Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.     

Uptake of biotin by human hepatoma cell line,Hep G2: A carrier-mediated process similar to that of normal liver

Little is known about the cellular and molecular regulation of the uptake process of the water-soluble vitamin biotin into liver cells, the major site of biotin utilization and metabolism. Such studies are best done using a highly viable and homogeneous cellular system that allows examination of prolonged exposure to an agent(s) or a particular condition(s) on the uptake process. Isolated hepatocytes when maintained in primary culture lose their ability to transport biotin by the specialized carrier system. The aim of the present study was, therefore, to examine the mechanism(s) of biotin uptake by the cultured human-derived liver cells, Hep G₂. Uptake to biotin by Hep G₂ cells was appreciable and linear for up to 10 min of incubation. The uptake process was Na⁺ gradient-dependent as indicated by studies of Na⁺ replacement and pretreatment of cells with gramicidin and ouabain. Biotin uptake was also dependent on both incubation temperature and intracellular energy. Unlabeled biotin and the structural analogs with free carboxyl groups (thioctic acid, desthiobiotin) but not those with blocked carboxyl group (biocytin, biotin methyl ester, and thioctic amide) caused significant inhibition of ³H-biotin uptake at 37°C but not 4°C. Initial rate of biotin uptake was saturable as a function of concentration at 37°C but was lower and linear at 4°C. Pretreatment of Hep G₂ cells with sulfhydryl group inhibitors (e.g., p-chloromer-curibenzene sulfonate) led to a significant inhibition in biotin uptake; this inhibition was effectively reversed by reducing agents (e.g., dithiothreitol). Biotin uptake was also inhibited by the membrane transport inhibitors probenecid (noncompetitively), DIDS and furosemide but not by amiloride. Pretreatment of Hep G₂ cells with valinomycin did not alter biotin uptake. The stoichiometric ratio of biotin to Na⁺ uptake in Hep G₂ cells was also determined and found to be 1:1. These findings demonstrate that biotin uptake by these cultured liver cells is mediated through a specialized carrier system that is dependent on Na⁺-gradient, temperature, and energy and transports the vitamin by an electroneutral process. These findings are similar to those seen with native liver tissue preparations and demonstrate the suitability of Hep G₂ cells for in-depth investigations of the cellular and molecular regulation of biotin uptake by the liver. © 1994 Wiley-Liss, Inc.

1 This article is a US Government work, and as such, is in the public domain in the United State of America

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A radioimmunoassay for chicken avidin. Comparison with a [14C]biotin-binding method.

A double-antibody solid-phase radioimmunoassay for chicken avidin is reported. Avidin was labelled with 125I by the chloramine-T method. The bound and free avidin were separated with a second antibody bound to a solid matrix. In the logit-log scale the standard curve was linear from 1-2 to 100-200ng of avidin/ml. Cross-reaction of ovalbumin was less than 0.015%. Saturation of biotin-binding sites of avidin with an excess of biotin decreased radioimmunoassay values by about 15%. Recovery studies indicated that avidin can be assayed from all chicken tissues studied with radioimmunoassay, whereas the [14C]biotin/bentonite method gave poor recoveries for avidin in the liver and kidney. Radioimmunoassay and the [14C]biotin/bentonite method gave similar concentrations for oviduct avidin.     

Further studies on hypothermia and the blood-brain barrier system.

Rats subjected to five episodes of recurrent, progressively deeper hypothermia showed no difference in cerebral deposition of rubidium tracer at 16 °C and/or apnea from animals lowered to this temperature and/or condition only once. Rats allowed to rewarm from 16 °C showed persisting increased cerebral deposition of tracer at 20 °C with gradual diminution at higher temperatures; at 37 °C, deposition of rubidium tracer in brains of rewarmed rats was not different from that of euthermic rats which were not subjected to hypothermia.     

Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures

Dalgliesh R. J. and Stewart N. P. 1979. Observations on the morphology and infectivity for cattle of Babesia bovis parasites in unfed Boophilus microplus larvae after incubation at various temperatures. International Journal for Parasitology9: 115–120. The temperature of incubation of unfed Boophilus microplus larvae infected with Babesia bovis influenced the morphology and infectivity of the Babesia within the tick. Incubation at 37°C for 1–3 days stimulated the development of parasites morphologically similar to those usually observed in fed larvae harvested from cattle; similar forms appeared more slowly in larvae incubated at 31°C or 25°C. Extracts prepared from larvae after incubation at 37°C for 3–5 days or 30°C for 8 days were consistently infective for cattle. Prior storage of larvae at 14°C for up to 28 days enhanced the development of infectivity at 37°C; infectivity could still be produced after 65 days storage at 14°C but not after 76 days. Larvae released on a host transmitted B. bovis sooner if they had been incubated at 37°C for 4 days. It was concluded that the development of B. bovis to an infective stage in B. microplus is temperature dependent and does not require the stimulus of feeding by the host.     

Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability.

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.     

Role of avidin and other biotin-binding proteins in the deposition and distribution of biotin in chicken eggs. Discovery of a new biotin-binding protein.

In addition to the previously characterized egg-yolk biotin-binding protein (BBP-I), we have discovered another BBP (BBP-II) in the plasma and yolk from laying hens. BBP-I is stable to 65 degrees C, whereas BBP-II is stable to 45 degrees C. Both proteins are normally saturated with biotin and together they account for most, if not all, of the biotin in hen plasma and yolk, except in hens fed excessive amounts of biotin (greater than 1 mg of biotin/kg of feed). The maximal production of BBP-I is attained at lower levels of dietary biotin (approximately 50 micrograms/kg) than for BBP-II (approximately 250 micrograms/kg); however, the maximal production of BBP-II is severalfold greater than for BBP-I. Consequently, as dietary biotin increases, the ratio of BBP-II to BBP-I increases and becomes constant at dietary intakes of biotin above 250 micrograms/kg. The observation that the amounts of these proteins are limited by biotin in the normal dietary range (less than 250 micrograms/kg) suggests that biotin is required for the synthesis, secretion or stability of these proteins. Although both plasma vitamin-protein complexes are transported to the oocyte and concentrated in the yolk, BBP-II is transferred more efficiently. Thus biotin deposition in the yolk is a function of the amounts and relative concentrations of the two proteins. Dietary biotin above 250 micrograms/kg exceeds the transport capacity of BBP-I and BBP-II in the plasma; however, unbound biotin does not accumulate. Rather it is efficiently scavenged by avidin in the oviduct and transferred to the egg albumen. Only when avidin becomes saturated at high dietary intake does free or weakly bound biotin accumulate in plasma and yolk. The synthesis of avidin is independent of dietary biotin. Small amounts of BBPs with the heat-stability of avidin or BBP-I respectively are present in the plasma of adult males or immature chickens. BBP-II, the major BBP in the plasma and yolk of laying hens, was not detected in the plasma of non-laying chickens.     

Determination of the quantity of acetyl CoA carboxylase by [14C]methyl avidin binding

Conditions are described under which monomeric [14C]methyl avidin binds to SDS-denatured biotin enzymes and remains bound through polyacrylamide gel electrophoresis. The location of radioactive proteins on the dried gel was determined by fluorography and their identity was established by subunit molecular weight. The relative quantity of bound radioactive avidin, stoichiometrically equivalent to the molar quantity of biotin protein, can be determined by scanning the fluorograph with a soft laser densitometer. To determine the absolute quantity of biotin protein, the radioactive areas of the dried gel were cut out, resolubilized, and assayed for radioactivity. Since the specific radioactivity of the [14C]methyl avidin was known, the quantity of avidin bound and therefore the quantity of biotin enzyme could be calculated. The method is illustrated by the analysis of purified acetyl CoA carboxylase and is applied to the analysis of biotin enzymes in isolated rat liver mitochondria.     

