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Biomedical subjects

Maribel Díaz-Ricart

Publications and source records attributed to Maribel Díaz-Ricart.

18 recordsLinked to original sources
PubMed2006

Effects of etamsylate on equine platelets: in vitro and in vivo studies.

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte-platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.

Animals↗
PubMed2006

Serotonergic mechanisms: a potential link between affective disorders and cardiovascular risk.

Cardiovascular disease and major depression are highly prevalent disorders in our society. Evidence has been found that confirms a reciprocal relationship between mechanisms of depression and those of cardiovascular pathology. This possible feedback between both pathologies is a subject of great concern. In recent years some studies suggest that platelets and serotonergic mechanisms could be involved in both conditions. The present review seeks a better understanding of the mechanisms that could link depression with an enhanced cardiovascular risk.

Journal Article↗
PubMed2005

Serotonergic mechanisms: a potential link between affective disorders and cardiovascular risk.

Cardiovascular disease and major depression are highly prevalent disorders in our society. Evidence has been found that confirms a reciprocal relationship between mechanisms of depression and those of cardiovascular pathology. This possible feedback between both pathologies is a subject of great concern. In recent years some studies suggest that platelets and serotonergic mechanisms could be involved in both conditions. The present review seeks a better understanding of the mechanisms that could link depression with an enhanced cardiovascular risk.

Antidepressive Agents↗
PubMed2005

Uremic platelet dysfunction: past and present.

Uremic patients develop an acquired platelet dysfunction that results in bleeding complications. The pathogenesis of this hemostatic dysfunction is multifactorial and includes effects of circulating toxins, alterations of the vessel wall, anemia, and other factors, complicated by unwanted effects of hemodialysis procedures. This review seeks to place in perspective the evolution of knowledge on uremic platelet dysfunction. It examines how investigations of the altered hemostasis in these patients have led to a better understanding of the mechanisms involved and how these advances have contributed to the development of effective therapeutic strategies. It also comments upon the fact that elevated rates of thrombotic complications are apparently emerging as the bleeding tendency is better controlled. Emphasized is the delicate balance of hemostasis in the uremic condition, in which deficient hemostasis paradoxically coexists with accelerated atherosclerosis and an enhanced risk of thrombosis.

Blood Platelet Disorders↗
PubMed2004

Decrease of the platelet 5-HT2A receptor function by long-term imipramine treatment in endogenous depression.

BACKGROUND: Animal studies have found that many antidepressants induce decreases in both the density and the functional activity of the serotonin 2A (5-HT2A) receptor subtype. However, the extrapolation of findings to humans has been inconclusive. A physiological platelet response mediated by this receptor, the serotonin-amplified platelet aggregation, was measured to study whether long-term antidepressant treatment induces changes in 5-HT2A receptor functioning in endogenous depressed patients. METHOD: The percentage of serotonin-amplified platelet aggregation to adenosine diphosphate (ADP) was studied in 15 untreated patients with major depressive disorder (DSM-IV) with endogenous features (Newcastle scale). This index was used as an indirect measurement of the functional status of platelet 5-HT2A receptors. Aggregation studies were repeated once remission of the symptoms was achieved during treatment with imipramine (150-300 mg/day). A group of 15 concurrent normal subjects was used as a control. RESULTS: A statistically significant decrease (p = 0.038) in the percentage of serotonin-amplified platelet aggregation to ADP was observed when remission was achieved (after 145 +/- 27 days). CONCLUSIONS: The results showed a decrease in a platelet functional response mediated by 5-HT2A receptors following effective imipramine treatment, suggesting that desensitization or down-regulation of the 5-HT2A receptor function could be linked to the therapeutic effect of some antidepressants. The data also support the use of platelet aggregometry as a surrogate measurement of antidepressant action, particularly in intra-subject designs.

Adult↗
PubMed2004

Granulocyte colony-stimulating factor increases expression of adhesion receptors on endothelial cells through activation of p38 MAPK.

