Deficiency of ARHGAP21 alters megakaryocytic cell lineage responses and enhances platelet hemostatic function

Agradecimentos: This study was supported by São Paulo Research Foundation (FAPESP processes 17/21801-2, 2013/13022-2 and 17/19674-2), National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES). The authors would like to thank Mariana Pissarra and Mariana Ozello Baratti for their valuable technical assistance. We also thank Raquel S Foglio for the English revision Abstract: Background: Microtubules, actin and Rho GTPase proteins are essential players in the megakaryocyte biology for platelet formation and function. Objectives: To investigate the role of ARHGAP21, a RhoGAP protein, in megakaryocytic differentiation and platelet function. Methods: Cytoskeletal proteins were investigated in HEL cells silenced for ARHGAP21 and submitted to megakaryocyte differentiation. The role of Arhgap21 in platelet function was accessed using haploinsufficient (Arhgap21+/-) mice. Arhgap21+/- platelet aggregation and p-selectin exposure were evaluated in response to thrombin. Vessel occlusion time and thrombus formation were detected after injury of the carotid artery. Platelet morphology was accessed by electronic microscopy. Results: ARHGAP21 was upregulated during megakaryocytic differentiation of the cell line and primary mouse cells. In the HEL cell model, ARHGAP21 was detected in the cytoplasmic protrusions, colocalized and associated with a-tubulin and was mostly detected in the protein cell fraction containing the polymerized tubulin. Silencing of ARHGAP21 decreased the expression of Glu-tubulin, suggesting microtubule instability, and enhanced cell spreading. Platelets from Arhgap21+/- mice presented enhanced thrombin-induced aggregation and p-selectin exposure associated with increased size of a-granules. Arhgap21+/- mice also showed increased CDC42 and RHOA activities, shorter tail-bleeding and accelerated thrombus formation. Conclusions: Our results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado