[Show abstract][Hide abstract] ABSTRACT: Background
Developmental haemostatic studies may help identifying new elements involved in the control of key haemostatic proteins like antithrombin, the most relevant endogenous anticoagulant.
In this study, we showed a significant reduction of sialic acid content in neonatal antithrombin compared with adult antithrombin in mice. mRNA levels of St3gal3 and St3gal4, two sialyltransferases potentially involved in antithrombin sialylation, were 85% lower in neonates in comparison with adults. In silico analysis of miRNAs overexpressed in neonates revealed that mir-200a might target these sialyltransferases. Moreover, in vitro studies in murine primary hepatocytes sustain this potential control.
These data suggest that in addition to the direct protein regulation, microRNAs may also modulate qualitative traits of selected proteins by an indirect control of post-translational processes.
[Show abstract][Hide abstract] ABSTRACT: The medical and socioeconomic relevance of thromboembolic disorders promotes an ongoing effort to develop new anticoagulants. Heparin is widely used as activator of antithrombin but incurs side effects. We screened a large database in silico to find alternative molecules and predicted d-myo-inositol 3,4,5,6-tetrakisphosphate (TMI) to strongly interact with antithrombin. Isothermal titration calorimetry confirmed a TMI affinity of 45 nM, higher than the heparin affinity (273 nM). Functional studies, fluorescence analysis, and citrullination experiments revealed that TMI induced a partial activation of antithrombin that facilitated the interaction with heparin and low affinity heparins. TMI improved antithrombin inhibitory function of plasma from homozygous patients with antithrombin deficiency with a heparin binding defect and also in a model with endothelial cells. Our in silico screen identified a new, non-polysaccharide scaffold able to interact with the heparin binding domain of antithrombin. The functional consequences of this interaction were experimentally characterized and suggest potential anticoagulant therapeutic applications.
[Show abstract][Hide abstract] ABSTRACT: 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.
Liver injury was induced by CCl(4) injection and bile duct ligation (BDL) in wild type (WT) and AT-deficient (AT(+/-)) mice. Twenty-four hours after CCl(4) treatment, aspartate-transaminase, alanine-transaminase, liver lesion size, leukocyte infiltration, and apoptosis were reduced in WT animals compared to AT(+/-) mice.
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 CCl(4). In the BDL model, increased liver injury was also evident in AT(+/-) compared to WT mice. An 85kDa covalent complex involving AT was identified in immunoblottings of liver lysates from CCl(4)-treated animals. This complex was also present in anoikis hepatocytes and H(2)O(2)-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, ΔR393 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.
Our study reveals a protective role of AT against liver injury due to its recognized anticoagulant and anti-inflammatory 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.
Journal of Hepatology 06/2012; 57(5):980-6. DOI:10.1016/j.jhep.2012.06.023 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metagenomics is an emerging field for mining the bioresources for new biomolecules for potential application in biotechnology and biomedicine. In the present study, a novel acetylhydrolase (Est13) was detected during the function-based screening of a metagenomic library established from the DNA extracted from the cellulose-depleting microbial community set up with an earthworm cast. Analysis showed that Est13 exhibited some similarities with a human and parasite platelet-activating factor acetylhydrolase (PAF-AH) belonging to the SGNH hydrolase superfamily. Biochemical characterization of the purified recombinant enzyme using substrates common for hydrolases of this superfamily demonstrated that Est13 hydrolysed p-nitrophenyl acetate quite efficiently, with a k(cat) /K(M) value of 3209 mM(-1) s(-1). The Est13 showed highest activity at pH 8.0 and 40°C, conditions in which it is relatively stable compared with known PAF-AHs. In vitro functional analysis of the platelet-activating factor hydrolysis showed a dose- and time-dependent inhibition of platelet aggregation in the range of 2-4 µM, making this enzyme a potential candidate for biomedical applications.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Dexamethasone is a potent, synthetic member of the glucocorticoid class of steroid drugs with pleiotropic effects on multiple signaling pathways, and has been widely used in many disorders during the last 50 years. Recent studies sustain a role of this drug in the heat stress response, increasing the levels of heat-shock proteins, particularly under certain stress conditions. More conflictive is the role of dexamethasone on the levels of endoplasmic reticulum chaperons. However, these effects may certainly contribute to explain the therapeutic benefits of dexamethasone in cardiac transplant, sepsis, cancer, and other pathologic disorders associated with stress affecting the folding of proteins. In this chapter, we review the methods that can be used to evaluate the effect of dexamethasone in the heat stress response both in patients and animal and cellular models.
