Evaluation of the role of platelets in sickle cell anemia

Author(s)

Hanan Chweih

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2019-10-01

Examining board members

Nicola Amanda Conran Zorzetto; Cristina Pontes Vicente; Alessandra Gambero; Claudia Regina Bonini Domingos; Sandra Fatima Menosi Gualandro

• Advisor: Nicola Amanda Conran Zorzetto

Abstract

Sickle cell anemia (SCA) results from a homozygotic mutation in the HBB gene, resulting in the production of the altered ß-globin (ßS) chain. The disease is characterized by chronic hemolytic anemia, painful vaso-occlusive crises, target organ damage and a chronic inflammatory state. Patients with SCA have high leukocyte counts, and evidence indicates the participation of leukocytes in the triggering and propagation of vaso-occlusive processes, as these cells are relatively large and rigid, causing their recruitment to blood vessel walls in the microcirculation and reducing blood flow. The role of platelets in the vaso-occlusive process is poorly understood, although we know that platelet activation occurs in SCA. Thus, this project aimed to evaluate the role of platelets in vaso-occlusive processes, stimulated by TNF cytokine, in chimeric mice with SCA, visualizing and differentiating the cells participating in this inflammatory process. Additionally, the effect of the depletion of platelets or the inhibition of their activity on the inflammatory response caused by TNF in mice with SCA was evaluated. For this project, we produced chimeric SCA mice by transplanting the bone marrow of SCA Berkeley transgenic mice into C56BL/6 mice (in order to increase the SCA mouse colony size), in addition to conventional intravital microscopy and intravital confocal microscopy of the cremaster muscle microcirculation, with fluorescent labeling for platelets and leukocytes. We found that, after stimulation of SCA animals with TNF, the platelets act as a primary response signal, covering the inflamed endothelium of microcirculation and subsequently recruiting leukocytes (with both high and low expressions of the Ly6G adhesion molecule), both in Control (CON) and SCA animals. Moreover, in association with increased platelet adhesion to the vascular wall, there was a reduction in blood flow velocity and animal survival. We found that treatments capable of decreasing platelet count or platelet activation (use of anti-CD42b antibody or prasugrel, respectively) were able to reduce, or even reverse, the recruitment of platelets and leukocytes in the microcirculation, and thus vaso-occlusive processes in SCA mice. Administration of hydroxyurea in SCA animals reduced platelet and leukocyte recruitment and the formation of platelet / leukocyte aggregates in the microcirculation, therefore improving blood flow rate. Finally, we compared the mechanism of vaso-occlusion, when stimulated by hypoxia and reperfusion processes, with TNF-induced vaso-occlusion in SCA mice; despite an increased recruitment of leukocytes in the microcirculation of the SCA mice subjected to hypoxia/reperfusion, we observed less platelet recruitment with a less significant decrease in blood flow. These data lead us to conclude that the platelet plays an important role in TNF-induced inflammatory processes, initiating and propagating vaso-occlusive mechanisms in animals with SCA. Despite the disappointing data from the first multicenter clinical trial of prasugrel in SCA, antiplatelet agents should still be studied as a form of therapy for the disease, in combination or not with hydroxyurea, to decrease the frequency of vaso-occlusive episodes or even for use in patients hospitalized for acute vaso-occlusive crisis (AU)

• FAPESP's process: 15/18369-6 - Evaluation of the role of platelets in sickle cell disease
• Grantee: Hanan Chweih
• Support Opportunities: Scholarships in Brazil - Doctorate