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Biomedical subjects

G Escolar

Publications and source records attributed to G Escolar.

At least 109 records · Page 6Linked to original sources
PubMed1995

Talin does not associate exclusively with alpha 2b beta 3 integrin in activated human platelets.

Talin is a high-molecular-weight protein that may stabilize connections between cytoplasmic actin and the submembrane portion of glycoprotein IIb-IIIa (GPIIb-IIIa) (alpha 2b beta 3 integrin) in thrombin-stimulated human platelets. Using morphologic and electrophoretic techniques, we have examined the association of talin with the cytoskeleton of platelets activated by thrombin in the presence of fibrinogen-coated gold particles (Fgn/Au). Ultrastructural studies confirmed the presence of Fgn/Au firmly bound to the outside membranes of detergent-extracted platelets. Immunoblots of protein bands showed GPIIIa, but not talin, associated with cytoskeletons of activated platelets. Immunogold cytochemical techniques were performed on ultrathin cryosections of whole platelets to localize talin at the ultrastructural level. Studies were performed on normal platelets and platelets defective in GPIIb-IIIa (Glanzmann's thrombasthenia) and GPIb (Bernard-Soulier syndrome). Talin was randomly distributed in the cytoplasm of resting platelets. Activation resulted in binding of Fgn/Au to the surface membrane and redistribution of talin to the submembrane region. However, no definitive colocalization between the two markers was noted. Activated thrombasthenic platelets failed to bind Fgn/Au, but talin was localized to the submembrane location. After activation, talin was confined to the submembrane zone of Bernard-Soulier syndrome platelets. No definitive colocalization was observed between large clusters of Fgn/Au-occupied receptors and talin distributed in the submembrane region. GPIb and GPIIb-IIIa are not necessary for talin to localize in the submembrane region of activated cells. Talin does not redistribute exclusively with GPIIb-IIIa, and it may stabilize connections with other glycoproteins.

Blood Platelets↗
PubMed1995

Dynamic redistribution of glycoprotein Ib/IX on surface-activated platelets. A second look.

The present study has re-evaluated the mobility of glycoprotein Ib/IX (GPIb/IX), the von Willebrand factor receptor, on surface-activated platelets. A previous report employing immunogold cytochemistry with monoclonal and polyclonal antibodies specific for GPIb/IX concluded that the receptor remained stabilized in plasma membranes and did not move during platelet attachment and spreading on formvar grids, despite the observation that immunogold particles marking GPIb/IX were missing from peripheral margins and pseudopods of the surface-activated platelets. Addition of thrombin to surface-activated, spread platelets freed GPIb/IX from its anchor to the membrane and stimulated movement of receptor-ligand complexes into caps over centers of spread platelets. In our investigation, surface-activated platelets, stimulated or not by thrombin, were fixed in a higher concentration of glutaraldehyde than used by the earlier workers before exposure to monoclonal or polyclonal antibody to GPIb/IX, after incubation with the antibody, but before treatment with the immunogold marker, protein A gold (PAG), or after both antibody and PAG. When fixed before exposure to antibody and PAG, GPIb/IX receptors were dispersed evenly over dendritic and spread platelets from edge to edge, including peripheral margins and pseudopods. Thrombin had no influence on distribution of the receptors. Exposure to antiglycocalicin antibody before fixation caused movement of GPIb/IX receptors from peripheral margins of spread cells and pseudopods of dendritic forms. Thrombin treatment did not enhance the movement. Fixation after exposure of surface-activated platelets, treated or not with thrombin, to antibody and PAG caused movement of GPIb/IX receptors into caps over cell centers. Results indicate that central movement of GPIb/IX receptors is unrelated to surface activation, spreading, or thrombin stimulation. Rather, the translocation is caused by the antiglycocalicin antibody and accentuated by PAG.

Antibodies, Monoclonal↗
PubMed1995

Effect of plasma from patients with primary antiphospholipid syndrome on platelet function in a collagen rich perfusion system.

