Inhibition of human platelet aggregation by gangliosides.

The content and composition of gangliosides is modified upon platelet stimulation, suggesting that these lipids may play functional roles in platelet physiology. Therefore, the effect of exogenously added gangliosides on human platelet aggregation was evaluated. The pretreatment of platelets with a mixture of total gangliosides from bovine brain and a series of purified mono-, di- and tri-sialogangliosides partially inhibit the collagen-induced aggregation process and ATP release and completely block the generation of the second aggregation wave when ADP is used as agonist. The inhibition was exerted at around 100 microM by G(TOT) as well as purified G(M1), G(M3), G(D1a), and G(T1b) gangliosides, whereas asialoG(M1) and sulphatide did not show a significant influence on platelet aggregation. Thrombin, Ca(2+) ionophores (A23187 and Ionomycin), arachidonic acid, and U46619 were unable to bypass the inhibitory effect exerted by gangliosides, suggesting that gangliosides inhibit platelet aggregation by inhibiting the synthesis or action of prostaglandins. Gangliosides inhibited U46619-induced aggregation, thus suggesting that they block the action of thromboxane A(2). Epinephrine induces a partial aggregation on gangliosides-treated platelets, similar to fluoroaluminate and phorbol myristate acetate, indicating that these platelets are still functional. To summarize, these results indicate that the major pathway(s), but not all, driving to the aggregation process following the interaction of ligand-receptor may be blocked by pretreatment of human platelets with gangliosides.

Calcium dependency of arachidonic acid incorporation into cellular phospholipids of different cell types.

Ca2+ -independent phospholipase A2 (iPLA2) is involved in the incorporation of arachidonic acid (AA) into resting macrophages by the generation of the lysophospholipid acceptor. The role of iPLA2 in AA remodeling in different cells was evaluated by studying the Ca2+ dependency of AA uptake from the medium, the incorporation into cellular phospholipids, and the effect of the iPLA2 inhibitor bromoenol lactone on these events. Uptake and esterification of AA into phospholipids were not affected by Ca2+ depletion in human polymorphonuclear neutrophils and rat fibroblasts. The uptake was Ca2+ independent in chick embryo glial cells, but the incorporation into phospholipids was partially dependent on extracellular Ca2+. Both events were fully dependent on extra and intracellular Ca2+ in human platelets. In human polymorphonuclear neutrophils, the kinetics of incorporation in several isospecies of phospholipids was not affected by the absence of Ca2+ at short times (<30 min). The involvement of iPLA2 in the incorporation of AA from the medium was confirmed by the selective inhibition of this enzyme with bromoenol lactone, which reduced < or =50% of the incorporation of AA into phospholipids of human neutrophils. These data provide evidence that suggests iPLA2 plays a major role in regulating AA turnover in different cell types.

Comparative study of the cytolytic activity of myotoxic phospholipases A2 on mouse endothelial (tEnd) and skeletal muscle (C2C12) cells in vitro.

A rapid in vitro cytolytic effect of some myotoxic phospholipases A2 (PLA2s) isolated from the venoms of Viperidae snakes has been previously described. This study was undertaken to investigate if cytolytic activity is a common property of the myotoxic proteins from this group. Murine endothelial cells (tEnd) and skeletal muscle myotubes (C2C12) were utilized as targets. The release of lactic dehydrogenase was quantified as a measure of cell damage, 3 h after exposure of cells to the different PLA2s, including representatives from the genera Bothrops, Agkistrodon, Trimeresurus, Crotalus (family Viperidae), and Notechis (family Elapidae). All of the group II myotoxic PLA2s tested displayed rapid cytolytic activity when tested in the micromolar range of concentrations (8-32 microM). In contrast, the group I myotoxic PLA2 notexin was devoid of this activity. Aspartate-49 and lysine-49 PLA2 group II variants showed a comparable cytolytic effect. Skeletal muscle myotubes, obtained after fusion and differentiation of C2C12 myoblasts, were significantly more susceptible to the cytolytic action of myotoxins than endothelial cells, previously reported to be more susceptible than undifferentiated myoblasts under the same assay conditions. Cytolytic activity appears to be a common characteristic of group II myotoxic PLA2s of the Viperidae. Bee venom PLA2, a group III enzyme of known myotoxicity, also displayed cytotoxic activity on C2C12 myotubes, being devoid of activity on endothelial cells. These results suggest that in vitro differentiated skeletal muscle myotubes may represent a suitable model target for the study of myotoxic PLA2s of the structural group II found in snake venoms.

A new phospholipase A2 isoform isolated from Bothrops neuwiedii (Yarará chica) venom with novel kinetic and chromatographic properties.

