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Biomedical subjects

G Escolar

Publications and source records attributed to G Escolar.

At least 145 records · Page 8Linked to original sources
PubMed1991

The platelet open canalicular system: a final common pathway.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were exposed to thrombin at 1 U/ml for 5, 60, or 180 seconds in the presence of fibrinogen molecules coupled to particles of colloidal gold (Fgn/Au). The samples were fixed in a low concentration of glutaraldehyde and embedded in L.R. White resin to preserve antigenicity. Thin sections were exposed to a rabbit polyclonal antibody to human fibrinogen followed by an anti-rabbit IgG coupled to 5-nm gold beads. Thrombin caused Fgn/Au particles to bind to platelets and enter channels of the surface-connected OCS. Endogenous fibrinogen detected by immunogold 5-nm beads were localized to alpha granules in resting platelets and 5 seconds after thrombin stimulation. At 60 seconds and 3 minutes Fgn/Au particles were present in swollen alpha granules, as well as OCS channels. Fibrinogen gold beads were evident in alpha granules and OCS channels connected to the platelet surface. The 18- to 20-nm Fgn/Au particles were in the same channels of the OCS as fibrinogen gold beads. The OCS is a final common pathway for uptake of particulates and discharge of secretory products in thrombin-activated human platelets.

Biological Transport↗
PubMed1991

The blood platelet open canalicular system: a two-way street.

Channels of the surface-connected, open canalicular system (OCS) of human platelets serve as the pathway for transport of substances into the cells and as conduits for the discharge of alpha granule products secreted during the platelet release reaction. The purpose of the present study was to determine if both functions of the OCS can take place simultaneously. Suspensions of washed platelets were incubated with 18 to 20-nm colloidal gold particles coupled to fibrinogen molecules (Fgn/Au) for 5 min, then exposed to 0.2, 1 or 5 U of thrombin/ml for 5, 10, 15, 30, 45, or 300 s. The samples were fixed in glutaraldehyde and osmic acid containing tannic acid under conditions that stain alpha granule fibrinogen during secretion from thrombin-stimulated cells. Thrombin caused Fgn/Au particles to bind to platelets and be cleared to channels of the OCS at about the same rate, regardless of the thrombin concentration. Discharge of tannic acid-osmium stained fibrinogen from alpha granules to channels of the OCS and platelet exterior was, on the other hand, time and thrombin concentration dependent. Fgn/Au receptor complexes were observed in the same OCS channels as the tannic acid-osmium stained alpha granule secretion products. Thus, the platelet OCS appears to be a two-way street with different speed limits for incoming and outgoing traffic.

Biological Transport↗
PubMed1990

[Effects of the treatment with human recombinant erythropoietin on primary hemostasis in uremic patients].

The effect of human recombinant erythropoietin (r-EPO) on primary hemostasia was studied in 9 patients undergoing hemodialysis. The analysis was performed before and after the hematocrit reached a value of 30%. The most important complications observed during the study period were a death for acute myocardial infarction in a patient with previous severe ischemic cardiopathy and a thrombosis of the venous line. The bleeding time shortened in four patients although the mean value did not change significantly. Platelet count showed a non significant increase. There was a significant improvement in in vitro platelet aggregability with ADP (p less than 0.05), arachidonic acid (p less than 0.05), adrenaline (p less than 0.05), and ristocetin (p less than 0.05) as well as in the parameters that quantify the interaction between platelets and subendothelium in in vitro experiments using perfused models (p less than 0.05). There were no significant changes in coagulation and fibrinolysis tests. The treatment with r-EPO improved primary hemostasia in uremic patients. This beneficial effect was due to the increased hematocrit and to the improvement of platelet function induced by r-EPO.

Adult↗
PubMed1990

Effect of zinc acexamate on gastric lesions induced by aspirin: a morphological study.

