Rivera Pozo, José

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Rivera Pozo, José

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Rivera Pozo, José

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Universidad de Murcia. Departamento de Medicina

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Open Access
Novel loci involved on platelet function and platelet count identified by a genome-wide study performed in children
(Ferrata Storti Foundation, 2011-09) Guerrero López, José Antonio; Rivera Pozo, José; Quiroga, Teresa; Martínez Pérez, Ángel; Antón, Ana Isabel; Martínez, Constantino; Panes, Olga; Vicente García, Vicente; Mezzano, Diego; Soria, José Manuel; Corral de la Calle, Javier; Medicina Interna
Background Genome-wide association studies are currently identifying new loci with potential roles in thrombosis and hemostasis: these loci include novel polymorphisms associated with platelet function traits and count. However, no genome-wide study performed on children has been reported to date, in spite of the potential that these subjects have in genetic studies, when compared to adults, given the minimal degree of confounders, i.e., acquired and environmental factors, such as smoking, physical activity, diet, and drug or hormone intake, which are particularly important in platelet function.Design and Methods To identify new genetic variants involved in platelet reactivity and count, we performed a genome-wide association study on 75 children (8.5±1.8 years) using the Illumina Sentrix Human CNV370-Quad BeadChip containing 320,610 single nucleotide polymorphisms. Functional analyses included assessment of platelet aggregation and granule secretion triggered by different agonists (arachidonic acid, collagen, epinephrine, ADP), as well as platelet count. Associations were selected based on statistical significance and physiological relevance for a subsequent replication study in a similar sample of 286 children.Results We confirmed previously established associations with plasma levels of factors XII, VII and VIII as well as associations with platelet responses to ADP. Additionally, we identified 82 associations with platelet reactivity and count with a P value less than 10−5. From the associations selected for further replication, we validated two single nucleotide polymorphisms with mildly increased platelet reactivity (rs4366150 and rs1787566) on the LPAR1 and MYO5B genes, encoding lisophosphatidic acid receptor-1 and myosin VB, respectively; and rs1937970, located on the NRG3 gene coding neuroregulin-3, associated with platelet count.Conclusions Our genome-wide association study performed in children, followed by a validation analysis, led us to the identification of new genes potentially relevant in platelet function and biogenesis.
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Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor
(Elsevier, 2005) Lozano Almela, María Luisa; Castillo, J.; Benavente-García, O.; Vicente, Vincente; Rivera Pozo, José; Guerrero López, José Antonio; Medicina Interna; Facultad de Medicina
Background: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. Objective: The aim of this study was to Investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A2 (TxA2) generation and on receptor antagonism, and to analyze the structural requirements for such effects. Methods: The effect of several types of flavonoids on platelet aggregation, serotonin release, andTxA2generationwasinvestigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA2 receptors. Results: Flavones (apigeninand luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA2 synthesis. These compounds were identified as specific ligands of the TxA2 receptor in the lmol L)1 range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA2 receptor relies on structural features such as the presence of the double bond in C2–C3, and a keto group in C4. Conclusions: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA2receptor. Therefore, antagonism of this TxA2 receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.
Open Access
Flavonoids inhibit the platelet TxA2 signalling pathway and antagonize TxA2 receptors (TP) in platelets and smooth muscle cells
(Wiley, British Pharmacological Society, 2007-04-10) Guerrero López, José Antonio; Navarro-Nuñez, Leyre; Lozano Almela, María Luisa; Martínez, Constantino; Vicente García, Vicente; Gibbins, Jonathan M.; Rivera Pozo, José; Medicina Interna; Medicina; Facultad de Medicina
Aims: Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA2 receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Methods: Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPaand TPb-transfected HEK 293T cells was explored using binding assays and the TP antagonist 3H-SQ29548. Results: Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10–30 mm). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by >50% 3H-SQ29548 binding to different cell types. Conclusions: These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.
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Genetic variants of the extra-large stimulatory Gs protein alpha-subunit and risk of thrombotic and haemorrhagic disorders
(Wiley, 2004-04-27) González-Conejero Hilla, Rocío; Corral de la Calle, Javier; Guerrero López, José Antonio; Iniesta Valera, Juan Antonio; Rivera Pozo, José; Arriba de la Fuente, Felipe de; Vicente García, Vicente; Medicina Interna
A polymorphism of the gene encoding the extra-large stimulatory G-protein a-subunit (XLas), originally identified in three patients with a bleeding tendency, involved a 36-bp insertion and two missense changes. A paternallyinherited insertion displayed a moderate platelet Gsa over-expression, which lead to platelet hypo-reactivity. These data prompted us to investigate the genetic, functional and clinical relevance of this polymorphism in the Mediterranean population. We included 414 healthy subjects and three case/ control studies: 263 consecutive patients with a first episode of primary intracerebral haemorrhage, 195 patients with deep venous thrombosis, and 104 patients with cerebrovascular disease. Controls were selected by approximating criteria to match selected risk factors to patients. Moreover, we performed studies of platelet function. We developed a simple method to determine the methylated allele, by digestion of genomic DNA with Sma I before polymerase chain reaction amplification. We identified two new rare variants, resulting from the loss of repeat units 7 and 5. The AB genotype was present in 3Æ6% of healthy population and the prevalence of the B allele was similar among cases and controls. Accordingly, the non-methylated B allele did not modify either the expression of platelet Gsa or the platelet response to Gs-agonists. Thus, our study suggests a minor functional role of XLas polymorphism in thrombotic or in haemorrhagic disorders.
