José Rivera

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor.

Summary.  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, and TxA2 generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA2 receptors. Results: 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 TxA2 synthesis. These compounds were identified as specific ligands of the TxA2 receptor in the µmol 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 TxA2 receptor. Therefore, antagonism of this TxA2 receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.

The venous thrombosis risk factor 20210 A allele of the prothrombin gene is not a major risk factor for arterial thrombotic disease

A nucleotide change (G to A transition) at position 20210 has recently been demonstrated to be a risk factor for venous thrombosis. The relevance of this polymorphism to thrombotic disease was investigated by genotypic identification in three prospective case–control studies: 101 case patients with acute coronary heart disease (CHD), 104 patients with acute cerebrovascular disease (CVD), 82 patients with a confirmed diagnosis of deep venous thrombosis (DVT), and one control age‐ and sex‐matched for each patient. The prevalence of the genetic variation was significantly associated with the occurrence of DVT, but did not differ in patients with CHD or CVD from that in controls, suggesting that this allele should not be considered a major risk factor for arterial thrombotic disease.

Association of NOS3 gene with metabolic syndrome in hypertensive patients

Recent data from animal models indicate that the eNOS null mice present a phenotype that resemble the human metabolic syndrome (hypertension, insulin resistance and hypertriglyceridemia). In this work, we have studied whether NOS3 gene, previously related to endothelial dysfunction, might have a role in metabolic syndrome susceptibility in hypertensive patients. To carry out the study, we genotyped 105 hypertensive patients < or = 60 years old with two polymorphisms of NOS3 gene: 1132 T>C and 7164 G>T (GeneBank:AF519768.1). To check the allelic frequency of these polymorphisms in our geographical area, we also genotyped 94 unselected healthy controls (control group). To perform sample genotyping, we designed a novel FRET system coupled to real time PCR. There were no differences in genotypic distribution or allelic frequency between hypertensive patients and the control group. However, we observed that 786CC genotype was significantly more frequent in hypertensive patients with metabolic syndrome than in those without the syndrome (p=0.0022). When both polymorphisms were analyzed, we identified the 786C894G as the risk haplotype for metabolic syndrome susceptibility (p=0.011). These data suggest a role of the NOS3 gene in the pathogenesis of metabolic syndrome in hypertensive patients.

Stability of glycoproteins Ib/IX and IIb/IIIa during preparation and storage of platelet concentrates: detection by binding assays with epitope-defined monoclonal antibodies and physiological ligands.

Preservation of the glycoprotein (GP) complexes Ib/IX and IIb/IIIa, because of their role as specific platelet receptors for adhesive proteins, is essential for the haemostatic efficacy of transfusions of platelet concentrates (PCs). We have combined binding assays with epitope‐defined monoclonal antibodies, and with the physiological ligands von Willebrand factor and fibrinogen (Fo), to investigate the total expression and functional status of these receptors, in PCs stored for up to 9 days. Routine preparation and storage for up to 5 days have no apparent effect on the surface expression and the functional status of GPs Ib/IX and IIb/IIIa. However, after prolonged storage for up to 9 days we found a 50% decrease in the level of the total and functional GP Ib/IX content of platelets. This finding parallelled a significant rise in plasma glycocalicin, a proteolytic fragment of GP Ib. In addition, long‐term storage promoted an impairment in the exposure of GP IIb/IIIa to Fo, and a 50% decrease in Fo binding capacity, without affecting the complex quantiatitvely. Finally, we also noticed a storage‐induced increase in the platelet surface expression of GMP 140, and after 9 days of storage there was a rise in mean platelet volume, and a significant reduction in pH levels.

Clinical and analytical relevance of the combination of prothrombin 20210A/A and factor V Leiden: results from a large family.

We analysed the clinical and analytical features of 18 subjects from a Spanish family who bear several combinations of two prothrombotic mutations, factor V Leiden (FVL) and prothrombin 20210A. We identified three subjects homozygous for the 20210A prothrombin mutation which additionally were heterozygous for FVL. The combination of both mutations increases the risk of developing venous thrombotic episodes at the earlier age. However, even in association with FVL, the homozygous condition of the prothrombin 20210A mutation requires additional risk factors to induce a thrombotic event. Finally, the plasma level of factor II showed a significant relationship with the prothrombin genotype.