The effect on platelet function of plasma from 9 patients with primary antiphospholipid syndrome (PAS) with previous thrombotic episodes was investigated under flow conditions. Five asymptomatic individuals with antiphospholipid antibodies (aPL) (A-aPL) and 14 normal controls were also studied. Patients and controls plasmas were added (1:20 v/v) to anticoagulated blood and perfused through annular chambers containing collagen rich vessel segments. The interaction of platelets with vessel subendothelium was morphometrically evaluated in thin sections. An increase in both covered surface and thrombi formation was observed in perfusions in the presence of PAS-plasma (mean +/- SD: 34.2% +/- 9.6% and 23.2% +/- 10.0% respectively) compared with control plasmas (21.4% +/- 7.3% and 10.1% +/- 7.7%, p < 0.01). Affinity purified anticardiolipin antibodies from one PAS patient showed a similar effect when added to normal blood. In contrast, A-aPL plasma had no effect on platelet-subendothelium interaction. In parallel studies, the same plasmas were incubated with isolated normal platelets before and after activation with ADP or collagen and the binding of immunoglobulins (Ig) was determined by flow cytometry. A significantly increased binding of Ig was observed in 8 out of 9 plasmas from PAS patients when platelets had been activated with collagen but not when resting or ADP activated platelets were used. No increased Ig binding to platelets was seen using A-aPL individuals plasma. These observations might help to explain the pathophysiology of the thrombotic events occurring in patients with PAS.

Adult↗
PubMed1995

[Expression of von Willebrand's disease in endothelial cord cell from a type 1 patient. Discrepancies between clinical manifestations and laboratory tests].

PURPOSE: The reactivity of subendothelium generated by endothelial cells attained from the umbilical cord of a newborn girl from a patient with type I von Willebrand's disease was evaluated. MATERIAL AND METHODS: Platelet adhesiveness was assessed by means of "ex vivo" blood perfusion systems (shearing coefficients from 300 to 1,300 s-1), on umbilical artery subendothelium and over the extracellular matrix (ECM) generated by cultured endothelial cells attained from the umbilical cord of a newborn baby from a type I von Willebrand mother. RESULTS: By means of an ELISA technique, a significant reduction of both the vWF secreted into the culture (p < 0.05) and of that associated with ECM (p < 0.05) was noticed. The two subendothelial surfaces examined supported a lesser extent of platelet adhesion than the control surfaces from healthy individuals in the perfusion tests at 1,300 s-1 (p < 0.05). The experimental results did not agree with the lack of clinical manifestations and laboratory abnormalities in the propositus at age 3. CONCLUSIONS: These findings suggest that the endothelial cells cultured from the tissues of a newborn baby from a type I von Willebrand woman expressed less vWF, at the same time confirming the importance of vWF bound to connective tissue in sustaining platelet adhesion. The data attained here illustrate the complex model of inheritance of moderate von Willebrand's disease, along with the technical limitations of cell culture studies. Such observations must be borne in mind whenever any experimental approach is performed with material attained from ill-differentiated tissues such as the umbilical cord vein.

Endothelium, Vascular↗
PubMed1994

Combined use of immunocytochemical techniques and ligand-gold complexes for investigation of platelet membrane responses to surface activation.

Exposure of blood platelets to foreign surfaces results in dramatic changes in physical appearance and conversion from a non-sticky to an adhesive state. Membrane glycoproteins and cytoskeletal assembly play a pivotal role in these interactions. Cytochemical techniques commonly applied for demonstration of macromolecules in tissues have been used for the localization of target glycoproteins on spread cells. The present review examines different experimental strategies and immunocytochemical techniques that can be combined to better understand the organization of platelet receptors during surface activation. Glycoprotein IIb-IIIa (GPIIb-IIIa) was localized by immunocytochemical techniques on fixed, surface-activated platelets. The distribution of functional fibrinogen receptors expressed on GPIIb-IIIa was revealed by incubation of fixed platelets with fibrinogen-gold conjugates (Fgn/Au). The movement of receptor complexes was investigated in additional experiments in which surface-activated platelets were interacted with Fgn/Au and then fixed at different periods. The overall impression of these observations suggests that fibrinogen receptors on surface-activated platelets do not redistribute spontaneously and that particulates (gold particles), rather than fibrinogen, may trigger the movement. These results are presented in detail and their significance discussed in the light of current theory. Applications and limitations of such techniques are also discussed.

