Publications (24)73.46 Total impact
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Article: Differences and similarities in tyrosine phosphorylation of proteins in platelets from human and pig species.
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ABSTRACT: Pigs have been widely used as animal models to study hemostasis. However, there are significant differences when comparing the hemostatic behavior of pig and human platelets. To investigate signaling through tyrosine-phosphorylation of proteins in pig platelets after activation in suspension or by adhesion under flow conditions, in comparison with human platelets. Activation of platelet suspensions was performed with thrombin (T; 0.1 and 1 U mL(-1)) and type I collagen (Col-I; 20 microg mL(-1)), at two different time points (30 and 90 s). Activation by adhesion was carried out on Col-I-coated coverslips, using citrated whole blood samples perfused through a parallel-plate chamber. Significant differences between pig and human platelets were detected before and after activation. Activation of pig platelets required higher concentrations of thrombin, as well as increased activation times, to achieve similar levels of tyrosine phosphorylation. Proteins p160, p140, p85 and pp62, present in human platelets, were not detected in profiles corresponding to activated pig platelets. A protein of 70 kDa appeared only in pig platelet profiles, p55 was highly phosphorylated, and the phosphorylation levels of some proteins were significantly different from those found in human platelet profiles. In profiles corresponding to adhered pig platelets, p85 and p62 were absent, and p115 appeared highly phosphorylated. As observed in suspension studies, p70 and p55 appeared specifically in adhered pig platelets. Our study shows that the phosphotyrosine proteins involved in the activation of pig platelets are significantly different from those observed in activated human platelets. These findings may help to explain the differing adhesive and cohesive properties of platelets from both species, which should be considered when extrapolating results.Journal of Thrombosis and Haemostasis 12/2003; 1(11):2411-8. · 5.73 Impact Factor -
Article: A von Willebrand factor/factor VIII complex restores platelet adhesion and fibrin formation in type 3 von Willebrand syndrome.
Journal of Thrombosis and Haemostasis 05/2003; 1(4):865-6. · 5.73 Impact Factor -
Article: [Uremic media affects hemostatic properties of human endothelial cells in culture and increases the production of von Willebrand factor].
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ABSTRACT: We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effect of the uremic media on the morphology of ECs, and their resistance to flow was analyzed. The reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic media resulted in abnormal morphology and signs of accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (22% vs 13%). Platelet deposition and formation of aggregates were significantly elevated on ECMs generated in the presence of uremic media (40.23 +/- 6.43% vs 25.42 +/- 2.69%, p < 0.05, n = 5). Immunocytochemical methods detected an enhanced expression of von Willebrand factor antigen on uremic ECMs (uremic 17.1 +/- 4.2% vs control 13.57 +/- 3.98%, p < 0.05) and its mRNA expression in endothelial cells (uremic 213.24 +/- 6.13 vs control 200.77 +/- 7.52, p < 0.05). These results suggest that uremic medium alters endothelial function and impairs the antithrombotic functions of cultured endothelial cells. This effect may contribute to the increased cardiovascular and thrombotic risk reported in ESRD patients.Nefrologia: publicacion oficial de la Sociedad Espanola Nefrologia 01/2002; 22(1):33-41. · 1.00 Impact Factor -
Article: State-of-the-art knowledge on thrombosis and hemostasis.
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ABSTRACT: The XVIII Congress of the International Society on Thrombosis and Haemostasis, held July 6-12, 2001, in Paris, France, covered the latest advances in the field of thrombosis and hemostasis, including vascular biology. Plenary conferences, state-of-the-art lectures, symposia, and oral and poster presentations focused most of the attention on the current research on the pathogenesis and treatment of thrombotic and hemorrhagic disorders and provided updates on trials currently under way or already completed.Drug News & Perspectives 11/2001; 14(8):508-12. · 2.21 Impact Factor -
Article: Uremic medium disturbs the hemostatic balance of cultured human endothelial cells.
