Correlation between electrophoretic mobility and extent of aggregation of human red blood cells

The extent of aggregation, electrophoretic mobility, and zeta potential of human erythrocytes were measured in the presence of some hormones and prostaglandins. Catecholamines, adrenomimetics, and prostaglandins were found to significantly increase the extent of aggregation; the electrophysiological characteristics of the cells were affected in different ways.     

Effect of some amphiphilic drugs on the membrane morphology and aggregation of rabbit platelets

Treatment of normal, disc-shaped rabbit platelets with lysophosphatidylcholine and chlorpromazine induced respectively spine formation and spherical transformation. In similar concentration ranges to those in which they induced these morphological changes, the drugs suppressed a series of events triggered by thrombin: pseudopod formation, arachidonate release from the membrane phospholipids, and aggregation. Washing the drug-treated platelets reversed the morphological changes and abolished the inhibitory effect on aggregation. These observations suggest that amphiphilic drugs perturb the plasma membrane structure of platelets, inducing the membrane shape change and inhibiting the stimulus-induced aggregation.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Effect of ethinylestradiol on arachidonic acid incorporation, release, and conversion to prostaglandins in rabbit platelets

Intramuscular administration to female rabbits of 2 mg/kg ethinylestradiol every other day for 10 days increased the uptake and incorporation of [14C]arachidonic acid into platelet lipids, and increased the proportion of [14C]arachidonic acid released from platelets after stimulation by thrombin. The conversion of [14C]arachidonic acid to thromboxane B2 did not differ between the control and ethinylestradiol-treated groups. Thus, the results of this study indicate that the major site in the prostaglandin metabolic pathway influenced by estrogen is the incorporation and release of arachidonic acid in platelet phospholipids.     

Identification of a "new" rabbit and human serum protein with fast electrophoretic mobility

A new protein has been identified in both rabbit and human serum. The salient characteristic of this protein is its high negative charge as revealed by its rapid anodal migration during electrophoresis at alkaline pH. This protein has tentatively been designated fast-moving protein because of its electrophoretic mobility. Molecular weight determination by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the molecular weight was 85,000 daltons. A goat antiserum made to the rabbit fast-moving protein cross-reacted with both rabbit and human serum albumin. Although no apparent structural relationship between fast-moving protein and albumin was found by peptide-mapping studies, a peptide with a molecular weight of 24,000 daltons and with antigenic determinants in common with rabbit fast-moving protein, was isolated from cyanogen bromide-treated human serum albumin. The structural relationship between fast-moving protein and albumin is discussed.     

Effects of cytochalasin B on the aggregation, electrophoretic mobility and surface morphology of chick neural retina cells.

Over a concentration range of o-5-10 mug/cm-3, cytochalasin B caused a biphasic change in the electrophoretic mobility of disaggregated neural retina cells. An initial rise in anodal mobility at low concentrations of the drug was transformed into a reduction in the mobility below that of the control at a concentration of 10 mug/cm-3. The effect of cytochalasin B was found to be reversible by washing treated cells in cytochalasin B-free media. This was investigated at a concentration of cytochalasin at which the greatest difference existed between the mobilities of the control and experimental cell suspensions. Reaggregation of cell dispersions failed to show any significant difference in the rate of aggregation between cytochalasin B-treated cells and the control. Scanning electron microscopy of cells fixed while in suspension also showed little significant change in the surface morphology upon application of cytochalasin B. In high concentrations of the drug cells appeared somewhat smoother in outline, but no correlation was found between changes in surface morphology and the variations in cell electrophoretic mobility. It is concluded that the observed changes in electrophoretic mobility may be attributed to a binding of cytochalasin B to the cell membrane. This lends support to the hypothesis that the primary site of action of cytochalasin B may be the plasma membrane.     

Effect of neuraminidase and proteolytic enzymes on electrophoretic mobility of erythrocytes and their aggregation induced by La(3+)

The effect of neuraminidase, trypsin and pronase on the electrophoretic mobility of human erythrocytes and their aggregation induced by La3+ was studied in vitro. The aggregation of intact red cells differs from that of cells treated with neuraminidase and proteolytic enzymes. The above differences are observed in the range of La3+ suspension concentration from 20 to 330 microM. A possible mechanism of aggregation is discussed.     

Cell electrophoretic mobility and cationic flux

The net negativity of the surfaces of Ehrlich ascites cells was reduced by treating them with either neuraminidase or ribonuclease. Neither enzyme treatment affected the Na⁺ or K⁺ content of the cells, before or after cooling at 4°C. Experiments with K⁴² revealed a reduction (9.5 to 17%) in unidirectional K⁺-fluxes following incubation with neuraminidase, but no change after ribonuclease-treatment. Our data suggest that surface anionic sites associated with RNA and sialic acid moieties are not of major quantitative importance in regulating either intracellular Na⁺ and K⁺ concentrations, or unidirectional transmembrane K⁺-flux. Our results do not enable us to determine whether ion-binding to anionic sites at the electro-kinetic surface is not an essential prerequisite to transmembrane movement, or whether it is essential, but occurs through the 40% of cell surface net negativity which is unaffected by ribonuclease- and neuraminidase-treatment.     

Inhibitory effect of sulfur-containing compounds in Scorodocarpus borneensis Becc. on the aggregation of rabbit platelets

The inhibitory effects of three pure compounds isolated from wood garlic, 2,4,5-trithiahexane (I), 2,4,5,7-tetrathiaoctane (II), and 2,4,5,7-tetrathiaoctane 2,2-dioxide (III), on rabbit platelet aggregation induced by collagen, arachidonic acid, U46619, ADP (adenosine 5'-diphosphate), PAF (platelet aggregating factor), and thrombin were studied in vitro. The anti-aggregating activity of 2,4,5,7-tetrathiaoctane 4,4-dioxide (IV) was also measured with collagen and arachidonic acid. I, II, III, and IV inhibited the platelet aggregation induced by all tested agonists. I, II, and III exhibited a stronger inhibitory effect against the thrombin-induced aggregation of GFP (gel-filtered platelets) than against the aggregation induced by the other agonists. Notably, the IC50 value for III was 4 microM, which is approximately 2.5 times stronger than MATS (methyl allyl trisulfide), a major anti-platelet compound isolated from garlic. In inhibiting collagen-induced aggregation, II was as potent as MATS and aspirin, with a marked disaggregation effect on the secondary aggregation by arachidonic acid, at the rate of 47.05%/min at a concentration of 10(-4) M. I, II, and III also suppressed U46619-induced aggregation. These results suggest that sulfur-containing compounds in wood garlic not only inhibit arachidonic acid metabolism but also suppress aggregation in association with the function of the platelet plasma membrane.     

Influence of short term dietary supplementation of different lipids on aggregation and arachidonic acid metabolism in rabbit platelets

Semisynthetic diets containing 8% by weight of either corn oil or butter were fed to male New Zealand rabbits for three weeks. The plasma cholesterol values were determined, the threshold concentrations for aggregation of platelet rich plasmas were measured for collagen and Na arachidonate, and the conversion of ¹⁴C arachidonic acid to thromboxane B₂ and hydroxy fatty acids (HETE and HHT) at 10, 20 and 40 μM substrate concentrations were studied. The thresholds for arachidonate induced aggregation were lower and the amplitudes of collagen induced aggregations were greater in the butter fed than in the corn oil fed rabbits. Conversions of arachidonic acid to thromboxane B₂ but not to hydroxy fatty acids were greater in the butter fed rabbits at 10 and 20 μM substrate. The observed changes were accompanied by only slight modifications of plasma cholesterol levels.