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Biomedical subjects

R Apitz-Castro

Publications and source records attributed to R Apitz-Castro.

At least 37 records · Page 2Linked to original sources
PubMed1989

Interaction of antiaggregant molecule ajoene with membranes. An ESR and 1H, 2H, 31P-NMR study.

The structure of ajoene, a molecule extracted from garlic, has been studied by 1H-NMR and its interaction with model membranes by 1H-, 2H-, 31-P-NMR and ESR experiments. This study clearly shows that the ajoene molecule is located deep in the layer and is close to the interlayer medium. Moreover while NMR experiments show that the membrane structure is only slightly affected by the presence of ajoene, ESR experiments reveal significant modifications in phospholipid dynamics. This interaction, observed before with the phenothiazine derivative, promazine, results in an increase of the membrane fluidity in its hydrophobic part and could be related to clinical properties of ajoene.

Deuterium↗
PubMed1988

Galactosylation of endogenous proteins from human platelets.

Human platelets have been shown to contain the enzyme glycoprotein:galactosyltransferase that catalyzes the transfer of galactose to an endogenous protein acceptor present in the platelet. Galactosylation of added ovalbumin also occurs. The activity was extracted with 30 mM Tris buffer (pH 7.5). The endogenous activity was enriched 1.4-fold (compared with the crude homogenate) in the fraction, 105,000 g pellet, and the exogenous enzyme was retained in the respective supernatant. The two galactosyltransferase activities showed proportionality to time, protein, and substrate concentration, and were identical in pH dependence and Mn+2 requirement. The effect of Triton X-100 (range 0-1.5%) in the assay system appeared to be different for both activities: with the optimum concentration of detergent (0.15%) the endogenous activity increased by 50% whereas the exogenous activity was augmented 5-fold. From a number of sugar nucleotides tested as glycosyl donor into the endogenous proteins, the optimum substrate was UDP-Glc (100%), followed by UDP-Gal (80%), GDP-Man (24%), UDP-Glc-NAc (21%), UDP-Xyl (19%), and ADP-Glc (5%). An appropriate exogenous acceptor for UDP-Glc as donor was not found. The different solubilization of galactosyl- and glucosyltransferase activities by Triton X-100 suggests that they are distinct enzymes. In addition, the exogenous galactosyltransferase activity achieved after the treatment was much higher (940%) than the endogenous (26%). It is suggested that these differences on both galactosyltransferases could reflect changes in the accessibility of the exogenous substrate to the enzyme.

Blood Platelets↗
PubMed1988

In vivo platelet hyperreactivity, another risk factor for patients under continuous ambulatory peritoneal dialysis.

A high mortality rate due to thromboembolic accidents has been described in patients undergoing chronic haemodialysis. This type of complications, although recognized, has not been appropriately evaluated in continuous ambulatory peritoneal dialysis patients. The present study demonstrates that continuous ambulatory peritoneal dialysis patients present in vivo platelet hyperreactivity, as evidenced by enhanced platelet responses to epinephrine ex vivo and an increased MDA/MDAa index which traduces a decreased threshold for activation of the arachidonate pathway and subsequent thromboxane production. Since the etiopathogeny of this platelet abnormality seems to be related to abnormalities in lipid metabolism, compounds such as fish oil must be beneficial in the management of this risk factor.

Adult↗
PubMed1988

Reversible prevention of platelet activation by (E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide (ajoene) in dogs under extracorporeal circulation.

Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), an organosulfur compound derived from garlic inhibits platelet activation induced ex vivo by all known agonists. The effect of ajoene on the thrombocytopenia associated with the circulation of blood through extracorporeal devices such as dialyzers or oxygenators was studied under the following conditions: Ex vivo using fresh, heparinized human blood, circulating through a dialyzer or oxygenator, and in vivo, in dogs subjected to extracorporeal circulation. In both experimental conditions ajoene proved very efficacious in preventing platelet loss (60-65% loss in controls vs. 15-20% loss in the presence of ajoene, p less than 0.01). Moreover, recuperation of platelet function was achieved after 3-4 h in the in vivo experiments.

Adenosine Diphosphate↗
PubMed1987

Glycoprotein biosynthesis by human normal platelets.

Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation.

Bacitracin↗
PubMed1986

The molecular basis of the antiplatelet action of ajoene: direct interaction with the fibrinogen receptor.

