Identification of miRNAs as potential modulators of tissue factor expression in patients with systemic lupus erythematosus and antiphospholipid syndrome.

BACKGROUND: Tissue factor (TF) is the main initiator of the coagulation cascade and elements that may upregulate its expression might provoke thrombotic events. Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are autoimmune diseases characterized by a high TF expression in monocytes. OBJECTIVES: To examine the role of microRNAs (miRNAs) in TF expression and to evaluate their levels in SLE and APS patients. METHODS: An in silico search was performed to find potential putative binding sites of miRNAs in TF mRNA. In vitro validation was performed transfecting cells expressing TF (THP-1 and MDA-MB-231) with oligonucleotide miRNA precursors and inhibitors. Additionally, reporter assays were performed to test for the binding of miR-20a to TF mRNA. Levels of miRNAs and TF were measured by quantitative (qRT-PCR) in patients with APS and SLE. RESULTS: Overexpression of miRNA precursors, but not inhibitors, of two of the members of cluster miR-17∼92, for example miR-19b and miR-20a, in cells expressing TF decreased TF mRNA, protein levels, and procoagulant activity between 30% and 60%. Reporter assays showed that miR-20a binds to TF mRNA. Finally, we measured levels of miR-19b and miR-20a in monocytes from patients with APS and SLE and observed significantly lower miRNAs levels in comparison with healthy subjects inversely correlated with the levels of TF. CONCLUSIONS: Down-regulation of miR-19b and miR-20a observed in patients with SLE and APS could contribute to increased TF expression and thus provoke the hypercoagulable state characteristic of these patients.

Pharmacogenetics of acenocoumarol in patients with extreme dose requirements.

SUMMARY BACKGROUND: There is currently intense debate as to whether pharmacogenetic algorithms for estimating the initial dose of coumarins provide a more accurate dose than the fixed-dose approach. Recently, it has been suggested that the greatest benefit of pharmacogenetic algorithms is observed in patients with extreme dose requirements. OBJECTIVES: To identify clinical and genetic factors that better characterize patients who need extreme acenocoumarol doses for steady anticoagulation state. PATIENTS/METHODS: We reviewed 9538 patients with a steady acenocoumarol dose from three Spanish hospitals, selecting 83 who took or= 30.00 mg week(-1) (p95). We also selected patients matched by gender and age taking 13.50-14.00 mg week(-1) (p50). We genotyped VKORC1 (rs9923231), CALU (rs1043550), GGCX (rs699664), CYP2C9 (rs1799853; rs1057910), CYP4F2 (rs2108622) and F7 (rs5742910) single-nucleotide polymorphisms (SNPs). RESULTS: Comparison between p5 and p95 revealed five parameters with significant differences: body surface area (BSA) (P = 0.006), age, VKORC1, CYP2C9 and CYP4F2 genotypes (all P < 0.001). First VKORC1, and second, CYP2C9 SNPs played a strong effect by determining extreme doses, particularly in p95. Only one out of 203 p95 had the VKORC1 A-1639A genotype, but this subject was CYP2C9*1/*1. In contrast, nine out of 83 p5 carried the VKORC1 G-1639G genotype, although six of them were CYP2C9*3 homozygotes and another two were heterozygotes. Surprisingly, CYP4F2 V433M SNP displayed prevalences that suggest that its influence might only be evident when patients are treated with high doses. CONCLUSION: Two clinical data, age and BSA, and three SNPs in the VKORC1, CYP2C9 and CYP4F2 genes strongly predict outlier patients treated with acenocoumarol.