Epidemiological studies have revealed that modification of the levels of individual components of the haemostatic system may have repercussion in the development of thrombosis or haemorrhage. To maintain the necessary equilibrium, the haemostatic system is finely regulated. It is known that acquired factors and/or alterations in genes (SNPs or mutations) may be the cause of inter-individual differences or exacerbated levels of haemostatic proteins in plasma, but there are still many non-characterized factors that provoke such variations. The search for new elements, such as microRNAs (miRNAs), a family of small non-coding RNAs that are novel regulators of protein expression, may reveal an additional layer to investigate the causes of haemostatic diseases. In this review, we discuss the latest developments in research into the role of miRNAs in the regulation of several haemostatic factors and the potential use of miRNAs as prognostic or diagnostic tools in haemostasis and thrombosis. This article is protected by copyright. All rights reserved.
High on-treatment platelet reactivity (HTPR), referred to as a higher than expected platelet reactivity in patients under antiplatelet therapy, could influence outcome in cerebrovascular disease (CVD), but its prevalence and its stability over time is uncertain. Platelet reactivity was assessed in 18 patients with ischemic stroke/transient ischemic attack (TIA) 7 days (D7) and 90 days (D90) after prescription of clopidogrel, using four methods: light transmission aggregometry with 5 ?mol/l ADP (LTA-ADP), vasodilator-stimulated phosphoprotein (VASP), Verify Now P2Y12 and platelet function analyzer (PFA) P2Y. HTPR was defined as LTA-ADP more than 46%; PFA-100-P2Y closure time less than 106 s; VerifyNow P2Y12, PRU greater than 235, VASP, PRI greater than 50%. Patients displayed, both at D7 and D90, a marked inhibition of platelet reactivity towards ADP in all tests as compared with reference levels. Correlations between the results obtained with all the tests at D7 and D90 and between measurements on each day in each test were low-to-moderate. The prevalence of HTPR for all the tests was 40% at D7 and 42% at D90. There was a moderate degree of agreement (k statistic < 0.5) between tests with regard to categorizing patients as HTPR/No-HTPR (D7 and D90). The on-clopidogrel platelet reactivity phenotype, HTPR/No-HTPR, remained stable in 55-72% of patients, depending on the test. A high prevalence of HTPR is found among CVD patients treated with clopidogrel and this platelet reactivity phenotype remains over time. There is poor agreement between the different platelet function tests for categorizing the platelet reactivity phenotype in these patients. The new PFA-100 P2Y equals other platelet function assays for evaluating HTPR in CVD.
There are few biomarkers able to forecast new thrombotic events in patients with AF. In this framework, microRNAs have emerged as critical players in cardiovascular biology. In particular, miR-146a-5p is recognised as an important negative regulator of inflammation. This study aims to evaluate the prognostic role and biological effect of functional MIR146A polymorphisms, rs2431697 and rs2910164, in non-valvular atrial fibrillation (AF) patients under oral anticoagulation.We studied 901 patients with permanent/paroxysmal AF stabilized for at least six months. Patients were followed-up for two years and adverse cardiovascular events (ACE) were recorded. In vitro studies were performed in monocytes from healthy homozygous for the two genotypes of rs2431697. Rs2910164 had no association with ACE. However, multivariate analysis (adjusted by CHA2DS2-VASc score) revealed that rs2431697TT was associated with adverse cardiovascular events [HR: 1.64 (1.09-2.47); p=0.017]. The predictive value of usefulness of the CHA2DS2-VASc+IL6+rs2431697 for predicting ACE, was statistically better than that predicted by CHA2DS2-VASc+IL6. Functional studies showed that after 24 hours incubation, monocytes from CC individuals showed a 65?% increase in miR-146a-5p levels, while TT individuals only showed a 28?% increase. Indeed, after 24 hours of LPS activation, TT monocytes showed a higher increase in IL6 mRNA expression than CC (52?% vs 26?%). Our study established MIR146A rs2431697 as a prognostic biomarker for ACE in anticoagulated AF patients. These data suggest that TT individuals, when submitted to an inflammatory stress, may be prone to a highest pro-inflammatory state due, in part, to lower levels of miR-146a-5p.