Ajoene, the major antiplatelet compound derived from garlic inhibits the fibrinogen-supported aggregation of washed human platelets (ID50 = 13 microM) and, inhibits binding of 125I-fibrinogen to ADP-stimulated platelets (ID50 = 0.8 microM). In both cases, the inhibition is of the mixed non-competitive type. Furthermore, fibrinogen-induced aggregation of chymotrypsin-treated platelets is also inhibited by ajoene in a dose-dependent manner (ID50 = 2.3 microM). Other membrane receptors such as ADP or epinephrine receptors are not affected by ajoene. Ajoene strongly quenches the intrinsic fluorescence emission of purified glycoproteins IIb-IIIa (ID50 = 10 microM). These results indicate that the antiaggregatory effect of ajoene is causally related to its direct interaction with the putative fibrinogen receptor.

Adenosine Diphosphate↗
PubMed1986

Ajoene, the antiplatelet principle of garlic, synergistically potentiates the antiaggregatory action of prostacyclin, forskolin, indomethacin and dypiridamole on human platelets.

Ajoene, the major antiplatelet compound derived from garlic, synergistically potentiates the antiaggregatory action of prostacyclin, forskolin, indomethacin and dypiridamole. For collagen-induced platelet aggregation in human PRP, the ID50 for ajoene is 95 +/- 5 microM. However, in the presence of the antiaggregatory drugs mentioned above, the ID50 for ajoene decreases more than what would be predicted on the basis of simple additive effects. Similarly, the ID50 for prostacyclin decreases from 1 nM to 0.15 nM in the presence of 80 microM ajoene. Isobolic curves for the various combinations of ajoene with prostacyclin or indomethacin exhibit departure from linearity, as predicted for a potentiated synergism between ajoene and these drugs. Dypiridamole, which in PRP has very little effect on the dose-response curve for ajoene, when assayed in whole blood decreases the ID50 for ajoene by a factor of four. These results demonstrate that the antithrombotic potential of ajoene is substantially increased in the presence of physiologically and pharmacologically active antiplatelet agents.

Blood Platelets↗
PubMed1985

Protein and glycoprotein abnormalities in platelets from human Chediak-Higashi syndrome: polyacrylamide gel electrophoretic study of platelets from five patients.

Polyacrylamide electrophoretic analysis of proteins and Tritium-labelled glycoproteins of the platelets from five patients with Chediak-Higashi Syndrome shows the existence of marked quantitative differences when compared to normal platelets. While the glycoprotein abnormalities are solely related to the plasma membrane, some of the abnormalities detected in the Coomasie blue pattern are probably representative of defects related to the dense bodies and the alpha-granules. Some of the abnormalities found may, in part, explain the variability of aggregatory responses described in these patients, as well as the marked tendency towards desaggregation exhibited by platelets from humans with the Chediak-Higashi Syndrome.

Blood Platelets↗
PubMed1985

The storage pool deficiency in platelets from humans with the Chédiak-Higashi syndrome: study of six patients.

Functional and biochemical studies of platelets from human Chédiak-Higashi syndrome (CHS) are scarce and/or incomplete. In the present report, the aggregation response to a variety of inducers of platelet aggregation, the content of the dense granule constituents ATP, ADP, serotonin and calcium, the secretion of ATP, ADP, and calcium induced by thrombin, the total content of magnesium, the incorporation of 14C-adenine in the cytoplasmic pool of adenine nucleotides, as well as the content of intracellular cyclic-AMP, have been quantitated in six patients with CHS. Furthermore, data is presented on the kinetics of uptake of radiolabelled serotonin and its storage in human CHS platelets. An abnormal aggregation behaviour was found in all patients. However, the response of CHS platelets to the different inducers studied did not show a uniform pattern. The total content and the maximal amounts of the dense granule constituents secretable by thrombin were greatly decreased in all six patients. Total magnesium content was similar to that of normal platelets. The ATP/ADP ratio was higher than in controls. Uptake of radiolabelled serotonin by CHS platelets closely followed the uptake by normal platelets; during the first 2-3 min, however, incorporation of the amine by CHS platelets came rapidly to a plateau which contrasts with the steady, linear increase in uptake found in controls. CHS platelets loaded with radiolabelled serotonin and gel-filtered, showed a spontaneous release of radioactivity not observed in normal platelets under the same conditions. The cyclic-AMP content of CHS platelets was similar to that of normals. In contrast to platelets from patients with storage pool disease, the secretable calcium from CHS platelets represents a 67% of total platelet calcium (61% in normals), suggesting that the absolute values for the non-secretable portion in CHS platelets must be very low. The results reported confirm the existence of a true storage pool deficiency of the dense granule constituents as a common defect in CHS platelets. The variety of responses among patients, to the different aggregatory stimuli studied, can not be solely ascribed to the storage pool deficiency described.

Adenine Nucleotides↗
PubMed1983

Effects of garlic extract and of three pure components isolated from it on human platelet aggregation, arachidonate metabolism, release reaction and platelet ultrastructure.

