelial cells with baboon platelets and to thereby identify molecular mechanisms and therapies.

Methods

Fresh pig hepatocytes, liver sinusoidal and aortic endothelial cells were isolated by collagenase digestion of livers and processing of aortae from GTKO and Gal+ MGH-miniature swine. These primary cell cultures were then tested for the differential ability to induce baboon or pig platelet aggregation. Phagocytosis was evaluated by direct observation of CFSE labeled-platelets, which are incubated with endothelial cells under confocal light microscopy. Aurintricarboxylic acid (GpIb antagonist blocking interactions with von Willebrand factor/vWF), eptifibatide (Gp IIb/IIIa antagonist), and anti-Mac-1 Ab (anti-α_Mβ₂ integrin Ab) were tested for the ability to inhibit phagocytosis.

Results

None of the pig cells induced aggregation or phagocytosis of porcine platelets. However, pig hepatocytes, liver sinusoidal and aortic endothelial cells (GTKO and Gal+) all induced moderate aggregation of baboon platelets. Importantly, pig liver sinusoidal endothelial cells efficiently phagocytosed baboon platelets, while pig aortic endothelial cells and hepatocytes had minimal effects on platelet numbers. Anti-MAC-1 Ab, aurintricarboxylic acid or eptifibatide, significantly decreased baboon platelet phagocytosis by pig liver endothelial cells (P<0.01).

Conclusions

Although pig hepatocytes and aortic endothelial cells directly caused aggregation of baboon platelets, only pig liver endothelial cells efficiently phagocytosed baboon platelets. Blocking vWF and integrin adhesion pathways prevented both aggregation and phagocytosis.