The morphology of gastric lesions induced by aspirin in the rat and their modification by pretreatment with zinc acexamate (100 mg/kg) were studied by scanning electron microscopy. The influence of mucosal levels of prostaglandin E2 (PGE2) on the development of these lesions was also investigated. High (200 mg/kg) or low (50 mg/kg) doses of aspirin inhibited PGE2 production similarly, but the morphology of these lesions differed considerably. While gross exfoliation of extensive areas of gastric mucosa was observed after 200 mg/kg aspirin, only ultrastructural lesions of surface epithelial cells were present after 50 mg/kg aspirin. Regardless of the dose of aspirin administered, pretreatment with zinc acexamate raised PGE2 levels and increased the presence of mucus. Our results showed that after zinc acexamate, the development of deep erosions appearing with high doses of aspirin was prevented and the ultrastructural lesions induced by low doses of aspirin were not observed. The fact that zinc acexamate did not modify the anti-inflammatory action of aspirin in the carrageenin-induced oedema model suggests that the protective effect of zinc acexamate is exerted locally on the gastric mucosa.

Aminocaproates↗
PubMed1990

Uremic platelets have a functional defect affecting the interaction of von Willebrand factor with glycoprotein IIb-IIIa.

Uremic patients have an impaired platelet function that has been related to membrane glycoprotein (GP) abnormalities. Using a perfusion system, we have studied the interaction of normal and uremic platelets with vessel subendothelium (SE) under flow conditions. Reconstituted blood containing washed platelets, purified von Willebrand factor (vWF) (1 U/mL), and normal washed red blood cells was exposed to de-endothelialized rabbit segments for 10 minutes at two different shear rates (800 and 1,600 seconds-1). In some experiments a monoclonal antibody to the GPIIb-IIIa complex (EDU3) was added to the perfusates. With normal platelets, the percentage of the vessel covered by platelets (%CS) was 23.1% +/- 3.7% at 800 seconds-1 and 30% +/- 4.3% at 1,600 seconds-1. Platelets were observed in contact or forming monolayers on vessel SE. EDU3 inhibited the spreading of normal platelets. The %CS (11.1% +/- 3.3%) was statistically decreased (P less than .01) and most of the platelets were observed in contact with the vessel surface. These data indicate that, under flow conditions, the interaction of vWF with GPIIb-IIIa can support the spreading of normal platelets in the absence of exogenous fibrinogen. Under the same experimental conditions, the interaction of uremic platelets with SE was markedly impaired at both shear rates studied (P less than .01 v normal platelets). The presence of EDU3 did not modify the interaction of uremic platelets. These results confirm the impairment of the platelet adhesion observed in uremic patients. Furthermore, they indicate the presence of a functional defect in the interaction of vWF with GPIIb-IIIa. The fact that perfusions with normal and uremic platelets in the presence of an antibody to the GPIIb-IIIa complex did not show any differences gives indirect evidence on a functionally normal interaction vWF/GPIb in uremic patients.

Blood Platelets↗
PubMed1990

Differential localization of von Willebrand factor, fibronectin and 13-HODE in human endothelial cell cultures.

Von Willebrand factor (vWF), fibronectin (FN) and 13-hydroxy-octadecadienoic acid (13-HODE) are known to influence the regulation of the adhesive properties of vascular surfaces. In the present study vWF, FN and 13-HODE were comparatively localized in endothelial cells (EC) and in the extracellular matrix (ECM) produced by EC. An indirect immunofluorescent technique was applied to coverslips containing human EC cultures previously fixed and permeabilized following different procedures: A. Alcohol/acetone; B. Paraformaldehyde alone and C. Paraformaldehyde followed by Triton X-100. vWF was observed inside EC (A), on the ECM produced by EC (B) or in EC and ECM (C) depending on the fixation procedures used. FN was mainly localized in the ECM despite the fixation procedures employed. FN was only seen in relation to cell bodies after strong permeabilization (A). Under our experimental conditions 13-HODE was never found in ECM. This latter antigen was observed randomly dispersed in those preparations fixed with alcohol/acetone, indicating that it is probably extracted by this fixative. 13-HODE was detected in granular shaped structures in EC after permeabilization with detergent (C). These results suggest that the cellular localization of vWF and FN is compatible with an adhesive role related to the abluminal side of ECs. 13-HODE was readily observed after mild permeabilization. This finding would be morphologically consistent with its contribution to the regulation of the vessel wall thromboresistance.