High levels of factor XI (FXI) increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs) in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK) demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.
Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease resulting from mutations in the LYST/CHS1 gene, which encodes for a 429 kDa protein, CHS1/LYST, that regulates vesicle trafficking and determines the size of lysosomes and other organelles. To date, 60 different mutations have been characterized, and a reasonably straightforward phenotype-genotype correlation has been suggested. We describe two patients on opposite ends of the CHS clinical spectrum with novel missense mutations. We characterized these patients in terms of their mutations, protein localization and expression, mRNA stability, and electrostatic potential. Patient 1 is the first report of a severe early-onset CHS with a homozygous missense mutation (c.11362 G>A, p.G3725R) in the LYST/CHS1 gene. This molecular change results in a reduction at the CHS1 protein level, not due to an mRNA effect, but maybe a consequence of both, a change in the structure of the protein and most likely attributable to the remarkable serious perturbation in the electrostatic potential. Patient 2, who exhibited the adolescence form of the disease, was found to be homozygous for a novel missense mutation c.961 T>C, p.C258R, which seemed to have minor effect on the structure of the CHS1/LYST protein. Reexamining accepted premises of missense mutant alleles being reported among patients with clinically mild forms of the disorder should be carried out, and attempts to link genotype and clinical phenotype require identifying the actual molecular effect of the mutation. Early and accurate diagnosis of the severity of the disease is extremely important to early differentiate patients who would benefit from premature enrollment into a transplantation protocol.
Regulation of key proteins by microRNAs (miRNAs) is an emergent field in biomedicine. Vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1) is a relevant molecule for cardiovascular diseases, since it is the target of oral anticoagulant drugs and plays a role in soft tissue calcification. The objective of this study was to determine the influence of miRNAs on the expression of VKORC1. Potential miRNAs targeting VKORC1 mRNA were searched by using online algorithms. Validation studies were carried out in HepG2 cells by using miRNA precursors; direct miRNA interaction was investigated with reporter assays. In silico studies identified two putative conserved binding sites for miR-133a and miR-137 on VKORC1 mRNA. Ex vivo studies showed that only miR-133a was expressed in liver; transfection of miRNA precursors of miR-133a in HepG2 cells reduced VKORC1 mRNA expression in a dose-dependent manner, as assessed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) as well as protein expression. Reporter assays in HEK293T cells showed that miR-133a interacts with the 3UTR of VKORC1. Additionally, miR-133a levels correlated inversely with VKORC1 mRNA levels in 23 liver samples from healthy subjects. In conclusion, miR-133a appears to have a direct regulatory effect on expression of VKORC1 in humans; this regulation may have potential importance for anticoagulant therapy or aortic calcification.
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.
MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs that are negative regulators in a crescent number of physiological and pathological processes. However, their role in haemostasis, a complex physiological process involving multitude of effectors, is just beginning to be characterized. We evaluated the changes of expression of miRNAs in livers of neonates (day one after birth) and adult mice by microarray and qRT-PCR trying to identify miRNAs that potentially may also be involved in the control of the dramatic change of hepatic haemostatic protein levels associated with this transition. Twenty one out of 41 miRNAs overexpressed in neonate mice have hepatic haemostatic mRNA as potential targets. Six of them identified by two in silico algorithms potentially bind the 3UTR regions of F7, F9, F12, FXIIIB, PLG and SERPINC1 mRNA. Interestingly, miR-18a and miR-19b, overexpressed 5.4 and 8.2-fold respectively in neonates, have antithrombin, a key anti-coagulant with strong anti-angiogenic and anti-inflammatory roles, as a potential target. The levels of these two miRNAs inversely correlated with antithrombin mRNA levels during development (miR-19b: R?=?0.81; p?=?0.03; miR-18a: R?=?0.91; p<0.001). These data suggest that miRNAs could be potential modulators of the haemostatic system involved in developmental haemostasis.