Methods in enzymology 01/2011; 490:121-35. DOI:10.1016/B978-0-12-385114-7.00007-6 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Haematologica 2011 [Epub ahead of print] Citation: Guerrero J, Rivera J, Quiroga T, Martinez-Perez A, Anton-Garcia I, Martínez C, Panes O, Vicente V, Mezzano D, Soria JM, and Corral J. Novel loci involved on platelet function and platelet count identified by a genome-wide study performed in children. Haematologica. 2011; 96:xxx doi:10.3324/haematol.2011.042077 Publisher's Disclaimer. E-publishing ahead of print is increasingly important for the rapid dissemination of science. Haematologica is, therefore, E-publishing PDF files of an early version of manuscripts that have completed a regular peer review and have been accepted for publication. E-publishing of this PDF file has been approved by the authors. After having E-published Ahead of Print, manuscripts will then undergo technical and English editing, typesetting, proof correction and be presented for the authors' final approval; the final version of the manuscript will then appear in print on a regular issue of the journal. All legal disclaimers that apply to the journal also pertain to this production process. Haematologica (pISSN: 0390-6078, eISSN: 1592-8721, NLM ID: 0417435, www.haemato-logica.org) publishes peer-reviewed papers across all areas of experimental and clinical hematology. The journal is owned by the Ferrata Storti Foundation, a non-profit organiza-tion, and serves the scientific community with strict adherence to the principles of open access publishing (www.doaj.org). In addition, the journal makes every paper published immediately available in PubMed Central (PMC), the US National Institutes of Health (NIH) free digital archive of biomedical and life sciences journal literature.
[Show abstract][Hide abstract] ABSTRACT: We studied the effect of acrolein, an alpha,beta-unsaturated aldehyde that causes adduct-modification of lysine, cysteine, and histidine residues, on antithrombin, a key anticoagulant serpin. Intrinsic fluorescence, functionality (anti-FXa and anti-IIa activity), heparin affinity and conformational features of plasma and purified antithrombin were evaluated. In vivo experiments were carried out in mice. Intrinsic fluorescence showed a two-step conformational change. Acrolein, even at low dose, impaired the anticoagulant function of purified antithrombin by affecting its heparin affinity. However, higher concentrations of acrolein and long incubations are required to cause mild functional effects on plasma antithrombin and mice.
[Show abstract][Hide abstract] ABSTRACT: 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.
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 (125)I-thrombin.
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.
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.
British Journal of Pharmacology 10/2009; 158(6):1548-56. DOI:10.1111/j.1476-5381.2009.00440.x · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) 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.
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 TPalpha- and TPbeta-transfected HEK 293T cells was explored using binding assays and the TP antagonist (3)H-SQ29548.
Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC(50) 10-30 microm). 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% (3)H-SQ29548 binding to different cell types.
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.