Cells, Cultured↗
PubMed1990

Receptor-ligand complexes are cleared to the open canalicular system of surface-activated platelets.

Human platelets were incubated with gold particles coupled to fibrinogen to label the glycoprotein IIb-IIIa (GPIIb-IIIa) receptor after initial activation of the cells by contact with formvar-coated grid and glass surfaces. Fibrinogen-gold (Fgn-Au) markers were absent on discoid platelets, but diffusely spread over the surface and extended pseudopods of early dendritic cells. Conversion to spread platelets resulted in movement of ligand-receptor complexes away from the cell margin toward cell centres. However, Fgn-Au gold did not concentrate in the central region. Rather, the Fgn-Au, GPIIb-IIIa complexes in the middle of spread platelets appeared to move toward a belt-like, intermediate zone, as did the ligand receptor complexes from the cell margin and pseudopods. The ultimate destination of the mobile receptor-ligand complexes, however, appeared to be channels of the surface-connected open canalicular system (OCS). Fgn-Au was concentrated in OCS channels of most dendritic and a small proportion of spread platelets. The decreased frequency of Fgn-Au filled channels in more transformed platelets may have been due to collapse or evagination of the OCS. Examination of platelets exposed to Fgn-Au after spreading on glass and then prepared for thin sections confirmed that the OCS was the final destination for mobile ligand receptor complexes on surface-activated platelets. Findings of this study are consistent with previous work showing clearance of mobile receptor-ligand complexes to the OCS of platelets activated in suspension.

Blood Platelets↗
PubMed1990

Zinc acexamate inhibits gastric acid and pepsinogen secretion in the rat.

Pretreatment with zinc acexamate (25-100 mg kg-1 i.p.) inhibited acid and pepsinogen secretion in the pylorus-ligated rat. Zinc acexamate (5-50 mg kg-1 p.o.) also inhibited the increases in acid secretion induced by carbachol (10 micrograms kg-1) and 2-deoxy-D-glucose (200 mg kg-1) in the perfused stomach of the anaesthetized rat. A delayed antisecretory effect was observed with this drug on histamine induced responses. High concentrations of zinc acexamate (10(-5) - 10(-2) M) did not modify the in-vitro activity of pepsin. Administration of zinc acexamate resulted in an increase in the presence of pepsinogen at the mucosal level. A morphological examination of the gastric mucosa confirmed an accumulation of zymogen-containing granules in the gastric chief cells of zinc acexamate-treated rats (50 mg kg-1 p.o.). These results indicate that zinc acexamate decreases acid and pepsinogen secretion in-vivo, and this may explain its antiulcer activity.

Aminocaproates↗
PubMed1990

Effect of zinc acexamate and ranitidine on chronic gastric lesions in the rat.

Using the rat as an experimental model we have studied the healing of chronic gastric lesions and the modifications of these lesions by antiulcer agents. Gastric injuries were induced by submucosal injection of 0.05 ml of 5% acetic acid. Placebo, ranitidine (RNT) or zinc acexamate (ZAC) were administered orally. The evolution of gastric injuries was macro- and microscopically evaluated at 6, 12 and 21 days after acetic acid injection. The administration of either RNT (30 mg/kg) or ZAC (200 mg/kg) was followed by a marked improvement of the healing process with respect to control groups. The size of experimental ulcers at 21 days was 3.1 +/- 0.8 mm2 for the control group, 1.8 +/- 1.1 mm2 for RNT-treated animals and 0.3 +/- 0.6 mm2 for ZAC-treated rats (p less than 0.05, vs. control). A similar tendency was observed when lesions were microscopically analyzed. Indices of microscopical lesions (0-6) at 21 days were 3.8 +/- 0.8 for the control group, 3.0 +/- 0.8 for rats receiving RNT and 2.3 +/- 0.4 for rats receiving ZAC (p less than 0.05, vs. control). The statistical analysis of the distribution of microscopical indices of lesions showed significant differences in favour of ZAC at days 6 (p less than 0.01) and 21 (p less than 0.05). Our study indicates that the evolution of gastric damage induced by acid acetic injection was consistently better in rats treated with ZAC than in those receiving RNT. Data obtained in our experiments suggest that the blockade of H2 receptors does not guarantee the optimal healing of chronic gastric lesions induced in rats.