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ABSTRACT: We have investigated the ability of serum from uremic patients to modify the thrombogenic properties of the endothelium. The effects of uremic medium on the morphology of endothelial cells (ECs), and their resistance to flow was analyzed. The influence of uremic media on the reactivity of the extracellular matrix (ECM) generated by ECs towards normal platelets was evaluated in a parallel-plate perfusion chamber. Exposure of ECs to uremic medium resulted in abnormal cell morphology and signs of an accelerated growth. Detachment of ECs exposed to circulating blood was increased when cells had been grown with media supplemented with uremic serum (21% vs. 14% non exposed). Platelet deposition was significantly elevated on ECMs generated in the presence of uremic media (uremicECMs) (p<0.01 vs. control studies). Effects of uremic serum were not observed at short incubation periods (5 h) but were evident after 24 or 72 h of incubation. Northern blot analysis revealed increased expression of tissue factor (TF) mRNA in ECs exposed to uremic conditions. Immunocytochemical methods detected an augmented expression of TF antigen on uremic ECMs. Incubation of ECMs with an antibody to human tissue factor prevented the increase in platelet deposition observed in uremic ECMs, suggesting that the presence of TF in ECM could be responsible for the enhanced platelet deposition. Results from our study indicate that uremic medium impairs the antithrombotic functions of cultured endothelial cells.Thrombosis and Haemostasis 10/2001; 86(4):1099-105. · 5.04 Impact Factor -
Article: Latest advances from basic and clinical research in hematology.
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ABSTRACT: New treatments in hematological malignancies were a focal point of sessions and presentations at the 42nd Annual Meeting of the American Society of Hematology, held December 15, 2000, in San Francisco, California, U.S.A. The meeting also provided discussion on pathogen inactivation in blood banking, stem cell transplantation in leukemia as well as nonmalignant diseases, the reparative potential of stem cells, a new oral antithrombotic therapy and a new class of highly selective factor Xa inhibitors.Drug News & Perspectives 03/2001; 14(1):50-3. · 2.21 Impact Factor -
Article: Alterations in cytoskeletal organization and tyrosine phosphorylation in platelet concentrates prepared by the buffy coat method.
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ABSTRACT: Numerous morphologic and biochemical changes occurring during platelet storage may result in the impairment of platelet function. The effect of preparation and storage conditions on platelet function was analyzed through evaluation of cytoskeletal organization and signaling mechanisms involved in the activation of platelets by thrombin. Samples of platelets prepared by the buffy coat method were obtained before and after the platelet concentrates were prepared during storage for 1, 3, and 5 days. Thrombin-induced aggregation was monitored, and changes in the organization of proteins in the cytoskeleton were analyzed by gel electrophoresis. For the analysis of tyrosine phosphorylation, proteins were transferred to nitrocellulose membranes and probed with a specific antibody. The aggregation and the cytoskeletal organization induced by thrombin activation were markedly impaired immediately after preparation of platelet concentrates, although they normalized after the first 24 hours of storage and decreased progressively after 3 days of storage. Results in tyrosine phosphorylation paralleled those obtained with cytoskeletal organization, except for samples obtained immediately after processing to obtain platelet concentrates. These data indirectly suggest that the stress induced by the preparation method has an activating effect on platelet function that may imply a delayed platelet response to further stimuli. This effect may result in a deficient redistribution of signaling molecules within platelets.Transfusion 06/2000; 40(5):535-42. · 3.22 Impact Factor -
Article: Abnormal platelet cytoskeletal assembly in hemodialyzed patients results in deficient tyrosine phosphorylation signaling.
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ABSTRACT: Uremic patients have a bleeding tendency associated with a platelet dysfunction. We evaluated the impact of a repeated hemodialysis procedure on primary hemostasis by analyzing different aspects of platelet activation in uremic patients. Studies were performed in (1) eight patients with end-stage renal disease before the hemodialysis program was initiated and after initiating hemodialysis treatment, and in (2) eight patients on maintenance hemodialysis who were transferred to continuous ambulatory peritoneal dialysis. Studies included routine platelet aggregations and evaluation of platelet-subendothelium interactions under flow conditions. Contractile proteins and tyrosine phosphorylation associated with the cytoskeleton were analyzed, before and after thrombin activation of platelets, by electrophoresis after Triton X-100 extraction. No changes in the clinical parameters analyzed were observed among the different study groups. Aggregation and platelet adhesion only improved when patients were shifted from hemodialysis to continuous ambulatory peritoneal dialysis (P < 0.05 for both percentage of surface covered by platelets and aggregate formation). The association of cytoskeletal proteins in platelets from patients under hemodialysis treatment was statistically decreased with respect to the corresponding values in platelets from patients not subjected to dialysis (P < 0.01 for actin). However, after two months on peritoneal dialysis, these values increased to almost control values (P < 0.001 for actin, vs. hemodialysis). Similarly, translocation of tyrosine-phosphorylated proteins to the cytoskeletal fraction was impaired in platelets from hemodialyzed patients, and it recovered partially after the patients transferred to continuous ambulatory peritoneal dialysis. Our present data support the concept that repeated platelet stress during hemodialysis has a deleterious effect on the organization of platelet cytoskeleton, which seems to impair the translocation of signal transduction proteins within platelets compromising the platelet function in uremia.Kidney International 05/2000; 57(5):1905-14. · 6.61 Impact Factor -
Article: Thrombin facilitates primary platelet adhesion onto vascular surfaces in the absence of plasma adhesive proteins: studies under flow conditions.