We studied the effect of the methanol extract of garlic bulbs (EOG) and of three pure components isolated from it (F1, F2, F3), on human platelet aggregation induced by ADP, epinephrine, collagen, thrombin, arachidonate, PAF, and the ionophore A-23187. Incubation of PRP with EOG, either in methanol or in homologous PPP, inhibits platelet aggregation induced by all of the above mentioned agonists. F1, F2, and F3 also inhibit platelet aggregation, however, F3 was about four times more potent. Addition of EOG or F3 to platelets that have already been irreversibly aggregated by 10 microM ADP, induces rapid deaggregation. Inhibition of aggregation was still present after three hours. The inhibitory effect persisted even after the treated platelets were Gel-Filtered (GFP) or separated from plasma through a metrizamide gradient and resuspended in new homologous PPP. Thrombin-induced release of ATP from GFP was inhibited by 75-80% after EOG or F3 treatment. Incorporation of [3-H]-arachidonate by intact platelets was decreased by 50-60% in treated platelets. However, platelets incubated with the inhibitors after incorporation of radiolabeled arachidonate, although did not aggregate, produced, after thrombin activation similar amounts of radiolabeled TXB2 and lipoxygenase products as the controls. Electron microscopy of inhibited platelets, in the presence of thrombin, showed no degranulation but an increase of spherical forms. Our results suggest that the effects described might be mediate by a perturbation of the physicochemical properties of the plasma membrane rather than by affecting arachidonate or calcium metabolism in the cells. Chemical structures of F1, F2 and F3 have been provisionally assigned: F1 is diallytrisulfide, F2 is 2-vinyl-1,3-dithiene, and F3 is most probably allyl 1,5-hexadienyltrisulfide.

Adenosine Diphosphate↗
PubMed1982

Interaction of phospholipase A2 and phospholipid bilayers.

Binding of phospholipase A2 from porcine pancreas and from Naja melanoleuca venom to vesicles of 1,2-di(tetradecyl)-rac-glycero-3-phosphocholine (diether-PC14) is studied in the presence and absence of 1-tetradecanoyl-sn-glycero-3-phosphocholine and myristic acid. The bound enzyme coelutes with the vesicles during gel filtration through a nonequilibrated Sephadex G-100 column, modifies the phase transition behavior of bilayers, and exhibits an increase in fluorescence intensity accompanied by a blue shift. Using these criteria it is demonstrated that the snake-venom enzyme binds to bilayers of the diether-PC14 alone. In contrast, the porcine enzyme binds only to ternary codispersions of dialkyl (or diacyl) phosphatidylcholine, lysophosphatidylcholine and fatty acid. Binding of pig-pancreatic enzyme to vesicles of the diether-PC14 could not be detected even after long incubation (up to 24 H) below, at, or above the phase-transition temperature, whereas the binding in the presence of products is almost instantaneous and observed over a wide temperature range. Thus incorporation of the products in substrate dispersions increases the binding affinity rather than increase the rate of binding. The results are consistent with the hypothesis that the pancreatic enzyme binds to defect sites at the phase boundaries in substrate bilayers induced by the products. The spectroscopically obtained hyperbolic binding curves can be adequately described by a single equilibrium by assuming that the enzyme interacts with discrete sites. The binding experiments are supported by kinetic studies.

Animals↗
PubMed1982

Origin of the latency phase during the action of phospholipase A2 on unmodified phosphatidylcholine vesicles.

The reaction progress curve for the action of pig-pancreatic phospholipase A2 on dimyristoylphosphatidylcholine vesicles is characterized under a variety of conditions. The factors that regulate the rate of hydrolysis during the presteady-state phase determine the latency period. The results demonstrate that the accelerated hydrolysis following the latency phase of the reaction progress curve is due to the product-assisted binding of the enzyme to the substrate bilayer by chaning the number of bindings sites and therefore the binding equilibrium. A critical mole fraction of products appears to be formed in the substrate bilayers before the steady-state phase of hydrolysis begins. The latency phase shows a minimum at the phase-transition temperature of the substrate vesicles; however, we did not observe a significant binding of the enzyme to pure substrate bilayers even at the phase-transition temperature. The rate of binding of the enzyme is found to be fast and the rate of desorption of the bound enzyme is very slow compared to the latency phase. The rate of redistribution of products between substrate bilayers is rather slow. These observations demonstrate that during the latency phase of the action of phospholipase A2, a critical mole fraction of products is formed in the substrate bilayer.

Animals↗
PubMed1981

Inhibition of the platelet reaction by a high molecular weight phosphoglycoprotein isolated from human platelet plasma membranes.