Acetates↗
PubMed1990

Mobility of GPIIb-IIIa receptors within membranes of surface- and suspension-activated platelets does not depend on assembly and contraction of cytoplasmic actin.

Investigations using fibrinogen coupled to colloidal gold (Fgn/Au) have shown that glycoprotein IIb-IIIa (GPIIb-IIIa) receptors are mobile and undergo centripetal reorganization on spread platelets following surface activation. The assembly of cytoplasmic actin and its constriction into an inner filamentous zone have been proposed as the mechanism driving the Fgn/Au receptor complexes across the plasma membrane. The present study has used cytochalasin B (CB), an agent known to inhibit actin assembly and cause breakdown of newly formed actin filaments to test that hypothesis. At a concentration which inhibited pseudopod formation and spreading, CB did not block movement of Fgn/Au receptor complexes toward platelet centers or into the open canalicular system (OCS). Channels of the OCS filled with Fgn/Au receptor complexes were evident in over 90% of the CB-treated, surface-activated platelets. The findings do not support the concept that assembly of cytoplasmic actin and its contraction move receptor-ligand complexes on the platelet plasma membrane.

Actins↗
PubMed1990

[Update on the role of vascular, plasmatic and cellular components in the regulation of the interaction of platelets with the subendothelium].

The interaction of platelets (Plts) with subendothelium (SE) or with extracellular matrices (ECM) generated by endothelial cells was studied under flow conditions. The role of: a) platelet membrane glycoproteins (GPS) or subunits; b) plasma adhesive proteins such as von Willebrand factor (VWF) and fibronectin (Fn); and c) structural proteins of the SE such as laminin (Lm). Specific antibodies or purified proteins were added to the perfusates. GPS. Monoclonal antibodies (MoAb) directed against distinct epitopes of the GPIIb-IIIa affected in different manner the deposition of platelets. MoAb EDU3 decreased (p less than 0.01) the surface of the vessel covered by platelets (% CS) and increased the presence of platelets in contact (% C) with the SE. MoAb C17 did not modify the % CS but altered the morphological characteristics of the aggregates. A monoclonal antibody against GPIIb, decreased the % CS (p less than 0.01) without influencing % C; VWF. A linear progression of the % CS was observed for VWF levels ranging from 0.1 to 0.6 U/mL. A plateau was reached for concentrations of vWF above 0.6 U/mL; Fn. Perfusions in which blood was reconstituted with Fn-depleted plasma showed the cooperation of this protein facilitating Plt-Plt interaction. No statistical differences were observed in the % CS when ECM were incubated with a MoAb against Fn (3E3); Lm. The % CS decreased statistically (p less than 0.01) when SE or ECM were incubated with an antibody to Lm. These results show the critical role of vascular, plasmatic and cellular components in the maintenance of and adequate platelet function.

Animals↗
PubMed1990

A rapid embedding procedure for the study of platelet interactions with extracellular matrices in a flowing system. Effect of aspirin on platelet activity.

The cultured endothelial cell (EC) is currently used as a model for the study of the interaction of platelets with the vascular wall. Described is a method for rapid quantitative and qualitative evaluation of platelet interactions with extracellular matrices (ECM) produced by human cultured ECs growing on plastic coverslips. Morphometric calculations can be performed on the same perfused coverslips. A very good correlation (r = 0.96) was found between results of a morphometric method en face and those obtained from analysis of cross sections of the perfused coverslips. A shear rate-dependent increase on platelet deposition onto ECMs was observed with both morphometric procedures. The method is sensitive enough to detect drug-related changes of platelet function. An impairment of the interaction of platelets with the ECM was observed when blood obtained from healthy volunteers who took 500 mg aspirin/day for five days was perfused. Aspirin showed a marked effect, decreasing platelet spreading onto the subendothelium (p less than 0.05). The embedding method described benefits from the use of plastic coverslips that are easily detected from the glycol methacrylate compound used for the embedding procedure. Quantitative analysis en face (covered surface) and qualitative evaluation of platelet interactions (contact, adhesive and aggregated platelets) in cross sections are performed on the same coverslip. This embedding procedure provides a useful tool for the study not only of platelet interactions with ECMs but also for the investigation of interactions of blood elements with other cultured cells.