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ABSTRACT: The effect of local and circulating thrombin on platelet adhesion onto vascular surfaces was explored in the absence of plasma adhesive proteins using flow conditions. To study the local effects of thrombin, denuded rabbit aorta segments were incubated with thrombin concentrations of 0.001, 0.01 and 0.1 U/mL. To evaluate the effects of circulating thrombin, the same concentrations were added to perfusates consisting of washed platelets and washed red blood cells suspended in a human albumin solution (5%). In some experiments, purified von Willebrand's factor (vWF) (Haemate-P) was added to the perfusates (0. 8 U/mL of vWF, final concentration). A humanized chimeric antibody to the GPIIb-IIIa complex (Reopro) was used to determine the role of this glycoprotein on platelet adhesion under the conditions described. The effect of blocking GPIb was also assessed. Perfusions were carried out at 800 s(-1) for 10 min. The interaction of platelet with the vessel surface was morphometrically evaluated and expressed as percentage of surface coverage (%SC). Changes in the surface expression of the major platelet antigens were also analyzed by flow cytometry. Incubation of subendothelial surfaces with thrombin enhanced platelet deposition with respect to control levels (increases in SC of 64%, 79% and 86% with 0.001, 0.01 and 0.1 U/mL of thrombin, respectively). Low concentrations of thrombin (0.001 and 0.01 U/mL) incorporated in the perfusates resulted in a similar pro-adhesive effect (increases in SC of 64% and 71%, respectively) while the highest concentration (0.1 U/mL) failed to produce a pro-adhesive effect due to the augmented formation of platelet aggregates with subsequent thrombocytopenia (15+/-1 vs. 160+/-5x10(9) plt/L in the perfusates). Similar results were obtained when VWF was present in the perfusate. Reduction of platelet deposition by blockade of GPIIb-IIIa (to 5.3+/-0.7%) was partially restored by thrombin. Blockade of GPIb prevented platelets from adhering even when thrombin was present (%SC of 2.0+/-0.8%). No significant changes in the distribution of platelet membrane glycoproteins during perfusion experiments were detected. Our results suggest that thrombin facilitates primary platelet adhesion onto vascular surfaces even in the absence of plasma adhesive proteins. This effect seems to be mainly dependent on the GPIb/vWF axis.Haematologica 04/2000; 85(3):280-8. · 6.42 Impact Factor -
Article: Adherence of platelets under flow conditions results in specific phosphorylation of proteins at tyrosine residues.
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ABSTRACT: Collagen is a powerful platelet activating agent that promotes adhesion and aggregation of platelets. To differentiate the signals generated in these processes we have analyzed the tyrosine phosphorylation occurring in platelets after activation with collagen in suspension or under flow conditions. For the suspension studies, washed platelets were activated with different concentrations of purified type I collagen (ColI). Studies under flow conditions were performed using two different adhesive substrata: ColI and endothelial cells extracellular matrix (ECM). Coverslips coated with ColI or ECM were perfused through a parallel-plate perfusion chamber at 800 s(-1) for 5 min. After activation of platelets either in suspension or by adhesion, samples were solubilized and proteins were resolved by electrophoresis. Tyrosine-phosphorylated proteins were detected in immunoblots by specific antibodies. Activation of platelet suspensions with collagen induced tyrosine phosphorylation before aggregation could be detected. Profiles showing tyrosine-phosphorylated proteins from platelets adhered on ColI or on ECM were almost identical and lacked proteins p95, p80, p66, and p64, which were present in profiles from platelets activated in suspension. The intensity of phosphorylation was quantitatively weaker in those profiles from platelets adhered on ECM. Results from the present work indicate that activation of platelets in suspension or by adhesion induces differential tyrosine phosphorylation patterns. Phosphorylation of proteins p90 and p76 may be related to early activation events occurring during initial contact and spreading of platelets. Considering that adhesion is the first step of platelet activation, studies on signal transduction mechanisms under flow conditions may provide new insights to understand the signaling processes taking place at earliest stages of platelet activation.Cell adhesion and communication 02/2000; 7(4):349-58. -
Article: Modifications in accessibility of membrane glycoproteins, binding of specific ligands and coagulation factor V during the activation of platelets in blood emerging from bleeding time wounds.