British Journal of Clinical Pharmacology 08/2007; 64(2):133-44. DOI:10.1111/j.1365-2125.2007.02881.x · 3.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Antithrombin, the most potent anticoagulant in vivo, displays a significant conformational flexibility. The native five-stranded anticoagulant form transforms under different conditions or mutations to inactive six-stranded conformations: latent or polymer. However, the function, potential deleterious effects, and clearance of these forms are not completely known. The dimerization of latent antithrombin with a native molecule has been suggested to have thrombotic potential. We have assessed the potential thrombogenicity of high amounts of latent and polymeric antithrombin by experiments performed in mice and human plasma. Moreover, we have analyzed the clearance of (125)I-labeled native, latent, polymer, and thrombin-complexed antithrombins in rat, as well as the clearance of latent antithrombin from plasma of patients treated with commercial concentrates. Our results show that high plasma levels of latent or polymeric antithrombin do not interfere with the anticoagulant function of native antithrombin. Moreover, we confirm that all monomeric forms of antithrombin have similar turnover. Finally, we show that polymers have the longest half-life of all conformers, being in circulation for prolonged periods of time. In conclusion, our data support that latent and polymeric antithrombin would not likely have a thrombotic effect, thus dispelling doubts about the potential harmful effect of latent antithrombin present in commercial concentrates for therapeutic use. Moreover, the suggested antiangiogenic role of latent antithrombin, together with its stability in plasma and its negligible thrombogenicity raises the possibility of its use as a new antiangiogenic drug.
Experimental Biology and Medicine 03/2007; 232(2):219-26. · 2.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction/Aims
Non-enzymatic glycation of proteins can impair their function by incorporating sugars into their lysine and arginine residues. Glycation of antithrombin, a powerful anticoagulant, might be associated with the thrombotic risk observed in hyperglycemic conditions such as diabetes mellitus. Our aim was to study the effects of antithrombin glycation and determine its significance in the thrombotic complications observed in diabetes.
1) In vitro study of non-enzymatic glycation of purified and plasma antithrombin by their incubation with methylglyoxal and glucose. 2) The effect of different compounds on the in vitro glycation of antithrombin was analyzed. 3) We studied 101 diabetic patients. Antigen levels, anti-FXa activity, conformational features and antithrombin affinity to heparin were determined.
In vitro non-enzymatic glycation of antithrombin with methylglyoxal or glucose caused no significant conformational change in the molecule, but induced the transformation to a low heparin-affinity form, which explains the significant loss of activity observed (< 40% of basal). This effect was prevented by heparin, aminoguanidine and catechin. Diabetic patients presented lower antigenic and antithrombin functional levels (80%) than controls. However, no correlation between activity or antigen levels of antithrombin and glycemia or glycosylated hemoglobin was found in diabetic patients.
In vitro glycation of lysine and arginine residues located in the heparin-binding site of antithrombin significantly reduces its anticoagulant activity. Interestingly, heparin, aminoguanidine and catechin prevented this effect. However, the non-enzymatic glycation of antithrombin in diabetic patients seems to be mild, since the action of glucose is very slow and the half life of antithrombin in plasma is short.
Clínica e Investigación en Arteriosclerosis 10/2005; 17(5). DOI:10.1016/S0214-9168(05)73344-1
[Show abstract][Hide abstract] ABSTRACT: 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.
We evaluated the biological efficacy of aspirin in healthy subjects.
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 mumol/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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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.
The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects.
The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors.
Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4.
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 TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.
Journal of Thrombosis and Haemostasis 03/2005; 3(2):369-76. DOI:10.1111/j.1538-7836.2004.01099.x · 5.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 GPIbalpha (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.
[Show abstract][Hide abstract] ABSTRACT: A polymorphism of the gene encoding the extra-large stimulatory G-protein alpha-subunit (XLalphas), originally identified in three patients with a bleeding tendency, involved a 36-bp insertion and two missense changes. A paternally-inherited insertion displayed a moderate platelet Gsalpha 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 3.6% 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 Gsalpha or the platelet response to Gs-agonists. Thus, our study suggests a minor functional role of XLalphas polymorphism in thrombotic or in haemorrhagic disorders.
British Journal of Haematology 07/2004; 125(5):621-8. DOI:10.1111/j.1365-2141.2004.04947.x · 4.71 Impact Factor