BACKGROUND AND OBJECTIVES: Granulocyte colony-stimulating factor (G-CSF) is specific for the granulocytic cell line, although receptors for this cytokine have been found in other cell types including endothelial cells. These observations prompted us to investigate the potential effect of G-CSF on the endothelium. DESIGN AN METHODS: Endothelial cell monolayers were exposed to G-CSF to evaluate: i) signal transduction mechanisms, ii) expression of adhesion receptors at the cell surface, and iii) leukocyte adhesion on EC monolayers. RESULTS: Exposure of human umbilical vein endothelial cells (EC) in culture to G-CSF resulted in the activation of the signal transduction pathways JAK/STAT (JAK-1, STAT-1 and STAT-3) and RAS/MAPK (MAPK p42/44 and p38 MAPK). We also observed significantly increased expression of the adhesion receptors, E-selectin (ELAM-1), vascular endothelial cell adhesion molecule-1 and intracelleular adhesion molecule-1 at the cell surface in response to G-CSF, increases that were followed by an augmented adhesion of leukocytes on the previously exposed EC monolayers. These effects were blocked by the presence of SB203580, a p38 MAPK inhibitor, by U0126, a MAPK p42/44 inhibitor, and by inhibiting the G-CSF receptor with a specific antibody. INTERPRETATION AND CONCLUSIONS: Our results demonstrate that G-CSF increases the expression of adhesion receptors on EC, promoting leukocyte adhesion. This effect seems to be triggered by the signaling events that follow receptor binding. Results from experiments using specific inhibitors suggest that activation of p38 MAPK is required to promote expression of adhesion receptors in endothelial cells and the recruitment of leukocytes in response to G-CSF.

Cell Adhesion↗
PubMed2003

Glycoproteins expression on platelet membrane in inherited macrothrombocytopenias.

INTRODUCTION: Inherited giant platelet disorders (IGPD) are a heterogeneous group of rare diseases characterized by thrombocytopenia, large platelets and variable bleeding symptoms. Glycoprotein (GP) expression on platelet surface in these conditions is poorly characterized. We have investigated the expression of constitutively expressed platelet membrane GP and the response to TRAP activation in a group of patients with different forms of inherited macrothrombocytopenias. MATERIALS AND METHODS: Two patients diagnosed Epstein syndrome (ES), two with Bernard-Soulier syndrome (BSS) and eight with Mediterranean macrothrombocytopenia (MM) were studied using flow cytometry combined to monoclonal antibodies (MoAbs). Mean platelet volume was also measured. RESULTS: In general, there was an increase in the binding of MoAbs directed against platelet membrane GPs in the patients studied (except, obviously, in BSS patients). The observed increase ranged between 0.9 and 3.36 times the value of the control for GPIbalpha, between 1.36 and 7.2 times for GPIIb-IIIa and 0.94 and 8.07 times for GPIV. However, when the observed value was divided by the measured platelet volume, the estimated density was similar to controls in the case of GPIbalpha but were increased for GPIIb-IIIa and GPIV. TRAP activation provoked significant increments in the number of copies of GPIIb-IIIa complexes present on platelet surface in controls (91%), MM patients (49%), but almost no changes in BSS platelets (6%). CONCLUSION: In the group of macrothrombocytopenia studied, an increase in the expression of platelet membrane GP was observed, although a wide variability exists even in patients with the same disorder.

Adolescent↗
PubMed2003

Uraemic medium accelerates proliferation but does not induce apoptosis of endothelial cells in culture.

BACKGROUND: Chronic renal failure patients exhibit accelerated atherosclerosis, which is associated with a high incidence of cardiovascular death. We investigated the potential effect of uraemic medium on cell proliferation and apoptosis of endothelial cells in culture (ECs), two key processes in the development of atherosclerosis. Phosphorylation kinetics of the mitogen-activated protein kinase (MAPK) p42/44 and p38 were also evaluated. METHODS: ECs were cultured with growth media supplemented with pooled sera from healthy donors. Semiconfluent ECs were incubated for 24 h with media supplemented with pools of control or uraemic sera. Cell proliferation was assessed through morphometric analysis and by flow cytometry evaluation of cell cycle. To investigate if uraemic medium induces apoptosis in ECs, we used a combination of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay and activation of caspase-3 using flow cytometry. Changes in the phosphorylation levels of MAPK were evaluated in cell lysates by western blotting. RESULTS: Exposure to uraemic media caused an alteration in the morphology of ECs, showing irregular shape and size. The number of ECs at S+G(2)M phase in the cell cycle was found to be increased when exposed to uraemic media for 24 h (28.4+/-2.9 vs 20.2+/-2.6% in control ECs). There was a transient increase in levels of phosphorylation of MAPK in both cells, although these levels were significantly higher in ECs exposed to uraemic media, especially after 5 min. In contrast, no signs of apoptosis were observed in ECs incubated with uraemic medium at the conditions applied. CONCLUSIONS: Under our experimental conditions, uraemic medium accelerates proliferation of ECs, but it does not seem to induce apoptosis. The increased proliferation observed could be related to a higher MAPK activity in these cells. Although the enhanced atherosclerosis cannot be explained on the basis of an apoptotic process, the proliferative status could contribute to intimal proliferation, which is considered to be an earlier step in the development of atherosclerosis.