Antibodies, Monoclonal↗
PubMed1994

Influence of combined thrombin stimulation, surface activation, and receptor occupancy on organization of GPIb/IX receptors on human platelets.

Down-regulation and clearance of as many as 60-80% of GPIb/IX receptors from exposed surfaces on thrombin-activated platelets to channels of the open canalicular system (OCS) is considered to be a fundamental mechanism regulating platelet adhesivity in vitro and in vivo. The present study has combined thrombin stimulation in suspension, surface activation on formvar grids, receptor occupancy by von Willebrand factor (vWF) and exposure to anti-vWF antibody in an effort to demonstrate the removal of GPIb/IX receptors from activated cells. Individually the stimuli failed to cause any change in the frequency of GPIb/IX receptors. Combined, the stimuli were no more effective than when each was used alone. The only way to cause GPIb/IX to move was to add anti-vWF to thrombin-activated platelets allowed to spread on formvar grids and covered with multimers of ristocetin-activated human or bovine vWF. Translocation of GPIb/IX-vWF-anti-vWF complexes from peripheral margins into caps over cell centres, however, did not clear the peripheral zone of vWF binding capacity. Exposure of capped platelets after fixation to a second incubation with vWF demonstrated as many multimers extending from the central cap to the peripheral margins as were seen on platelets exposed a single time to vWF. Antibodies to GPIb, but not to GPIIb/IIIA, prevented the second labelling by vWF. Down-regulation or clearance of GPIb/IX, in light of this study, does not appear to be a fundamental mechanism modulating platelet adhesivity.

Blood Platelets↗
PubMed1994

Platelet activation during hemodialysis measured through exposure of p-selectin: analysis by flow cytometric and ultrastructural techniques.

P-selectin is a platelet protein in alpha-granules that is expressed on their membranes after platelet activation. Using a specific monoclonal antibody (RUU-2.17), we have studied platelet activation during hemodialysis with a cuprophan membrane in eight patients with uremia. Blood samples were obtained before hemodialysis from venipuncture (sample I), at the beginning of the hemodialysis from the arterial (sample II) and venous lines (sample III), and after 2 hours of hemodialysis from the venous line (sample IV). Exposure of P-selectin on platelet surface was studied by flow cytometry, and morphologic and immunocytochemical studies were performed. No differences in P-selectin expression were observed by single-labeling flow cytometry in samples I, II, and IV. However, a significant increase in fluorescence occurred in sample III (percentage of positive platelets [%PP], 18.0% +/- 6.1%) with respect to sample I (%PP, 6.9% +/- 2.0%; p < 0.01). An apparently decreased fluorescence was seen in sample IV (%PP, 8.8% +/- 3.9%) with respect to sample III. Double-labeling flow cytometry showed statistically significant differences between sample I (%PP, 7.1% +/- 2.6%) and sample III (%PP, 19.4% +/- 9.4%; p < 0.01) and IV (%PP, 19.0% +/- 10.1%; p < 0.01), but not between samples III and IV. The ultrastructural study revealed an increasing number of platelet morphologic signs of activation throughout the period of hemodialysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

[Interaction of platelets with an artificial polystyrene surface under flow conditions: comparative study versus collagen].

PURPOSE: Platelets are known to interact with artificial surfaces, which leads to thrombosis that in turn may hamper repairing surgery. In order to evaluate such interaction, platelet adhesiveness on a polystyrene surface (Thermanox) was studied in an in vitro perfusion model. MATERIAL AND METHODS: Anticoagulated human blood was recirculated through a plain perfusion chamber containing fragments of the material under study, uncovered and covered with collagen. The studies were performed at a shearing coefficiente of 800 s-1 and with different timing. Additional blood samples were incubated with an anti- GPIIb-IIIa antibody. Platelet deposition on the perfused material was morphometrically evaluated with a computerized system of image analysis. RESULTS: The values attained, expressed as percent surface covered by platelets over the uncovered polystyrene surface, at 2, 5, 10 and 20 minutes, were, respectively, as follows: 10.4% +/- 1.83%, 27.04% +/- 2.32%, 36.04% +/- 3.09% and 61.48% +/- 8.86%; for the collagen-covered material these figures were: 35.2% +/- 1.3%, 47.8% +/- 4.2%, 71.7% +/- 2.4% and 73% +/- 3.2%, for similar timing (n = 7 for each group). CONCLUSIONS: The perfusion method proved useful for evaluating the interaction of platelets with artificial surfaces. These data show their polystyrene surfaces have high reactivity toward platelets and confirm that GPIIb-IIIa plays an important role in this reactivity.