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ABSTRACT: Dual color flow cytometric techniques were applied to micro-aliquots of whole blood obtained from bleeding time (BT) wounds. Modifications in platelet activation markers (P-selectin [CD62P]) and lysosomal related protein (UMPS [CD63]), presence of membrane glycoproteins (GPIb [CD42b], GPIIb-IIIa [CD41a], GDIV [CD36], binding of von Willebrand factor (vWF), fibrinogen (Fg) and factor V [FV]) were analyzed in normal donors (n = 10) and in a severe von Willebrand patient (type 3) von Willebrand disease [vWD]). Samples of blood (20 microl) were sequentially removed from BT wound edges for up to 5 min and fixed with paraformaldehyde. Antigens were detected using the corresponding tagged monoclonal antibodies (moAbs) and quantitative results were referred to those found on platelet samples obtained from venous blood obtained from the same individuals. A progressive increase in % of platelets positive for activation dependent antigens (CD62 from 7 +/- 2 to 48 +/- 19% and CD63 from 9 +/- 1 to 44 +/- 8%; initial vs. 4 min) was observed. Accessibility of GPIIb-IIIa epitopes on platelets from BT wounds remained slightly above levels observed in venous blood platelets, despite a progressive increase in the presence of platelets positive for Fgn. Binding of MoAb to GPIV increased at late stages of BT. A moderate decrease in the binding of a moAb to GPIb was observed on platelets obtained at late stages of the BT (14 +/- 9% and 20 +/- 6% at 4 and 5 min, respectively). This apparent decrease in GPIb epitopes paralleled an increased presence of platelets positive for vWF (26 +/- 12 and 38 +/- 15%). Binding of moAb to GPIb always remained above basal levels in platelets obtained from BTs performed in the patient with type 3 vWD. FV levels on platelets coming from the BT persisted at background levels in all the individuals and at all times studied.American Journal of Hematology 05/1999; 60(4):260-7. · 4.67 Impact Factor -
Article: Inhibition of platelet-vessel wall interactions by thromboxane receptor antagonism in a human in vitro system: potentiation of antiplatelet effects of aspirin.
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ABSTRACT: Pharmacological inhibition of arachidonic acid metabolism has proven therapeutically useful in the prevention of cardiovascular events. We have investigated the ability of Bay u 3405, a synthetic thromboxane antagonist, to interfere with platelet aggregation and arachidonic acid metabolism. The antiplatelet action was also analysed in a perfusion system in which vascular subendothelium was exposed to circulating human blood (10 min; shear rate = 800 s-1). Platelet interactions were morphometrically analysed and results compared with those obtained in studies with blood from donors taking aspirin (acetylsalicylic acid, ASA) (500 mg day-1). The additional effect of Bay u 3405 on the antiplatelet action of ASA was also evaluated. Bay u 3405 caused a dose-dependent inhibition of platelet aggregation induced by U46619 with a maximal effect at concentrations > or = 0.01 microgram mL-1. Higher concentrations (> or = 0.05 micrograms mL-1) also inhibited aggregations induced by ADP or collagen. Bay u 3405 did not interfere with platelet arachidonic acid metabolism. In perfusion studies, Bay u 3405 (0.01 microgram mL-1) significantly decreased the total surface of the vessel covered by platelets (%CS = 18.7 +/- 1.09 vs. 24.4 +/- 1.94; P < 0.05) and the formation of large aggregates %T = 7.5 +/- 0.87 vs. 19.3 +/- 1.61; P < 0.01). ASA treatment reduced platelet aggregate formation (%T = 13.7 +/- 2.06; P < 0.05) but did not affect the total surface covered by platelets. The in vitro addition of Bay u 3405 to blood from ASA-treated donors further reduced the formation of large aggregates (%T = 2.7 +/- 0.79; P < 0.01 vs. ASA). In vitro effect of Bay u 3405 on platelet function were superior to those observed with ASA. The thromboxane antagonism antagonism provided by Bay u 3405 further enhanced the inhibition of platelet aggregate formation found after ASA treatment.European Journal of Clinical Investigation 07/1998; 28(7):562-8. · 3.02 Impact Factor -
Article: Frozen extracellular matrix preserves its thrombogenicity after thawing, while Matrigel induces a poor platelet response.
