Guerrero López, José Antonio

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Guerrero López, José Antonio

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Medicina

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Inhibición de la función de las plaquetas por flavonoides : implicación de la ruta de señalización del tromboxano A2 / José Antonio Guerrero López; [directores, José Rivera Pozo y María Luisa Lozano Almela].
(Murcia : Universidad de Murcia, Departamento de Bioquímica y Biología Molecular B e Inmunología,, 2005) Guerrero López, José Antonio
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In vivo relevance for platelet glycoprotein Ibα residue Tyr276 in thrombus formation
(Elsevier, 2008) Guerrero López, José Antonio; Shafirstein, G.; Russell, S.; Varughese, K. I.; Kanaji, T.; Liu, J.; Gartner, T. K.; Bäumler, W.; Jarvis, G. E.; Ware, J.; Medicina Interna; Facultad de Medicina
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Protective role of antithrombin in mouse models of liver injury
(Elsevier, 2012) Teruel, Raul; Martínez, Constantino; Arcas, Isabel; Martínez-Martínez, Irene; Morena-Barrio, María Eugenia de la; Vicente, Vicente; Corral, Javier; Guerrero López, José Antonio; Medicina
Background & Aims: Coagulopathy caused by an imbalance of hemostatic factors is associated with the pathophysiology of liver disease. We have investigated the role of antithrombin (AT), a key anticoagulant serpin, in the onset of liver disease. Methods: Liver injury was induced by CCl4 injection and bile duct ligation (BDL) in wild type (WT) and AT-deficient (AT+/ ) mice. Twenty-four hours after CCl4 treatment, aspartate-transaminase, alanine-transaminase, liver lesion size, leukocyte infiltration, and apoptosis were reduced in WT animals compared to AT+/ mice. Results: Administration of exogenous AT in AT+/ animals did not restore the values observed in WT mice, suggesting that intrahepatic AT might also offer protection against CCl4. In the BDL model, increased liver injury was also evident in AT+/ compared to WT mice. An 85 kDa covalent complex involving AT was identified in immunoblottings of liver lysates from CCl4-treated animals. This complex was also present in anoikis hepatocytes and H2O2-treated HepG2 cells, suggesting a role for AT in apoptosis. Expression of recombinant WT–AT by HEK-EBNA cells increased cell survival while expression of AT mutants, DR393 and R47C, did not modify viability. Finally, plasma anti-FXa activity was attenuated by liver injury, with AT+/ animals showing a greater reduction than WT mice. Conclusions: Our study reveals a protective role of AT against liver injury due to its recognized anticoagulant and antiinflammatory action. AT may also act via a previously unrecognized antiapoptotic effect. The clinical implications of AT deficiency in patients with liver disease should be further addressed. 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
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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Platelet glycoprotein Ibα supports experimental lung metastasis
(National Academy of Sciences, 2007-05-22) Guerrero López, José Antonio; Jain, Shashank; Zuka, Masahiko; Liu, Jungling; Russell, Susan; Dent, Judith; Forsyth, Jane; Maruszak, Brigid; Gartner, T. Kent; Felding-Habermann, Brunhilde; Ware, Jerry; Medicina Interna; Facultad de Medicina
The platelet paradigm in hemostasis and thrombosis involves an initiation step that depends on platelet membrane receptors binding to ligands on a damaged or inflamed vascular surface. Once bound to the surface, platelets provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin-rich network produced by coagulation factors. The platelet-specific receptor glycoprotein (GP) Ib-IX, is critical in this process and initiates the formation of a platelet-rich thrombus by tethering the platelet to a thrombogenic surface. A role for platelets beyond the hemostasis/thrombosis paradigm is emerging with significant platelet contributions in both tumorigenesis and inflammation. We have established congenic (N10) mouse colonies (C57BL/6J) with dysfunctional GP Ib-IX receptors in our laboratory that allow us an opportunity to examine the relevance of platelet GP Ib-IX in syngeneic mouse models of experimental metastasis. Our results demonstrate platelet GP Ib-IX contributes to experimental metastasis because a functional absence of GP Ib-IX correlates with a 15-fold reduction in the number of lung metastatic foci using B16F10.1 melanoma cells. The results demonstrate that the extracellular domain of the α-subunit of GP Ib is the structurally relevant component of the GP Ib-IX complex contributing to metastasis. Our results support the hypothesis that platelet GP Ib-IX functions that support normal hemostasis or pathologic thrombosis also contribute to tumor malignancy.
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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.
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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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
Visualizing the von Willebrand factor/glycoprotein Ib-IX axis with a platelet-type von Willebrand disease mutation
(American Society of Hematology (ASH Publications), 2009-12-24) Kyei, Mark; Russell, Susan; Liu, Junling; Gartner, T Kent; Storrie, Brian; Ware, Jerry; Guerrero López, José Antonio; Medicina Interna; Facultad de Medicina
Platelet-type von Willebrand disease (PT-VWD) is a bleeding disorder of the platelet glycoprotein Ib-IX/von Willebrand factor (VWF) axis caused by mutations in the glycoprotein Ib-IX receptor that lead to an increased affinity with VWF. In this report, platelets from a mouse expressing a mutation associated with PT-VWD have been visualized using state-of-the art image collection and processing. Confocal analysis revealed that VWF bound to the surface of single platelets and bridging micro-aggregates of platelets. Surface-bound VWF appears as a large, linear structure on the surface of 50% of the PT-VWD platelets. In vivo thrombus formation after chemical injury to the carotid artery revealed a severe impairment to occlusion as a consequence of the PT-VWD mutation. In vitro stimulation of PT-VWD platelets with adenosine diphosphate or thrombin demonstrates a significant block in their ability to bind fibrinogen. The impairment of in vivo thrombus formation and in vitro fibrinogen binding are more significant than might be expected from the observed platelet binding to VWF polymers over a small portion of the plasma membrane. Visualization of the receptor/ligand interaction and characterization of a severe antithrombotic phenotype provide a new understanding on the molecular basis of bleeding associated with the PT-VWD phenotype.
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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.