Blood Platelets↗
PubMed1994

Persistence of mobile receptors on surface- and suspension-activated platelets.

We examined the distribution of glycoprotein Ib (GPIb) and glycoprotein IIb-IIIa (GPIIb-IIIa) receptors on suspension- and surface-activated platelets before and after exposure to thrombin (1 U/ml, 10 minutes). Frozen thin sections prepared from fixed suspension-activated platelets or grids containing fixed surface-activated platelets were stained with specific antibodies to GPIb (antiglycocalicin) and GPIIb-IIIa (AP2 or 7E3), incubated with the corresponding gold-labeled secondary antibody, and examined in the electron microscope. GPIb and GPIIb-IIIa were evenly distributed on membranes of resting and suspension-activated platelets. GPIb and GPIIb-IIIa were present in the open canalicular system (OCS) of resting and, more prominently, in dilated OCS channels of thrombin suspension-activated platelets. On surface-activated platelets more intense labeling for GPIb was observed along pseudopods of dendritic cells whereas GPIIb-IIIa receptors were slightly increased over the peripheral zone. Morphometric study of labeling on fully spread, surface-activated platelets revealed that the density of GPIb increased significantly after thrombin treatment (60.7 +/- 13.1 vs 40.9 +/- 8.3 gold particles/microns 2, p < 0.05). A flow cytometry assay employing the same antiglycocalicin antibody revealed no down-regulation or clearance of GPIb after exposure of platelets to thrombin. GPIIb-IIIa distribution on spread platelets after exposure to thrombin remained basically unchanged (28.4 +/- 10.5 vs 32.6 +/- 10.9 particles/microns2 in nonactivated platelets). These findings indicate that clearance of GPIIb-IIIa and GPIb on suspension-activated platelets does not take place to the extent suggested in previous studies and does not occur spontaneously or after thrombin activation on surface-activated platelets. Although the presence of mobile receptors on platelets is essential for spreading on immobile surfaces and each other, their clearance to the OCS is not a fundamental mechanism regulating adhesion.

Flow Cytometry↗
PubMed1993

Gastroprotective effect of zinc acexamate against damage induced by nonsteroidal antiinflammatory drugs. A morphological study.

The gastroprotective effect of zinc acexamate against gastric damage induced by different nonsteroidal antiinflammatory drugs (indomethacin, diclofenac, and piroxicam) was morphologically assessed in the rat glandular stomach by light and scanning electron microscopy. In addition, the capability of these antiinflammatory drugs to inhibit gastric prostaglandin E2 production was compared with their ability to induce gastric lesions. Microscopically, disappearance of mucus glycoprotein and exfoliation of the mucosal surface were the most common findings. Surface ultrastructural lesions varied from minimal lesions of the surface epithelial cells to deep erosions of the gastric mucosa with release of associated cellular elements and sloughing of the denuded lamina propria. Diclofenac elicited the most powerful inhibitory activity on mucosal prostaglandin E2 (98% inhibition vs control), closely followed by piroxicam (97.8%) and indomethacin (91.05%). Pretreatment of animals with zinc acexamate significantly increased the presence of mucus glycoprotein, maintained the continuity of the surface epithelial cells, and decreased the depth of the mucosal erosions. The degree of protection exerted by zinc acexamate varied with the antiinflammatory, but was always evident.

Aminocaproates↗
PubMed1993

Prothrombin fragment 1 + 2 and thrombin-antithrombin complex measurements indicate continuous and progressive intraoperative thrombin generation in liver transplantation.

Prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complex (TAT) have been measured in the different surgical phases in 40 patients undergoing 43 orthotopic liver transplantations (OLT), in order to study thrombin generation throughout the surgical procedure. F1 + 2 and TAT progressively rose from preoperative values (1.47 +/- 0.92 nmol/l and 22.41 +/- 19.15 ng/ml, respectively) to the end of anhepatic phase (5.97 +/- 2.20 nmol/l, 64.24 +/- 11.30 ng/ml), whereas changes in circulating antithrombin III (ATIII) were negligible in this period. Thrombin generation continued to slightly but significantly increase immediately after liver graft reperfusion (F1 + 2 6.87 +/- 1.65 nmol/l, TAT 89.32 +/- 7.54 ng/ml), and ATIII levels decreased from 70.61 +/- 12.28 to 57.16 +/- 9.57%. Higher preoperative values of F1 + 2 were associated with larger requirements of both packed red blood cells and fresh frozen plasma (FFP) in the host liver explantation phase, whereas the lowest basal levels of ATIII were related to a higher plasma expense in the whole OLT procedure. The activation of circulating prothrombin begins early in OLT, and adequate FFP infusion is capable of maintaining appropriate circulating thrombin-inhibitory activity.

Adolescent↗
PubMed1993

Organization of von Willebrand factor on surface-activated platelets.

The distribution and organization of von Willebrand factor (vWF) multimers on platelets after surface activation have not been fully characterized. In the present study, washed human platelets were allowed to interact with Formvar-coated, electron microscope grids for 20 minutes at 37 degrees C and then fixed. After fixation, cells were washed and then incubated with buffer alone, human plasma, human plasma preincubated with ristocetin (1.2 mg/mL), purified human vWF plus ristocetin, or bovine plasma. Macromolecular complexes were revealed by ultrastructural immunocytochemistry employing a polyclonal antibody against vWF and protein A-gold (PAG) as the electron-dense probe. vWF multimers were not present in discoid platelets but appeared on the central zone of dendritic cells and over larger central areas of fully spread platelets. Exposure to human plasma alone did not affect the distribution of electron-dense probes for vWF in central regions of surface-activated cells. Incubation of spread platelets with ristocetin-activated human plasma or bovine plasma resulted in the appearance of randomly dispersed, mottled areas of increased density covering the surface from edge to edge. Exposure to vWF antibody and PAG resulted in specific labeling of the dense areas in a serpentine, linear array. The gold-probe distribution suggested that the vWF multimers were not superimposed and were distributed in a random, irregular manner from edge to edge with label-free, clear areas between them. The results extend previous observations demonstrating that glycoprotein Ib-IX receptors are not spontaneously cleared from the plasma membranes of surface-activated platelets by showing that the receptor function of glycoprotein Ib-IX complex remains unchanged.

Animals↗
PubMed1993

Influence of heat on platelet biochemistry, structure, and function.

The present investigation has evaluated the influence of temperatures ranging from 37 degrees C to 45 degrees C for intervals of 30, 60, and 90 minutes on the biochemistry, morphology, and function of human platelets. Exposure to temperatures up to 43 degrees C for an hour did not significantly alter platelet morphology or physiologic response to aggregating agents. Samples of platelets heated at 43 degrees for 60 minutes lost their ability to aggregate in response to arachidonate, but sensitivity was restored by pretreatment with epinephrine. Platelets heated to 45 degrees C for 90 minutes were converted from discs to spheres and failed to aggregate in response to all agonists, whether or not they were pretreated with epinephrine. The platelets heated at 45 degrees C for 90 minutes could adhere to formvar or denuded subendothelium but were unable to extend pseudopods or to spread. They maintained normal levels of adenine nucleotides and serotonin but failed to secrete these products on stimulation. Studies with fibrinogen coupled to gold (Fgn/Au) revealed only a few particles bound to glycoprotein IIb-IIIa (GPIIb-IIIa) on platelets heated at 43 degrees C for 90 minutes. Ligands that did bind to GPIIb-IIIa were transported to the open canalicular system. Biochemical studies demonstrated normal synthesis of thromboxane B2 and calcium flux by platelets heated at 45 degrees C for 90 minutes. Polyacrylamide gels of platelets heated at 45 degrees C for 90 minutes showed an increase in talin incorporation into heated platelet cytoskeletons but no increase in filamentous actin. The findings indicate that impaired function of heated platelets is due to the influence of heat on cytoskeletal proteins important for pseudopod extension, shape change, expression of GPIIb-IIIa, or other surface membrane receptors and secretion, but that impaired function is not due to inhibition of biochemical systems involved in activation events.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