The effect of phospho-glycoprotein (HMW-GP), obtained from human platelet plasma membranes, on the aggregation and secretion of human platelets was studied. Incubation of PRP with 4 to 16 micrograms/ml of HMW-GP results in inhibition of ADP-, Epinephrine-, Collagen-, and Thrombin-induced platelet aggregation. The effect is mainly reflected on the secondary wave of aggregation. The inhibitory effect is partially overcome by higher concentrations of inducers, however, even under these conditions, a clear tendency towards disaggregation is observed. 5HT release (Col-induced) is strongly decreased from 50% to 4.5. The inhibitory effect on Thrombin-induced aggregation is markedly dependent on external calcium, being maximal at 5 mM calcium. The HMW-GP does not bind ADP or Thrombin. Membrane conformation is markedly affected, as evidenced by the effect of HMW-GP on the iodination of surface polypeptides of intact platelets. It is suggested that interaction of HMW-GP with the platelet membrane blocks the signal(s) transmission that links stimulus to activation. The inhibition observed might just represent an experimental amplification of the endogenous modulatory function that has been proposed for this high molecular weight phosphoglycoprotein.

Blood Platelets↗
PubMed1981

The phospholipase A2 from human platelets.

Studies on a purified phospholipase A2 (PLA2) from human platelets show that the enzyme, which is copurified with the plasma membrane fraction, has a MW of approximately 50 K Dalton, requires Ca++, and has a pH optimum of 9.4. Under optimal conditions, PLA2 activity corresponds to at least 13 nmol/min/10(9) platelets. Unsaturated PL are preferred substrates and the enzyme is considerably more active on the aggregated form of the substrate than on the monomers. The specific activity is markedly affected by the quality of the interface, showing variations of more than 10-fold between different substrate forms. In the absence of detergents, a 4-fold increase in rate is observed when both products are present. Maximal rates are obtained at 20 mole percent of products to substrate. 1,2-Diglyceride and phosphatidic acid stimulate the hydrolysis of PC by the purified enzyme, however, in these forms of the substrate, neither of them are hydrolyzed. Activation of this enzyme by some intermediate of the phospholipase C pathway might play a role in the stimulus-linked release of platelet arachidonic acid.

Arachidonic Acid↗
PubMed1979

The effect of colchicine on human blood platelets under conditions of short-term incubation.

The effects of colchicine on ADP-induced aggregation and on the phosphorylation of tubulin-like protein from human blood platelets were studied. Colchicine at 2mM concentration completely inhibits ADP-induced aggregation after 8min incubation. Under the same inhibitory conditions, phosphorylation of tubulin-like materials in intact platelets was also impaired whereas the endogenous kinase activity of tubulin, isolated through polymerization--depolymerization cycles, was not affected. It was also shown that, under conditions of maximal inhibition of both aggregation and tubulin phosphorylation, colchicine does not penetrate into the cells. The results obtained suggest that the effect of colchicine on platelet aggregation might be mainly, although not exclusively, due to a non-specific effect of the alkaloid on the plasma membrane, rather than to a direct action of the drug on the microtubular protein subunits.

Adenosine Diphosphate↗
PubMed1978

Modulation of platelet responsiveness through selective phosphorylation of plasma membrane proteins.

32P phosphorylation of plasma membranes from human blood platelets, under conditions that closely resemble physiological ones (endogeneous phosphate donors and intact platelets in homologous plasma), result in the incorporation of the label mainly in a membrane glycoprotein of apparently high molecular weight (greater than 400 000). Dibutyryl cyclic AMP, an inhibitor of platelet aggregation, specifically increases the degree of phosphorylation of this glycoprotein. Moreover, it has been found that prostaglandin E1 one of the most potent inhibitors of platelet aggregation which also increases phosphorylation of the same glycoprotein, is significantly more effective than cyclic AMP. Cyclic GMP does not have any apparent effect on platelet aggregation. However, incubation of platelet-rich plasma with both cyclic GMP and cyclic AMP results in a partial recovery of the platelet responsiveness towards ADP-induced aggregation. Coincidently, the degree of phosphorylation of the high molecular weight glycoprotein under these conditions, although still higher than in controls (no nucleotides added), is significantly decreased as compared with cyclic AMP-treated cells. Furthermore, cyclic GMP inhibits the cyclic AMP-dependent protein kinase activity in isolated platelet plasma membranes. These results suggest a central role for this membrane phosphoglycoprotein in the triggering of platelet aggregation and, furthermore, suggest that modulation of its degree of phosphorylation may be exerted through some cyclic AMP/cyclic GMP relationship, which in the basal state might be critical for platelet responsiveness.

Blood Platelets↗