Apoptosis↗
PubMed2002

Primary arrest of circulating platelets on collagen involves phosphorylation of Syk, cortactin and focal adhesion kinase: studies under flow conditions.

After a vessel wall injury, platelets adhere to the subendothelium following a sequence of events: arrest of single platelets on the surface, progression to platelet spreading and final aggregation. Primary arrest of circulating platelets on subendothelial components occurs through platelet glycoprotein (GP) Ib and collagen receptors; then platelets spread and aggregate through a GPIIb-IIIa-dependent mechanism. A series of strategies were applied to analyse the tyrosine-phosphorylation mechanisms occurring at the different stages of platelet adhesion on subendothelial components under flow conditions, with special attention to primary arrest. To evaluate spread platelets, samples were exposed to acetylsalicylic acid, which blocks aggregate formation. To study single platelets in contact, a monoclonal antibody specific for GPIIb-IIIa was used to prevent platelet spreading and further aggregation. This experimental situation was also investigated using blood from two patients with Glanzmann's thrombasthenia (i.e. lacking GPIIb-IIIa). Results demonstrated that blockade of both spreading and aggregation results in significant changes in the tyrosine-phosphorylation patterns. Arrest of single platelets on collagen-rich surfaces resulted in phosphorylation of p125, identified as focal adhesion kinase (FAK), the 80/85 kDa doublet (cortactin), and p72, identified as Syk. Arrest of single platelets on von Willebrand factor as adhesive substrate showed that interaction through GPIb induces Syk phosphorylation, but not that of cortactin and FAK. Our data indicate that the initial arrest of platelets on subendothelial components involves Syk phosphorylation, which seems to be GPIb-dependent, and this is followed by activation and phosphorylation of cortactin and FAK. These processes seem to occur before GPIIb-IIIa becomes activated.

Blood Platelets↗
PubMed2002

Platelet 5-HT2A-receptor-mediated induction of aggregation is not altered in major depression.

BACKGROUND: Studies of the 5-HT(2A) receptor subtype in major depression have focused on the density of these receptors in neuronal cells and platelets, showing an up-regulation secondary to a deficit in serotonergic activity in major depression. However, their functional state has often been disregarded. The aim of the study was to investigate whether depressed patients show abnormalities in the function of the 5-HT(2A) receptor pathway in platelets. METHOD: The percentage of serotonin-amplified platelet aggregation to adenosine diphosphate (ADP) was assessed in 30 untreated patients with major depressive disorder and in 15 controls. Since 5-HT(2A) platelet receptors mediate the serotonin-induced platelet aggregation response, this index was used as a measure of the functional status of the platelet 5-HT(2A) receptor pathway. RESULTS: There was no significant difference in the percentage of serotonin-amplified platelet aggregation to ADP between depressed patients and controls. No correlation with the severity of depression, as assessed by the Hamilton scale, was found. CONCLUSION: The results showed no consistent changes in the platelet aggregating responses to serotonin in the depressed patients. Therefore this study does not support the hypothesis of an alteration of the functional status of platelet 5-HT(2A) receptors in major depression.

Adenosine Diphosphate↗
PubMed2002

TRAP induces more intense tyrosine phosphorylation than thrombin with differential ultrastructural features.