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ABSTRACT: Two putative substitutes for fresh endothelial cell (EC) extracellular matrix (ECM), frozen ECM and Matrigel, were studied using a parallel-plate perfusion system and platelet deposition was evaluated morphometrically. Coverslips covered with ECM were stored frozen at-30 C for 0 (fresh ECM), 1, 2, 3 or 4 weeks. The ability of frozen ECM to support platelet adhesion after freezing was analyzed under three experimental approaches, perfusing blood: (i) at different shear rates; (ii) on a highly reactive ECM obtained from stimulated EC; and (iii) on ECM incubated with a monoclonal antibody against laminin (LM). Matrigel, alone or mixed with different fractions of wet cryoprecipitate, was layered on coverslips as a thin uniform coat, and perfused, as was done with the frozen ECM. Platelet deposition onto fresh ECM was 21.3 1.5%, 25.5 2.1% and 30.8 2.4% (at shear rates of 300, 800 and 1300/s, respectively) and 40.0 3.8% in PMA stimulated ECM perfused at 800/s. Values obtained on frozen ECM did not vary from those obtained using fresh ECM. Results from perfusion studies using ECM preincubated with an anti-laminin antibody and observations from immunofluorescence studies indicated that the presence and distribution of the adhesive proteins in frozen ECM were similar to those observed on fresh ECM. Platelet deposition on Matrigel was practically absent. Addition of a 20% cryoprecipitate fraction partially restored its thrombogenicity. Our results indicate that when ECM is kept frozen for up to 4 weeks, it behaves as fresh ECM in perfusion studies. On the contrary, Matrigel is not a suitable substrate to support platelet attachment under flow conditions.Platelets 02/1998; 9(5):279-85. · 1.85 Impact Factor -
Article: Desmopressin (DDAVP) enhances platelet adhesion to the extracellular matrix of cultured human endothelial cells through increased expression of tissue factor.
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ABSTRACT: The effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DDAVP-treated ECMS were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin, 20 U/ml). Perfusions with run for 5 min at a shear rate of 800 s-1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p < 0.05 and p. < 0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p < 0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.Thrombosis and Haemostasis 05/1997; 77(5):975-80. · 5.04 Impact Factor -
Article: Redistribution of membrane glycoproteins in platelets activated under flow conditions.
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ABSTRACT: A reduction in the ability of GPIb to bind specific MoAbs or ligands (vWF) has been reported in platelets exposed to thrombin in suspension. We have analyzed modifications in the presence of glycoproteins (GPs) on platelets activated under flow conditions in a system which allows limited thrombin and fibrin generation. Normal blood anticoagulated with low molecular weight heparin (LMWH, Dalteparin 20 IU/ml) was recirculated for up to 10 min at 800 s-1 through annular chambers containing denuded arterial segments. Aliquots of blood were removed from the reservoir at 0, 1, 5 and 10 min and immediately mixed with paraformaldehyde. Membrane glycoproteins: GPIb (CD42b), GPIIb-IIIa (CD41a), GPIV (CD36); and activation dependent antigens: P-selectin (CD62P) and lysosomal glycoprortein (CD63), were detected in whole blood by dual color flow cytometry. Circulation of through the perfusion system resulted in platelet activated as demonstrated by the increased percentage of platelets positive for antigens CD62P and CD63. A gradual increase in the binding of MoAbs directed against GPIb, GPIIb-IIIa, and GPIV epitopes was noted during the entire perfusion period. Observed differences in mean fluorescence intensities at all the observation times were statistically significant (P < 0.001). Our results obtained on platelets in an experimental thrombosis system indicate that GPIb, GPIIb-IIIa and GPIV remain on the surface of activated platelets and actually increase their expression. Alterations detected at the level of GPIb in platelets activated by thrombin in suspension may not take place under in vivo situations.Blood Coagulation and Fibrinolysis 03/1996; 7(2):214-7. · 1.24 Impact Factor -
Article: Correction of uremic platelet dysfunction after renal transplantation.