Aspirin↗
PubMed1990

Platelet membrane responses to surface and suspension activation.

Exposure of blood platelets to foreign surfaces or to potent agonists in suspension results in dramatic changes in physical appearance and conversion from a nonsticky state. The transformation to the sticky state is associated with exposure of the fibrinogen receptor, GPIIB-IIIa, which is hidden in resting, discoid platelets. Recent studies employing fibrinogen coupled to gold (Fgn-Au) as an electron-dense probe have suggested that GPIIb-IIIa receptors not only become exposed in surface-activated platelets, but undergo a reorganization not observed in suspension-activated cells. Discoid platelets do not bind Fgn-Au; however, the bodies and extended pseudopods of dendritic forms are covered with Fgn-Au particles. Conversion of dendritic platelets to spread forms is accompanied by movement of receptor-ligand complexes away from peripheral margins into a concentrated mass in cell centers over the inner filamentous zone of the cytoplasm. Movement of the Fgn-Au particles to cell centers during spreading was considered due to the transmembrane action of the newly assembled actin filaments. We have carried out similar experiments on surface- and suspension-activated platelets with Fgn-Au and latex particles. GPIIb-IIIa receptors move Fgn-Au particles on outer membranes of surface- and suspension-activated platelets to channels of the open canalicular system. Treatment with cytochalasin B prevents assembly of actin filaments in surface- and suspension-activated platelets, and dissociates residual actin from the cell membranes, circumferential microtubules and organelles with which they interact. However, cytochalasin B does not prevent removal of Fgn-Au to channels of the open canalicular system. Thus, reorganization of fibrinogen receptors on surface- and suspension-activated platelets is due to the particles, and not to the fibrinogen, although fibrinogen is required to link gold to the receptor. The surface membrane has its own detergent and cytochalasin B-resistant cytoskeleton for directed transport of ligand-receptor complexes, independent of the internal assembly and contraction of actin into an inner filamentous zone.

Blood Platelets↗
PubMed1990

Induction of receptor clustering, patching, and capping on surface-activated platelets.

Interaction of selective ligands or specific antibodies with mobile glycoprotein IIb-IIIa receptors on suspended human platelets is associated with reorganization of receptor-ligand complexes. Words used to describe the reorganization have been adapted from similar, antigen-antibody redistribution on lymphocytes, and include descriptive terms such as clustering, patching, capping, and endocytosis. Studies on the interaction of fibrinogen coupled to colloidal gold (Fgn/Au) with glycoprotein IIb-IIIa on surface-activated cells failed to reveal clustering, patching, capping, and endocytosis observed by others on agonist-stimulated or immunoreacted platelets in suspension. The present study sought to induce these phenomena in order to distinguish them from the ligand-receptor interaction and translocation that do develop on surface-activated platelets. Treatment of platelets with cytochalasin B after spreading, but before exposure to Fgn/Au, resulted in clustering of electron-dense probes. Addition of cytochalasin B to fully spread platelets after they had been incubated with Fgn/Au produced patching. Allowing grids of fully spread platelets reacted with Fgn/Au for 5 minutes to rest on drops of Hanks' balanced salt solution for 10 minutes resulted in capping. Platelets exposed to cytochalasin B in suspension before interaction with Formvar grids failed to spread fully or develop pseudopods, but channels of the open canalicular system became filled with Fgn/Au-receptor complexes. The Fgn/Au-filled channels were concentrated in cell centers, and could be confused with endocytosis identified previously by immunofluorescence. Thus, phenomena resembling clustering, patching, capping, and endocytosis can be produced on plasma membranes of surface-activated platelets, but do not appear required for response to agonists or surface activation.

Blood Platelets↗
PubMed1990

[Variations in the thrombogenic properties of the extracellular matrix produced by endothelial cells in consecutive passages].

