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Study of the role of ARHGAP21 during megakaryocytic differentiation and in the hemostatic process

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Vanessa Aline Bernusso
Total Authors: 1
Document type: Doctoral Thesis
Press: Campinas, SP.
Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
Defense date:
Examining board members:
Sara Teresinha Olalla Saad; Jörg Kobarg; Nicola Amanda Conran Zorzetto; Daniela Sanchez Bassères; Luciene Borges
Advisor: Mariana Lazarini; Sara Teresinha Olalla Saad

Cytoskeleton reorganization mediated by the Rho family of GTPases is an essential part of megakaryocyte differentiation and platelet formation. The different functions of platelets also depend on the intense reorganization of the cytoskeleton. RhoGAP proteins, such as ARHGAP21, are negative regulators of Rho GTPases and accelerate their inactivation. Our research group has reported the participation of ARHGAP21 in the regulation of the functions of hematopoietic stem progenitor cells, including its involvement in megakaryocytic lineage differentiation. In the present study, we investigated the role of ARHGAP21 in megakaryocytic differentiation and platelet function. To this end, two models were used: 1) the human erythroleukemia cell line (HEL) silenced for ARHGAP21 and subjected to megakaryocytic differentiation in vitro and 2) heterozygous knockout mice for the Arhgap21 gene (Arhgap21+/-). The HEL cell line model was used to assess cytoskeleton proteins by confocal microscopy and western blotting as well as differentiation markers and ploidy by flow cytometry. The Arhgap21+/- mouse model was used in different experiments to investigate megakaryocytic differentiation of primary progenitor cells. Vessel occlusion time and thrombus formation were detected after injury of carotid artery with FeCl3. Arhgap21+/-platelet aggregation and exposure to p-selectin in response to thrombin were obtained using an aggregometer and platelet morphology was evaluated by electron microscopy. ARHGAP21 expression was increased during megakaryocytic differentiation both in the HEL cell line and in mice primary cells. In the HEL cell line, ARHGAP21 was detected in cytoplasmic protrusions, where it colocalized and associated with ?-tubulin, and mainly detected in the protein cell fraction containing polymerized tubulin. In those cells, ARHGAP21 silencing decreased the expression of glu tubulin suggesting microtubule instability and greater spreading with increased cell protrusions. Furthermore, ARHGAP21 silencing increased CDC42 activity in the HEL cell line, whereas no change in RHOA activity was observed. Using Arhgap21+/- mouse model, we observed increased platelet aggregation in vitro induced by thrombin and higher p-selectin exposure. There was higher RHOA and CDC42 activity in Arhgap21+/- bone marrow primary cells when they were subjected to megakaryocytic differentiation in comparison with wild-type cells. Arhgap21+/- platelets showed an increase in the size of ? granules. These results were associated with shorter tail bleeding time and accelerated thrombus formation after injury of carotid artery with FeCl3. Our results indicate that ARHGAP21 may be a critical protein in regulating platelet production and function through the control of cytoskeleton rearrangement (AU)

FAPESP's process: 13/13022-2 - Study of the role of ARHGAP21 protein in platelet formation and angiogenesis
Grantee:Vanessa Aline Bernusso
Support Opportunities: Scholarships in Brazil - Doctorate