Reduction of ARHGAP21 Alters Platelet Biogenesis in Vitro and Accelerates Hemostatic Response In Vivo

Megakaryocyte differentiation and platelet biogenesis require profound cytoskeleton rearrangement regulated by the Rho family of GTPases. ARHGAP21 is a RhoGAP protein that has been shown to negatively regulate the activity of RhoA, RhoC and Cdc42. We have previously demonstrated that ARHGAP21 knockdown in human common myeloid progenitors and in bipotent megakaryocyte-erythrocyte progenitors may bias the fate decision toward the megakaryocyte lineage. In addition, a mouse model with reduction of Arhgap21 expression (Arhgap21^+/-) present a slight reduction in platelet number and increased platelet volume. However, the participation of ARHGAP21 in platelet biogenesis and hemostatic response has never been investigated.

We studied the role of ARHGAP21 on cytoskeletal changes during megakaryocyte differentiation in HEL cell line. Differentiation was induced with 20ηM phorbol-13 myristate-12 acetate (PMA) for four days. ARHGAP21 protein expression was increased during the differentiation and was mostly detected in the protein cell fraction containing polymerized tubulin, in comparison with cell extracts containing soluble tubulin. ARHGAP21 co-localized (R ≥ 0.86 in all days of differentiation) and interacted with α-tubulin on day 2 of megakaryocyte differentiation, when ARHGAP21 expression was the highest. Silencing of ARHGAP21 with siRNA decreased the expression of Glu-tubulin and enhanced CDC42 activity on days 2 and 3 of differentiation. Increased cell size and spreading and alteration of the adhesion proteins p-p130Cas, vinculin, p-zyxin and p-FAK925 were also observed upon ARHGAP21 inhibition. There was no change in the acquisition of CD61, CD41 and CD42 megakaryocytic markers, neither in the polyploidy of HEL cells during differentiation.

We further investigated the effects of ARHGAP21 inhibition on platelet morphology and on the hemostatic response in vivo, using the C57BL/6 Arhgap21 heterozygous mouse model (Arhgap21^+/-). The study was approved by the Ethical Committee of our Institution. No differences were observed in CD61⁺CD41⁺ nor in CD41⁺CD42⁺ bone marrow cells from Arhgap21^+/- and wild type (WT) mice. However, transmission electron microscopy revealed that Arhgap21^+/- platelets presented increased alpha-granule size when compared to wild-type (WT). Tail bleeding time of Arhgap21^+/- mice was decreased compared to WT (P= 0.0008). Intravital microscopy of carotid artery injured by FeCl₃ showed increased adhesion of platelet and white blood cells on the vessel wall of Arhgap21^+/-, which reflected in accelerated occlusion time (twice as fast) compared to WT (P= 0.0150).

In conclusion, ARHGAP21 silencing may alter cell morphology and lead to increased microtubule dynamic instability during megakaryocyte differentiation in vitro, without compromising the acquisition of differentiation markers. In vivo, deficiency of Arhgap21 increases platelet granule size and accelerates hemostatic response. Together, these results indicate that ARHGAP21 may be a critical protein in the regulation of platelet production and function through the control of cytoskeletal rearrangement.

This study was supported by São Paulo Research Foundation (FAPESP) National Council for Scientific and Technological Development (CNPq) and Coordination for the Improvement of Higher Education Personnel (CAPES).