Timing of Bifidobacterium Administration Influences the Development of Allergy to Ovalbumin in Mice

C3H/HeJ mice were sensitized with ovalbumin (OVA) and choleratoxin (CT) for 5 weeks, and then Bifidobacterium bifidum BGN4 was administered continuously for 7 weeks, starting 2 weeks before (pre-treatment group) and 2 weeks after (post-treatment group) the initial sensitization. After sensitization, the OVA-induced (sham group) mice showed growth inhibition and had scab-covered tails which was associated with serum levels of 9887±175 ng OVA-specific IgE/ml and 758±525 ng IgG1/ml. The sera of the pre-treatment group had 4805±245 ng OVA-specific IgE/ml and 193±87 ng IgG1/ml, as well as less severe tail symptoms. The sera of the post-treatment group had 5723±207 ng OVA-specific IgE/ml but the IgG1 and IgG2a levels were the same as those of the sham group. In spleen cultures, both pre-treatment and post-treatment increased the levels of IFN-γ but decreased the levels of IL-6 and IL-18. Taken together, the in vivo and in vitro results show that treatment with Bifidobacterium before OVA sensitization suppresses or modulates the allergic response more effectively than treatment with Bifidobacterium following OVA sensitization.     

Disintegration of layer-by-layer assemblies composed of 2-iminobiotin-labeled poly(ethyleneimine) and avidin

A layer-by-layer thin film composed of avidin and 2-iminobiotin-labeled poly(ethyleneimine) (ib-PEI) was prepared and their sensitivity to the environmental pH and biotin was studied. The avidin/ib-PEI multilayer assemblies were stable at pH 8-12, whereas the assemblies were decomposed at pH 5-6 due to the low affinity of the protonated iminobiotin residue to avidin. The avidin/ib-PEI assemblies can be disintegrated upon addition of biotin and analogues in the solution as a result of the preferential binding of biotin or analogues to the binding site of avidin. The decomposition rate was arbitrarily controlled by changing the type of stimulant (biotin or analogues) and its concentration. The avidin/ib-PEI assemblies were disintegrated rapidly by the addition of biotin or desthiobiotin, whereas the rate of decomposition was rather slow upon addition of lipoic acid or 2-(4'-hydroxyphenylazo)benzoic acid. The present system may be useful for constructing the stimuli-sensitive devices that can release drug or other functional molecules.     

Ultramicroassay for plasma renin concentration in the rat using the antibody-trapping technique

Using the antibody-trapping technique, picogram quantities of angiotensin-I generated during 24 hr of incubation at 37°C were stable and fully protected against peptidases. The method employs purification of angiotensin-I antisera on DEAE-cellulose and purification of renin substrate by affinity chromatography using specific antirenin antibodies in order to remove endogenous renin. The assay was performed in a single tube without a transfer step in a total volume of 30 μl at pH 6,5 with incubation for 24 hr at 37°C. With a normal rat plasma renin concentration of 5 × 10^?4 GU ml^?1, the detection limit was 10 nl or a total of 5 × 10^?9 GU. In the range 20–125 nl, precision was ±10%.     

Localization of Endogenous Biotin-Containing Proteins in Mouse Bergmann Glial Cells

A peroxidase-conjugated avidin–biotin complex was used to detect endogenous biotin-containing proteins in mouse cerebellum. By this method, Bergmann glial cells were found to be strongly labelled in the adult mouse cerebellum. Developmentally, cells in the granular layer, probably astrocytes, appeared to be labelled around postnatal 10-day (P10). Their labelling decreased after P20, although the positive-labelling remained in the Bergmann glial cells up to the adult stage. The findings were confirmed by using a Alexa Fluor 488-conjugated streptavidin technique. The labelling was not affected by routine hydrogen peroxide treatment, but it was eliminated by avidin–biotin blocking. By another transblot method, the reactive proteins in the mouse cerebellum were found to be 120?kDa (the strongest one) and 75?kDa. For electron microscopy, a gold-conjugated anti-biotin antibody was immunoreacted to the mitochondria of Bergmann glial cells. These results suggest that endogenous biotin-containing proteins are abundant in the Bergmann glial cells. Therefore, the avidin–biotin complex method is useful for detecting Bergmann glial cells, probably because of the difference of biotin metabolism in the cerebellar glial cells.     