Transplantation Proceedings 09/1995; 27(4):2244-5. · 1.00 Impact Factor -
Article: Glycoprotein IIb-IIIa and glycoprotein IV expression on Bernard-Soulier syndrome platelets.
Blood 07/1995; 85(12):3763. · 9.90 Impact Factor -
Article: [Interaction of platelets with an artificial polystyrene surface under flow conditions: comparative study versus collagen].
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ABSTRACT: 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. 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. 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). 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.Sangre 11/1994; 39(5):337-41. -
Article: Abnormal cytoskeletal assembly in platelets from uremic patients.
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ABSTRACT: The mechanisms involved in the hemostatic abnormality of uremic patients remain obscure. We have explored the response of normal and uremic platelets to surface activation at the ultrastructural level and analyzed changes in the composition of proteins associated with normal and uremic platelet cytoskeletons after stimulation with thrombin (0.01 and 0.1 U/ml). Cytoskeletons were obtained by extraction with Triton X-100, processed by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and the presence of cytoskeletal proteins analyzed by densitometry. Under static conditions, uremic platelets spread with difficulty on formvar-coated grids. The percentage of platelets that spread fully on this polymer surface was statistically reduced compared with that of control platelets (11 +/- 1.4 vs. 21 +/- 1.6; P < 0.05). An impairment of cytoskeletal organization was observed in resting uremic platelets but abnormalities were more evident after thrombin activation. The incorporation of actin into the cytoskeletons of thrombin-stimulated uremic platelets was significantly reduced with respect to controls (6 +/- 3% vs. 29 +/- 5%; P < 0.01 after 0.01 U/ml and 28 +/- 9% vs. 59 +/- 10%; P < 0.05 after 0.1 U/ml). Decreased associations of actin-binding protein (P < 0.01), alpha-actinin (P < 0.05), and tropomyosin (P < 0.05) with the cytoskeletons of uremic platelets were also noted. No difference was observed for the incorporation of myosin into the cytoskeletons of activated uremic platelets. These results suggest functional and biochemical alterations of the platelet cytoskeleton in uremia, which may contribute to the impairment of platelet function observed in uremic patients.American Journal Of Pathology 09/1993; 143(3):823-31. · 4.89 Impact Factor -
Article: Dipyridamole induces changes in the thrombogenic properties of extracellular matrix generated by endothelial cells in culture.
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ABSTRACT: Dipyridamole (DIP) is a drug widely used as an antiplatelet agent, which also has effects on endothelial cells. In this study, the effects of treating confluent endothelial cell monolayers (EC) with DIP on EC viability (trypan blue exclusion test) and metabolic activity (3H-thymidine incorporation) were examined. Platelet reactivity of the extracellular matrix (ECM) produced by untreated and DIP-treated ECs was determined morphometrically by a perfusion technique. Levels of ECM-associated von Willebrand factor (vWF) and fibronectin (FN) were also quantified (ELISA). The present results indicate that treatment of EC with 10 microM DIP did not reduce EC viability but that the incorporation of labelled nucleotides was significantly decreased (p less than 0.01). Platelet deposition onto the ECM generated by DIP-treated cells, perfused at a shear rate of 1300 sec-1, differed significantly with respect to controls (p less than 0.05), and platelet adhesion was also reduced (25% less, p less than 0.05). This effect was shear rate dependent, as no differences were noted when the ECMs were perfused at 300 sec-1 shear rate. Levels of VWF and FN associated with ECM remained unchanged with respect to controls. These results suggest that treatment with DIP alters EC metabolic activity, which in turn, influences the reactivity of the ECM generated by treated cells.Thrombosis Research 12/1991; 64(3):341-53. · 2.44 Impact Factor
Top co-authors
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Institutions
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1990–2003
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University of Barcelona
- • Departament de Medicina
- • Facultad de Medicina
Barcelona, Catalonia, Spain
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1994–2001
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Hospital Clínic de Barcelona
- Servicio de Hemoterapia y Hemostasia
Barcelona, Catalonia, Spain
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1996–2000
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Southern Medical Clinic
San Fernando, San Fernando, Trinidad and Tobago
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