A new phospholipase A2 isoform, called P-3, isolated from Bothrops neuwiedii (Yarará chica) venom, showed different chromatographic, enzymatic and cytotoxic properties compared to the previously purified isoforms P-1 and P-2 but it had a similar edema-inducing activity. In contrast to previously reported B. neuwiedii phospholipase A2 isoforms, P-3 did not interact with the oligosaccharide matrix of gel filtration columns (Superose, Superdex). Its molecular weight was 15,000 and its N-terminal 14 amino acid sequence was Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Lys-Ile-Ala-Gly. Amino acid analyse revealed the presence of an unique histidine, presumably located at the active site, because a full inhibition of enzymatic activity was observed after treatment with p-bromophenacyl bromide. The new isoform also differentiated in its surface pressure activity profile when assayed in lipid monolayers. P-3 had an optimum activity towards dilauroylphosphatidylcholine monolayers of 27 mN/m and a cut-off pressure of 30 mN/m, whereas P-1 and P-2 had an optimum of 13 mN/m with a cut-off of 22 mN/m. P-3 retained its edema-inducing activity in the absence of hydrolytic activity, suggesting that the inflammatory activity was not dependent on the enzymatic activity. Neither the enzymatic nor the edema-inducing activity was affected by heparin. The new isoform was not lethal when a single dose of 5 micrograms/g body weight was injected intraperitoneally into mice. All of the isoforms displayed cytotoxic activity in vitro on B16F10 melanoma cells evaluated by direct MTT assay, with an EC50 of 31 micrograms/ml for P-3 and of 15 micrograms/ml for P-1 and P-2. The cytotoxic activity of P-3 was inhibited by p-bromophenacyl bromide treatment of the enzyme (up to 170 micrograms/ml), whereas the same treatment on P-1 and P-2 changed their EC50 to 60 micrograms/ml. The difference observed with inhibited enzymes suggests a different mechanism for the cytotoxic action of P-3 with respect to P-1 and P-2.

Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles.

The effect of complex glycosphingolipids (gangliosides) on the activity of phospholipase C from Bacillus cereus was studied using lipid monolayers, mixed micelles and small unilamellar vesicles containing phosphatidylcholine as substrate. In all artificial membrane systems assayed, gangliosides exhibit qualitatively similar inhibitory properties. Gangliosides decrease the enzyme activity irrespective of the aggregation structure in which the substrate is offered to B. cereus phospholipase C, and they do not affect the adsorption process of the enzyme. The modulatory effect of gangliosides occurs at the level of the interface, affecting both the maximum rate of catalysis of the enzyme already adsorbed and the availability of the substrate in a suitable organization for enzyme catalysis to take place.

Kinetic and pharmacologic characterization of phospholipases A2 from Bothrops neuwiedii venom.

Two phospholipases A2 (PLA2) (EC 3.1.1.4) were purified from Bothrops neuwiedii venom (isoenzymes P-1 and P-2). The molecular weights of P-1 and P-2 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 15,000 and 16,200 and the isoelectric points were 4.8 and 4.6, respectively. The N-terminal 14-amino-acid sequences determined were Asn-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly and Ser-Leu-Val-Gln-Phe-Glu-Thr-Leu-Ile-Met-Met-Ile-Ala-Gly for P-1 and P-2, respectively. Since both show sequence almost identical to that of a PLA2 from Crotalus atrox it was tentatively classified as being of group II. The enzymatic activity of P-1 and P-2 toward lipid monolayers was studied. The hydrolysis of dilauroylphosphatidylcholine (dlPC) shows a broad optimum between 7 and 18 mN m-1 and a cut-off pressure of 22 mN m-1. The activity toward dlPC displays a maximum at pH 8 and is dependent on the presence of Ca2+ with an apparent Kd of 0.1 mM, for both enzymes. P-1 and P-2 are heat-stable enzymes, unable to hydrolyze dilauroylphosphatidic acid monolayers. The enzymes are not lethal to mice at doses up to 5 micrograms/g body weight by intraperitoneal injection and they do not show myotoxic (up to 40 micrograms) or hemolytic activity (up to 8.5 micrograms/ml). Both lack anti-coagulant activity, determined by absence of changes in the recalcification time of platelet poor plasma (up to 100 micrograms/ml), and are not able to induce platelet aggregation (up to 50 micrograms/ml). However, both isoenzymes exhibit an important edema-inducing activity that is not altered at short times by the irreversible chemical inactivation of the hydrolytic activity with phenacyl bromide. P-1 and P-2 are able to release arachidonic acid from membrane phospholipids of neutrophils, property that is lost by the inactivation of the enzyme. This suggests that the edema-inducing activity of the active but not the inactive forms may be partly due to arachidonic acid-derived mediators. The edema-inducing activity of the active or inactive forms of the enzymes is inhibited by antagonists of histamine, suggesting that histamine plays an important role in both the active and the inactive B. neuwiedii PLA2s-induced edema. The results suggest that the inflammatory and the catalytic activity of these enzymes constitute separate properties.