Cultured endothelial cells are currently used as a model for the study of the interaction of platelets with the vascular wall under flow conditions. Little is known about the influence of cell-culture conditions on the preservation of the original characteristics of primary tissues. In our study, the reactivity of platelets towards extracellular matrices produced by endothelial cells from different passage numbers was morphometrically evaluated. The presence and distribution of the extracellular von Willebrand factor was analyzed by immunofluorescent techniques. Morphology of endothelial cells in culture varied dramatically along the culture and polymorphism increased clearly at fourth or fifth passages. Extracellular matrix thrombogenicity decreased significantly from the fifth passage number. Simultaneously, von Willebrand factor distribution in ECM changed its typical pattern. Our results confirm that the extracellular matrix produced by human umbilical vein derived-endothelial cells constitutes a good model for the study of platelet adhesion onto vascular subendothelium. The limited viability of ECs and the variation in the thrombogenic properties of ECM formed after multiple passages must be taken into account when these cells or their products are used in experimental research.

Cell Division↗
PubMed1989

The fate of the open canalicular system in surface and suspension-activated platelets.

We have examined the movement of fibrinogen-gold (fgn-Au) complexes in platelets activated in suspension and by surface contact. Fgn-Au probes did not react with resting cells but were bound to the external membrane of platelets in suspension 5 seconds after addition of 1 U/mL of thrombin. At intervals over a period of 5 to 20 minutes, fgn-Au probes moved from the cell surface to peripheral and then deep channels of the open canalicular system (OCS). When platelets were surface activated by exposure to carbon-stabilized, formvar-coated grids for 5 to 20 minutes and then exposed to fgn-Au complexes for 5 minutes, probes were also observed in the OCS. At 5 minutes, over 40% of the platelets had concentrated fgn-Au in their OCS. Results after 10 minutes revealed 25% with gold-filled channels, 16% after 15 minutes, and 5% after 20 minutes. The decrease in frequency of OCS staining correlated with the increasing frequency of spread platelets, suggesting that tension produced by spreading may cause collapse of the OCS or that the OCS may evaginate onto the platelet during spreading. To evaluate the latter hypothesis, platelets were initially exposed to grids for 5 minutes and then incubated with fgn-Au for intervals of 5 to 20 minutes. The frequency of platelets with fgn-Au concentrated in the OCS was greatest at 5 minutes (44%) and decreased at the same rate as the frequency of spread platelets increased. Only 14.7% of the cells contained fgn-Au in the OCS after 20 minutes. These were primarily dendritic in form, while fully spread platelets rarely contained an OCS filled with the probe. The study indicates that fgn-Au particles are cleared to channels of the OCS independent of the mechanism of platelet activation. Fgn-Au that has been concentrated in the OCS at early stages of surface activation can be externalized during platelet spreading but remain internalized in suspension-activated cells. The OCS represents a membrane reservoir that can be evaginated onto the platelet surface during interaction with surfaces.

Blood Platelets↗
PubMed1989

Role for platelet von Willebrand factor in supporting platelet-vessel wall interactions in von Willebrand disease.

Twelve infusions of plasma concentrates of von Willebrand factor (vWF) were given to four patients with severe (type III) von Willebrand disease (vWD). Their prolonged bleeding times were either completely or partially corrected after five infusions and had not changed after the remaining seven. In contrast, the low platelet coverage of the subendothelial surface of rabbit aorta perfused with normal washed platelets and red cells resuspended in preinfusion patient plasma was completely or partially corrected in ten instances by replacing preinfusion plasma with postinfusion plasma and remained unchanged in two. Postinfusion improvement in surface coverage was greater than that in bleeding time, suggesting that vWF from normal platelets is needed to support optimal platelet-vessel wall interactions in vWD. This possibility was further explored through other perfusion experiments. The subendothelial surface covered by platelets from an untreated patient with type III vWD (containing no measurable vWF) or from a type IIA vWD patient (containing dysfunctional vWF) resuspended in normal plasma was much smaller than that covered by normal platelets resuspended in normal plasma. These results establish that platelet vWF is important in supporting platelet-vessel wall interactions in vWD and also provide experimental support in favour of the therapeutic transfusion of normal platelets in addition to vWF concentrates to correct the bleeding time in vWD patients.

Blood Platelets↗