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Evaluation of refrigerated platelet concentrates supplemented with low doses of second messenger effectors
(Wiley, 2004-07-27) Pérez Ceballos, Elena; Rivera Pozo, José; Lozano Almela, María Luisa; Candela, M.J.; Corral de la Calle, Javier; Guerrero López, José Antonio; Vicente García, Vicente; Medicina Interna
With the goal of producing haemostatically effective platelet concentrates (PCs) with a longer shelf-life, we aimed to identify a simple combination of platelet inhibitors, with a low pharmacological load, which could avoid the unacceptable loss of platelets stored under refrigerated conditions. PCs stored with different combinations of second messenger effectors were analysed at days 5, 10 and 15 of storage and compared with those supplemented with ThromboSol – a combination of six platelet inhibitors that protects cells from cold damage. The following parameters were analysed: platelet counts, biochemical parameters (glucose, pH, bicarbonate, lactate), cell lysis (lactic dehydrogenase, LDH), membrane glycoproteins (GPs), platelet aggregation, fibrinogen binding and hypotonic shock response. We characterized the combination of amiloride and sodium nitroprusside (at 1/2 the dose included in ThromboSol). This was found to be similar to ThromboSol and superior to nontreated units in the prevention of cold-induced platelet aggregation at day 15 of storage (maintenance of 78% and 80% of initial platelet counts, respectively), preservation of GPIba (11% and 12% better maintenance of mean fluorescence intensity compared with control units, respectively), and reduced cell lysis (13% and 11% decrease in supernatant LDH, respectively). The reduced pharmacological load with the identified solution compared with ThromboSol is an argument in favour of the potential use of these agents when designing strategies to improve PC storage.
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Differential effects of quercetin, apigenin and genistein on signalling pathways of protease-activated receptors PAR(1) and PAR(4) in platelets
(Wiley, British Pharmacological Society, 2009-10-28) Guerrero López, José Antonio; Navarro-Núñez, I.; Rivera Pozo, José; Martínez, C.; Vicente García, Vicente; Lozano Almela, María Luisa; Medicina Interna
Background and purpose: The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors. Experimental approach: We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs). The ability of these flavonoids to bind to thrombin receptors was investigated by competitive radioligand binding assays using 125I-thrombin. Key results: Quercetin, apigenin and genistein impaired platelet aggregation, as well as 5-HT release and calcium mobilization, induced by thrombin and PAR-APs. Quercetin and apigenin were inhibitors of protein kinases, but genistein exhibited a minimal ability to suppress platelet phosphorylation. Binding assays did not establish any kind of interaction between thrombin receptors and any of the flavonoids tested. Conclusions and implications: Quercetin, apigenin and genistein did not inhibit thrombin responses by interacting with thrombin receptors, but by interfering with intracellular signalling. While inhibition by genistein may be a consequence of affecting calcium mobilization, subsequent platelet secretion and aggregation, for quercetin and apigenin, inhibition of kinase activation may also be involved in the impairment of platelet responses.
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Latent and polymeric antithrombin: Clearance and potential thrombotic risk
(Frontiers Media, 2007) Guerrero López, José Antonio; Corral de la Calle, Javier; Rivera Pozo, José; Miñano, Antonia; Alberca, Ignacio; Hernández Espinosa, David; Ordóñez, Adriana; Martínez, Constantino; Navarro-Núñez, Leyre; González-Conejero Hilla, Rocío; Lozano Almela, María Luisa; Vicente García, Vicente; Medicina Interna
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Biological assessment of aspirin efficacy on healthy individuals: heterogeneous response or aspirin failure?
(2004-12-16) González-Conejero Hilla, Rocío; Rivera Pozo, José; Corral de la Calle, Javier; Acuña, Carmen; Vicente, Vincente; Guerrero López, José Antonio; Medicina Interna; Facultad de Medicina
Background and Purpose— The widespread use of aspirin requires clarification of the aspirin resistance phenomenon. Most studies on this field are focused on patients which may affect the action of aspirin. Methods— We evaluated the biological efficacy of aspirin in healthy subjects. Results— Agonist–induced platelet aggregation was fully abrogated by 100 mg of aspirin in all individuals. By contrast, with the platelet function analyzer-100 device, 33.3% of the subjects displayed no response. This failure was overcome by 500 mg or by in vitro treatment of blood with 30 μmol/L acetylsalicylic acid. Intake of 100 mg of aspirin efficiently reduced by 75% the level of 11-dehydro thromboxane B2 (11-dTxB2) in all cases. However, variability on the pre-aspirin level (range 72.4 to 625.9 ng/mmol creatinine) led to substantial differences in the residual amount of the metabolite between subjects treated with aspirin (range 12.9 to 118.0 ng/mmol creatinine). Finally, there was no influence of platelet glycoprotein IIb/IIIa (Pro33Leu), platelet glycoprotein Ia/IIa, (C807T), and FXIII (Val34Leu) polymorphisms on the efficacy of aspirin. However, the cyclooxygenase (Cox)-1 50T allele associated with higher level of 11-dTxB2, both before and after aspirin. Moreover, the Cox-2 −765C variant displayed a slightly higher reduction in 11-dTxB2 level on treatment with aspirin. Conclusions— Our findings suggest that full resistance of healthy subjects to aspirin is rather unlikely. However, differences in aspirin absorption, or pharmacokinetic, or other unrecognized factors may lead to lack of effect of low dose of aspirin in some subjects when using tests like platelet function analyzer-100. Whether Cox polymorphisms are thrombotic risk factor for patients under aspirin will require further research.