We have analyzed modifications on platelet ultrastructural morphology, cytoskeletal assembly, and tyrosine phosphorylation developing in platelets activated by both thrombin and the thrombin receptor-activating peptide (TRAP). Washed platelets exposed to various concentrations of thrombin or TRAP, for different periods, were: fixed and examined by electron microscopy, or lysed and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under similar activating conditions, thrombin and TRAP induced different sequences of activation causing distinctive morphological and biochemical changes. Platelets exposed to thrombin showed centralized organelles encircled by constricted microtubule coils and granules secreting their contents through narrow channels of the open canalicular system. In contrast, activation by TRAP induced swelling of the open canalicular system with organelles remaining randomly dispersed and microtubules peripherally distributed. Compared to thrombin activation, TRAP induced higher rates of actin polymerization; increased association of actin-binding protein, myosin, and alpha-actinin; and higher association of tyrosine-phosphorylated proteins with the insoluble cytoskeletal fraction. Secretion of intragranule substances, measured as expression of P-selectin and lysosomal integral membrane protein at the surface level, were similar for both agonists at equivalent concentrations. Our biochemical observations indicate that TRAP causes more intense changes in signaling through tyrosine phosphorylation of proteins associated with the cytoskeletal fraction than thrombin. However, as derived from ultrastructural observations, TRAP seems to be less efficient in triggering cytoskeletal assembly and internal contraction in an organized manner in contrast with the natural protease.

Actinin↗
PubMed2002

Inhibition of cytoskeletal assembly by cytochalasin B prevents signaling through tyrosine phosphorylation and secretion triggered by collagen but not by thrombin.

Activation of platelets leads to cytoskeletal assembly that is responsible for platelet motility and internal contraction. We have evaluated the involvement of the cytoskeleton in platelet activation by two strong agonists, collagen and thrombin. Activation was assessed by measuring changes in cytoskeletal assembly, externalization of activation-dependent markers and expression of procoagulant activity, and tyrosine phosphorylation of proteins, in both the absence and the presence of cytochalasin B. Activation of platelets with collagen and thrombin induced morphological changes and increased the expression of CD62P, CD63, glycoprotein IV, and binding of annexin V to platelets. Moreover, both activating agents induced actin polymerization, increased the association of other contractile proteins, and promoted tyrosine phosphorylation of multiple proteins, some of which were associated with the cytoskeleton. The presence of cytochalasin B blocked the previous events when collagen was used as the activating agent, although binding of annexin V still occurred. In contrast, platelet response to thrombin was not completely prevented by the presence of cytochalasin B. Thus, activation by collagen requires a functional cytoskeleton to trigger signaling through tyrosine phosphorylation and secretion. This is not the case for thrombin, which is capable of activating signaling mechanisms in the presence of strong inhibitors of cytoskeletal assembly. Moreover, the expression of a procoagulant surface in platelets still occurs even when platelet motility has been inhibited.

Blood Platelets↗
PubMed2002

Platelet adhesion onto a polystyrene surface under static conditions: role of specific platelet receptors and effect of divalent cations.

An experimental model was used to elucidate the basic mechanisms involved in the interaction of platelets with an artificial surface. The role of divalent cations and the involvement of specific platelet membrane receptors were evaluated. Isolated platelets were allowed to interact with a polystyrene surface for 20 min in the presence of divalent cations (Ca2+, Mg2+ or Zn2+), a chelating agent (ethylenediaminetetraacetic, EDTA), and specific antibodies to the main platelet receptors, glycoproteins (GP) Ib and IIb-IIIa. The degree of platelet interaction was evaluated using light and electron microscopy. Morphometric analysis was performed to follow up the progression of platelet shape changes after surface activation. Neither Ca2+ nor Mg2+ influenced the number of adherent platelets or the degree of spreading on the polymer. Only Zn2+ induced a statistically significant increase in the rate of platelet adhesion (P<0.01) with higher proportion of fully spread platelets (P<0.01). Chelation of internal pools of divalent cations did not modify the rates of platelet adhesion but prevented platelet spreading. Presence of monoclonal antibodies to GPIb and GP IIb-IIIa did not result in significant differences in the studied parameters. These results suggest that platelet adhesion onto artificial surfaces, in the absence of flow and plasma proteins, is more dependent on cellular motility, where Zn2+ could play an important role, and less dependent on major receptorial mechanisms.

Antibodies, Monoclonal↗
PubMed2002

Phosphotyrosine proteins in platelets from patients with storage pool disease: direct relation between granule defects and defective signal transduction.