Whole exome sequencing identifies genetic variants in inherited thrombocytopenia with secondary qualitative function defects

Inherited thrombocytopenias are a heterogeneous group of disorders characterized by abnormally low platelet counts which can be associated with abnormal bleeding. Next-generation sequencing has previously been employed in these disorders for the confirmation of suspected genetic abnormalities, and more recently in the discovery of novel disease-causing genes. However its full potential has not yet been exploited. Over the past 6 years we have sequenced the exomes from 55 patients, including 37 index cases and 18 additional family members, all of whom were recruited to the UK Genotyping and Phenotyping of Platelets study. All patients had inherited or sustained thrombocytopenia of unknown etiology with platelet counts varying from 11×109/L to 186×109/L. Of the 51 patients phenotypically tested, 37 (73%), had an additional secondary qualitative platelet defect. Using whole exome sequencing analysis we have identified “pathogenic” or “likely pathogenic” variants in 46% (17/37) of our index patients with thrombocytopenia. In addition, we report variants of uncertain significance in 12 index cases, including novel candidate genetic variants in previously unreported genes in four index cases. These results demonstrate that whole exome sequencing is an efficient method for elucidating potential pathogenic genetic variants in inherited thrombocytopenia. Whole exome sequencing also has the added benefit of discovering potentially pathogenic genetic variants for further study in novel genes not previously implicated in inherited thrombocytopenia.

Differential effects of quercetin, apigenin and genistein on signalling pathways of protease-activated receptors PAR1 and PAR4 in platelets

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.

SLFN14 mutations underlie thrombocytopenia with excessive bleeding and platelet secretion defects

Inherited thrombocytopenias are a group of disorders that are characterized by a low platelet count and are sometimes associated with excessive bleeding that ranges from mild to severe. We evaluated 36 unrelated patients and 17 family members displaying thrombocytopenia that were recruited to the UK Genotyping and Phenotyping of Platelets (GAPP) study. All patients had a history of excessive bleeding of unknown etiology. We performed platelet phenotyping and whole-exome sequencing (WES) on all patients and identified mutations in schlafen 14 (SLFN14) in 12 patients from 3 unrelated families. Patients harboring SLFN14 mutations displayed an analogous phenotype that consisted of moderate thrombocytopenia, enlarged platelets, decreased ATP secretion, and a dominant inheritance pattern. Three heterozygous missense mutations were identified in affected family members and predicted to encode substitutions (K218E, K219N, and V220D) within an ATPase-AAA-4, GTP/ATP-binding region of SLFN14. Endogenous SLFN14 expression was reduced in platelets from all patients, and mutant SLFN14 expression was markedly decreased compared with that of WT SLFN14 when overexpressed in transfected cells. Electron microscopy revealed a reduced number of dense granules in affected patients platelets, correlating with a decreased ATP secretion observed in lumiaggregometry studies. These results identify SLFN14 mutations as cause for an inherited thrombocytopenia with excessive bleeding, outlining a fundamental role for SLFN14 in platelet formation and function.

Cryopreservation Modifies Flow‐Cytometric Analysis of Hemopoietic Cells

Although cryopreservation of human bone marrow has become very common in modern medicine, the knowledge on the effects of this procedure on hemopoietic cells is still limited. We herein have investigated whether the process of concentrating of human bone marrow to its buffy coat, the exposure to the cryoprotectant dimethyl sulfoide (DMSO), and/or the rate of controlled freezing/thawing procedures modifies the flow cytometric analysis of human bone marrow cells. We found that both the exposure of marrow cells to DMSO and/or the freezing procedure significantly modifies both the relative proportions of hemopoietic cell subsets and the intensity of expression of certain surface antigens. Thus, percentages of cells expressing CD7, CD13, CD33 and CD34, were found to be lower in both cryopreserved buffy coat and buffy coat merely exposed to DMSO, in comparison to those in untreated coat samples. Moreover, the intensity of the surface expression of CD33 decreased in cells exposed to DMSO, and further in cryopreserved samples. By contrast, the intensity of the CD19 antigen was higher in the last groups of samples than in the buffy coat or the unfractionated human bone marrow. Our present results suggest that flow‐cytometric analysis of cryopreserved human bone marrow cells is not fully equivalent to that corresponding to fresh bone marrow or its fractionated buffy coat.

Effect of quercetin on platelet spreading on collagen and fibrinogen and on multiple platelet kinases

alpha(2)beta(1) and alpha(IIb)beta(3) integrins, that support platelet adhesion to collagen and fibrinogen, respectively, share common signaling molecules. The effect of quercetin on platelet static adhesion to collagen and fibrinogen was assessed and correlated with its kinase inhibitory activity. Quercetin strongly abrogated PI3K and Src kinases, mildly inhibited Akt1/2, and slightly affected PKC, p38 and ERK1/2. Quercetin or the combined use of adenosine diphosphate and thromboxane A(2) inhibitors abrogated platelet spreading on these surfaces to a similar extent. We suggest that the inhibitory effect of quercetin on platelet kinases blocks early signaling events preventing a complete platelet spreading.