Investigation of new pathophysiological mechanisms of hypercoagulability in sickle cell disease

Author(s)

Bidossessi Wilfried Hounkpe

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2020-03-02

Examining board members

Erich Vinicius De Paula; Nicola Amanda Conran Zorzetto; Joyce Maria Annichino Bizzacchi; Marcelo Torres Bozza; Marcelo Larami Santoro

• Advisor: Magnun Nueldo Nunes dos Santos; Erich Vinicius De Paula

Abstract

Introduction: Sickle Cell Anemia (SCA) is a hereditary hemoglobinopathy characterized by chronic systemic inflammation and a propensity for venous thromboembolic events. Despite advances in the characterization of hypercoagulability, which is a well-characterized consequence of this disease, the relative contribution of discrete mediators of coagulation activation for the loss of hemostatic balance observed in these patients is still not clear. In this regard, recent studies have been suggested that heme and the generation of neutrophil extracellular traps (NETs) should be viewed as critical candidate elements of the pathophysiology of hypercoagulability in this disease. Although it has been shown that free heme can trigger vasoocclusion and acute chest syndrome via TLR4, its net effect on the activation of coagulation is still controversial. Furthermore, recent evidences have shown that PADI4 is a critical regulator of NETs formation, so that its inhibition has been considered a potential therapeutic strategy in the treatment of diseases whose pathophysiology involves the formation of NETs such as SCA. Although attractive, this possibility requires a more detailed knowledge of PADI4 contribution to the pathophysiology of SCA. Objectives: In the first part of the study, we evaluated the effect of heme on the activation of coagulation in whole blood and investigated whether this effect was dependent of tissue factor (TF) expression and/or activation through TF decryption in mononuclear cells. In relation to study that involves NETs regulation, we intended to characterize the expression PADI4, MPO and Elane in patients with SCA and to explore their correlations with biomarkers of hypercoagulability and disease severity. Methods: Thromboelastometry was used to evaluate the effect of heme on hemostasis activation in whole blood. In addition, the expression of TF was analyzed by qPCR and its activity was evaluated by a functional FX activity assay. PDI activity was evaluated using a commercial kit. Finally, the contribution of the TLR4 pathway and the PDI-mediated decryption mechanism were explored using specific inhibitors (Tak-242 and rutin for TLR4 and PDI respectively). Regarding the formation of NETs, two cohorts were included in this study. In the first one, gene expression of PADI4, MPO and Elane was investigated by qPCR. In the second cohort, PADI4 plasmatic activity was analyzed. Results: Heme was able to induce hemostasis activation in whole blood samples. This effect was further amplified by pre-treatment with TNF-'alfa' and was dependent on TF expression and activation, as demonstrated in mononuclear cells. TF activation was dependent on TLR4 pathway and at least partially dependent on PDI-induced TF decryption. Significant increase of PADI4, ELANE and MPO gene expression was observed during vaso-occlusive crisis. PADI4 activity also increased in plasma of patients with SCA during vaso-occlusive crisis. Together, these results reinforced the concept that heme and NETosis regulation, through PADI4 activity, should be regarded as two relevant elements for the pathophysiology of SCA (AU)

• FAPESP's process: 15/24666-3 - Investigation of new pathophysiological mechanisms of hypercoagulability in Sickle Cell Disease
• Grantee: Bidossessi Wilfried Hounkpe
• Support Opportunities: Scholarships in Brazil - Doctorate