BACKGROUND AND OBJECTIVES: Storage pool diseases (SPD) are heterogeneous disorders associated with an abnormal presence of intraplatelet granules, which cause mild to moderate bleeding diathesis. We investigated signaling through tyrosine phosphorylation of proteins occurring in platelets with total or partial absence of dense- and alpha-granules in response to activation. DESIGN AND METHODS: We included a patient with severe delta-SPD, a patient with severe alpha-SPD or gray platelet syndrome, and six patients with partial deficiency of dense or a-granules. SPD was confirmed by electron microscopy evaluation of platelet ultrastructure. Platelet function was evaluated by bleeding time determination and conventional aggregometry. Platelet suspensions were activated with collagen and thrombin to analyze changes in tyrosine phosphorylation of proteins by electrophoresis and Western-blotting. RESULTS: Bleeding times were prolonged in all the patients included. Aggregation responses were slightly decreased in delta-SPD and normal in the rest of patients. Tyrosine phosphorylation in platelets from patients with partial forms of SPD was equivalent to that observed in control platelets, absent in response to collagen and thrombin activation in delta-SPD, and deficient only to thrombin activation in alpha-SPD. INTERPRETATION AND CONCLUSIONS: Tyrosine phosphorylation of proteins in activated platelets is highly dependent on the substances contained in the dense-granules and moderately dependent on those contained in the alpha-granules. A minimum amount of intraplatelet granules ensures signaling through tyrosine phosphorylation of proteins.

Adolescent↗
PubMed2002

Erythropoietin triggers a signaling pathway in endothelial cells and increases the thrombogenicity of their extracellular matrices in vitro.

We demonstrate that exposure of cultured human endothelial cells to rHuEPO resulted in a dose-dependent increase in the tyrosine kinase activity, with phosphorylation of JAK-2 followed by rapid phosphorylation of STAT-5. Simultaneously, rHuEPO induced long-lasting phosphorylation of MAPK p42/44. Activation of this signaling pathways was directly associated with an increase in the thrombogenic properties of the extracellular matrix generated by these cells, when they were exposed to flowing blood. The enhancement in the reactivity of the resulting extracellular matrix towards platelets was associated with a higher expression of tissue factor. All these effects were blocked by an antibody to the EPO receptor and by specific inhibitors of tyrosine phosphorylation. The observed action of rHuEPO on endothelial cells seemed to be specifically triggered by the subsequent events that follow receptor binding, and occurred even at pharmacological concentrations of the cytokine. Our results indicate that rHuEPO has a direct action on the endothelium, increasing the reactivity of the underlying extracellular matrix towards platelets, effect that may be attributed to an increase in the expression of TF.

Blood Platelets↗
PubMed2002

Uremic medium causes expression, redistribution and shedding of adhesion molecules in cultured endothelial cells.

BACKGROUND AND OBJECTIVES: Patients with chronic renal failure show signs of accelerated atherosclerosis and high cardiovascular morbidity and mortality. Recent investigations indicate that uremia is associated with endothelial dysfunction and a microinflammatory state. We assessed changes in the expression of adhesion molecules [ELAM-1, VCAM-1 and ICAM-1], and proteins involved in hemostasis [von Willebrand factor (vWF) and thrombomodulin (TM)] in endothelial cells (ECs) and the corresponding extracellular matrices (ECM), respectively. DESIGN AND METHODS: Cultured human umbilical vein endothelial cells were incubated in the presence of a pool of normal or uremic sera. Immunocytochemical detection of related antigens was performed with specific antibodies coupled to colloidal gold. Concentrations of soluble adhesion molecules and TM in culture supernatants were evaluated by ELISA. Modifications in the transcription of the corresponding genes were also evaluated by Northern-blotting and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Exposure of ECs to uremic media caused an increase in the presence [ELAM-1, VCAM-1] and accessibility [ICAM-1] of the adhesion receptors on EC monolayers, as well as a higher presence of their soluble molecules in culture supernatants. We found a significant increase in the presence of vWF on the extracellular matrix derived from ECs grown in the presence of a uremic medium. The presence of TM on ECs and in the ECM remained unmodified, although there was a significant increase in the presence of TM in supernatants from ECs grown in the presence of uremic sera. Northern-blot or RT-PCR studies showed increased expression of the mRNA for all the corresponding genes (ELAM-1, VCAM-1, ICAM-1, vWF and TM). INTERPRETATION AND CONCLUSIONS: Uremic medium causes inflammatory changes in ECs, which are characterized by enhanced expression, redistribution and shedding of adhesion molecules and TM, with an increased incorporation of vWF on the extracellular matrix generated.

Blotting, Northern↗