Glutamic Acid Production in Biotin-rich Media by Temperature-sensitive Mutants of Brevibacterium lactofermentum,a Novel Fermentation Process

Temperature-sensitive mutants were derived from Brevibacterium lactofermentum strain 2256 in a search for mutants which would produce a large amount of L-glutamic acid in biotin- rich media at the nonpermissive temperature. A total of 159 mutant strains was selected which showed adequate growth at 30°C but showed little or no growth at 37°C on minimal medium. Twenty of these were found to produce glutamic acid in a biotin-rich medium after a temperature shift from 30°C to 37°C, while the wild-type strain 2256 did not produce it under the same cultural condition.

One of the typical mutant strains, Ts-88, produced approximately 2g/dl of glutamic acid from beet molasses (the yield > 55%) in the presence of 33 µg/liter of biotin when tempera- , ture was shifted from 30°C to 40°C during the cultivation. It was concluded that, by controlling only temperature during fermentation, glutamic acid production could be realized in media containing biotin-rich natural carbon sources, without any chemical control such as the addition of expensive surface-active agents or antibiotics. Characteristics and merits of the novel fermentation process are discussed.     

Impact of thermohaemolysis-associated membrane alteration on the passive ion permeability and life-span of erythrocytes

Suspending erythrocytes in medium containing sucrose prevented heat-induced lysis at its early stage. This allowed determination of the thermohaemolysis-related ion permeability by measuring the initial rate of the stipulated shrinkage of erythrocytes. Thus, correspondingly, the coefficient P of the ion permeability was calculated for heated human erythrocytes using ouabain-pretreated cells in 37–45°C range and intact cells in 50–58°C range. The values obtained for P obeyed a straight line Arrhenius plot over the entire 37–58°C range suggesting that the ion permeability was activated by a single mechanism earlier identified as relevant to thermohaemolysis. At the 37–58°C range, the activation energy of the P was 250±15 kJ/mol which was markedly different from the value of 56 kJ/mol known for the 10–37°C range. For erythrocytes from five mammals, similar temperature dependencies of the P were obtained over 45–60°C range. For erythrocytes from all species, excluding horse, the P, extrapolated at 37°C, had a value comparable with the known coefficient of the passive, ouabain-insensitive cation permeability at 37°C. For ouabain-treated human erythrocytes at 37°C, the period of thermohaemolysis-related shrinkage in sucrose containing media was found to be about six times shorter than the life-span of intact cells which substantiated the role of the active transport in balancing the thermohaemolysis-related diffusion of ions at 37°C. Consequently, the thermal resistance of erythrocytes, which was earlier related to their sphingomyelin content, was now found also to be in good correlation with their life-span in the circulation of 11 mammals.     

Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs,monensin, low temperature,and chloroquine

We studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for ¹²⁵I-asialo-orosomucoid binding, to measure receptor activity, or ¹²⁵I-anti-Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin-permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub-37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ? 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin-and chloroquine-treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.     

Biotin-binding protein from chicken egg yolk. Assay and relationship to egg-white avidin.

1. Biotin in chicken egg yolk is non-covalently bound to a specific protein that comprises 0.03% of the total yolk protein (0.8 mg/yolk). This biotin-binding protein is not detectable by the normal avidin assay owing to the biotin being tightly bound. Exchange of [14C]biotin for bound biotin at 65 degrees C is the basis of an assay for this protein. 2. Biotin-binding protein from egg yolk is distinguishable from egg-white avidin on Sephadex G-100 gel filtration, although the sizes of the two proteins appear quite similar. 3. Biotin-binding protein is denatured at a lower temperature and freely exchanges biotin at lower temperatures than does avidin. 4. The biotin-binding protein in egg yolk is postulated to be responsible for the deposition of biotin in egg yolk. D-[carboxyl-14C]Biotin injected into laying hens rapidly appears in the egg bound to yolk biotin-binding protein and avidin. Over 60% of the radioactivity is eventually deposited in eggs. The kinetics of biotin deposition in the egg suggests a 25 day half-life for an intracellular biotinyl-coenzyme pool in the laying hen.     

Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).     