Identification of mutations in the SERPINC1 gene has revealed different mechanisms responsible for antithrombin deficiency. Deletions and nonsense mutations associate with type I deficiency. Certain missense mutations cause type II deficiency by affecting the heparin binding site or the reactive center loop, while others result in type I deficiency by intracellular retention or RNA instability.
Support for the role of transmembrane and membrane-proximal domains of alpha IIb beta 3 integrin in the maintenance of receptor low affinity comes from mutational studies showing that activating mutations can induce constitutive bi-directional transmembrane signaling.
Polymorphisms affecting platelet receptors and intracellular proteins have been extensively studied in relation to their potential influence in thrombosis and haemorrhages. However, few reports have addressed their impact on platelet function, with contradictory results. Limitations of these studies include, among others, small number of patients, the platelet functional parameters analyzed and their known variability in the healthy population. We studied the effect of six polymorphisms [ITGB3 1565T > C (HPA-1), GPIBA variable number tandem repeat and 524C > T (HPA-2), ITGA2 807C > T, ADRA2A 1780A > G, and TUBB1 Q43P] on platelet function in 286 healthy subjects and their potential pathogenetic role in 160 patients with hereditary mucocutaneous bleeding of unknown cause. We found no effect of any of these polymorphisms on platelet aggregation, secretion, PFA-100, and thrombin generation in platelet rich plasma. Furthermore, patients and controls showed no significant differences in the frequency of any of these polymorphisms. Thus, our study demonstrated that polymorphisms in genes affecting platelet function do not influence significantly major platelet functions and appear irrelevant in the pathogenesis of bleeding disorders.
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.
Thromboxane A2 (TxA2) is a strong platelet agonist involved in the pathogenesis of thrombotic diseases that elicits platelet aggregation and vasoconstriction through the activation of its specific membrane receptor (TP). Previous studies have demonstrated that certain flavonoids, naturally occurring phytochemicals, inhibit platelet function through several mechanisms, including antagonism of TP in these cells. However, the steric and inductive or mesomeric requirements underlying this effect are not fully understood. In this study, the ability of 20 naturally occurring flavonoids belonging to different structural subtypes to inhibit [3H]-SQ29548 binding to platelet-rich plasma was compared to establish the structural basis explaining their TP antagonistic activity. The results show a key contribution of C7 and C8 carbons in the A ring, gamma-pyrone structure conjugated with a double bond between C2 and C3 carbons in the C ring, and C2, C3, and C4 carbons in the B ring as the structural determinants that create the active flavonoid skeleton in TP blockade. These data might help in the design of new TP antagonists with potential antithrombotic effects and provide additional evidence for the correlation between biological properties of flavonoids and their structure.
Cellular microtubules composed of ?-?-tubulin heterodimers that are essential for cell shape, division, and intracellular transport are valid targets for anticancer therapy. However, not all the conserved but differentially expressed members of the ?-tubulin gene superfamily have been investigated for their role in these settings. In this study, we examined roles for the hematologic isoform ?-tubulin VI and functional genetic variants in the gene. ?-tubulin VI was highly expressed in blood cells with a substantial interindividual variability (seven-fold variation in mRNA). We characterized DNA missense variations leading to Q43P, T274M, and R307H, and a rare nonsense variant, Y55X. Because variations in the hematologic target of microtubule-binding drugs might alter their myelosuppressive action, we tested their effect in cell lines stably expressing the different ?-tubulin VI full-length variants, finding that the T274M change significantly decreased sensitivity to paclitaxel-induced tubulin polymerization. Furthermore, patients treated with paclitaxel and carrying ?-tubulin VI T274M exhibited a significantly lower thrombocytopenia than wild-type homozygous patients (P = 0.031). Together, our findings define ?-tubulin VI as a hematologic isotype with significant genetic variation in humans that may affect the myelosuppresive action of microtubule-binding drugs. A polymorphism found in a tubulin isoform expressed only in hemapoietic cells may contribute to the patient variation in myelosuppression that occurs after treatment with microtubule-binding drugs.
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.
The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural ?-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified ?- and ?-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the ?-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the ?-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of ?-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the ?-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional ?-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.
Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.
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