Serum miR-34a as Indicator of Impaired Fibrinolytic Capacity in Pediatric Thrombosis Through Inadequate Regulation of the ACE/PAI-1 Axis

Background: Pediatric thrombosis (PT) represents a rare condition that can manifest from neonatal life to adolescence, encompassing life-threatening complications. Its pathogenesis is attributed to immature hemostasis in conjunction with environmental and genetic factors, predominantly including those resulting in increased levels of plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of fibrinolysis, subject to upstream regulation by ACE (angiotensin-converting enzyme). Although the implication of microRNAs (miRNAs), epigenetic modulators of gene-expression, is demonstrated in adult-thrombosis, evidence is lacking in the pediatric setting. Here, we investigated the involvement of two miRNA-regulators of PAI-1 in PT, in relation to clinical and genetic parameters that induce PAI-1 fluctuations. Methods: Following bioinformatic target-prediction, miRNA expression was assessed by quantitative real-time PCR, in serum-samples of 19 pediatric patients with thrombosis (1-18 months post-incident), and 19 controls. Patients were genotyped for the PAI-1-4G/5G and ACE-I/D polymorphisms by PCR-based assays. Genotypic and thrombosis-related clinical data were analyzed in relation to miRNA-expression. Results: Two miRNAs (miR-145-5p, miR-34a-5p) were identified to target PAI-1 mRNA, with miR-34a additionally targeting ACE mRNA. The expression of miR-34a was significantly decreased in patients compared to controls (p=0.029), while no difference was observed for miR-145. Within patients, miR-34a demonstrated a peak 1-3 months post-thrombosis and was diminished upon treatment-completion (p=0.031), followed by a slight long-term increase. MiR-34a expression differed significantly by thrombosis site (p=0.019), while a significant synergistic interaction between site and onset-type (provoked/unprovoked) was detected (p=0.016). Finally, an epistatic modification was observed in cerebral cases, since double homozygosity (4G/4G+D/D) led to miR-34a diminishing, with DD carriership reversing the 4G4G-induced upregulation of miR-34a (p=0.006). Conclusions: Our findings suggest that in pediatric thrombosis, downregulation of miR-34a is indicative of impaired fibrinolytic capacity, attributed to deficient regulation of the inhibitory ACE/PAI-1 axis.