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, which is subject to upstream regulation by angiotensin-converting enzyme (ACE). Although the implication of microRNAs (miRNAs), epigenetic modulators of gene expression, has been demonstrated in adult thrombosis, evidence is lacking in the pediatric setting. Here, we investigated the involvement of two miRNA regulators of PAI-1 (SERPINE1 gene) in PT, in relation to clinical and genetic parameters that induce PAI-1 fluctuations. 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 healthy controls. Patients were genotyped for the SERPINE1-4G/5G and ACE-I/D polymorphisms by PCR-based assays. Genotypic and thrombosis-related clinical data were analyzed in relation to miRNA-expression. Two miRNAs (miR-145-5p, miR-34a-5p) were identified to target SERPINE1 mRNA, with miR-34a additionally targeting the mRNA of ACE. The expression of miR-34a was significantly decreased in patients compared to controls (p = 0.029), while no difference was observed in miR-145 expression. Within patients, miR-34a expression 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 levels 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 a miR-34 decrease, with D/D carriership reversing the 4G/4G-induced upregulation of miR-34a (p = 0.006). 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.

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.

Background: Pediatric thrombosis (PT) is a rare clinical entity manifesting from neonatal life to adolescence with possibly severe clinical complications and increasing prevalence in the last decade. Its pathogenesis has been attributed to a multitude of factors, all of which share stress and inflammation as a common denominator. Although stress-induced inflammation, marked by the upregulation of proinflammatory interleukins, such as IL-1β, creates a prothrombotic environment, its relation with thrombosis remains incompletely defined, especially in the context of PT, which differs substantially from adult thrombosis. Here, we aimed to epigenetically explore the potential link between PT and stress-induced inflammation through a combinatorial approach. Methods: Initially, we bioinformatically identified candidate stress-specific miRNAs in childhood, followed by subsequent experimental assessment in serum samples of PT patients and healthy controls in relation to subjectively perceived stress levels and the functional +3953C/T genetic polymorphism of the IL1B gene, which affects the circulating levels of the proinflammatory and stress-induced interleukin 1beta (IL-1β). Results: The bioinformatic analysis identified miR-34a-5p as the most stress-specific candidate microRNA in childhood, exhibiting the highest gene-target coverage (80.6%) of the developed panel of adversity-related genes, but is also known to regulate IL1B expression. The expression of serum miR-34a-5p was not significantly influenced by subjectively perceived stress; however, its expression was significantly reduced in the presence of the T allele of the functional +3953C/T polymorphism of its target gene, IL1B, associated with increased levels of the stress-induced proinflammatory and simultaneously prothrombotic IL-1β cytokine (FC:0.57; p = 0.024). Conclusions: The observed downregulation of miR-34a-5p in the presence of the IL-1β-increasing T allele possibly indicates a dynamic regulatory relationship, which, alongside its previously established long-term downregulation in PT, renders miR-34a-5p a promising candidate molecule for bridging the biological processes of stress-associated inflammation and thrombosis. In addition, its bioinformatically yielded candidate stress-specific role holds great potential for the non-invasive evaluation of adversity-related stress in childhood.