The Impact of Temperature Stress on DNA and RNA Synthesis in Potentially Toxic Dinoflagellates <Emphasis Type="Italic">Prorocentrum minimum</Emphasis>

Biomarkers of temperature stress were studied as major characteristics crucial for the understanding complex processes that underlie the response of marine planktonic microorganisms to environmental factors and their sublethal effects. Using the potentially toxic dinoflagellates Prorocentrum minimum as a model object, the impact of temperature stress on viability, cell cycle, RNA synthesis and DNA replication in these protists was evaluated. It was shown by flow cytometry that stress evoked by a temperature increase from 25°C (control) to 37 or 42°C during 15 to 60 min did not cause any considerable alterations in the cell cycle, while cell death rate increased from ≤ 1% (control) to 2–12% at 37°C and 4–22% at 42°C. Along with a relatively low cell death rate, following a temperature increase to 37 and/or 42°C, P. minimum displayed the ability to boost the synthesis of DNA (1.7–1.9 and 1.2–1.6 times, respectively) and especially RNA (2.5–3.1 and 1.7–2.8 times, respectively) during the first 15–30 min after stress. At certain stages of the life cycle, this effect can be critical for maintaining the viability and normal development of the P. minimum population. The obtained results demonstrate that a significantly elevated synthesis of nucleic acids can serve as an indicator (biomarker) of sublethal environmental stress.     

Detergent solubilization of mammalian cardiac and hepatic beta-adrenergic receptors.

The solubilization of canine cardiac and hepatic β-adrenergic receptors was characterized with 19 different ionic and nonionic surfactants and surfactant combinations. The effects of alterations in the hydrophobic and hydrophilic moieties of the nonpolar detergents were examined in relation to their efficacy in solubilizing these receptor molecules. Within this group of surfactants the more effective agents contained an average of 9–10 polyoxyethylene units per molecule. The best degree of β-receptor solubilization for both heart and liver was obtained with 1% Brij 96 or a combination of 1% digitonin with 0.25% Triton X-100. Hepatic but not cardiac β-receptors were solubilized by polyoxyethylene ether W-1 or by Triton X-100. Solubilization time courses indicated that the maximum degree of receptor solubilization occurred within 5 min at 0–4 °C. Solubilization temperatures were varied from 0 to 37 °C. Temperatures up to 30 °C increased numbers of cardiac receptors solubilized by 30% over those obtained at 0 °C. The same temperature changes had no significant effects on liver β-receptor solubilization. Increasing the solubilization temperature to 37 °C decreased the detectable number of β-receptors by 91 (liver) and 72% (heart). β-Receptors solubilized in the absence of receptor ligand were extremely labile with a half-life on the order of 90 min at 4 °C for both heart and liver. Occupation of the receptors by [¹²⁵I]-iodohydroxybenzylpindolol prior to solubilization conferred a certain degree of stability on the receptors.     

On the susceptibility of human platelets to aggregation by concanavalin A and the effect of this lectin on their response to ADP

Concanavalin A aggregated gel-filtered platetes in 0.9% NaCl solution signifying cross-bridging by the lectin. Aggregation of these platelets by concanavalin A was temperature dependent; it did not occur at 0–4 °C unless the platelets were previously trypsinized. The level of aggregation of trypsinized platelets by concanavalin A at 0–4°C was similar to that of untreated platelets at 37°C. It is suggested that trypsin facilitates platelet aggregation by concanavalin A at 0–4°C by causing a configurational change in membrane glycoproteins which orientates concanavalin A receptor sites into positions that favour lectin cross-bridging. Concanavalin A failed to aggregate platelets in plasma. Radioisotope studies showed that the amount of [³H]concanavalin A which combined with platelets in plasma was extremely low compared with gel-filtered platelets in saline. The aggregation of Ehrlich ascites cells by concanavalin A was considerably reduced when platelet-free plasma was added to the medium suggesting that it was due to the presence of concanavalin A-reactive components in the plasma.Concanavalin A inhibited the ADP-induced aggregation of platelets suspended in plasma or in a salts solution supplemented with calcium and fibrinogen, although the inhibitory effect was more conspicuous in the latter case. The results suggests that concanavalin A produces its inhibitory effect on ADP-induced platelet aggregation by interacting with membrane glycoproteins, and this further suggests their involvement in aggregation.