Regulation of the
Asunaprevir cytoskeleton is a multimolecular process in which mDia1 plays a fundamental role. It has recently been recognized that many proteins move from a detergent-soluble cytosolic fraction to an insoluble cytoskeletal fraction in thrombin-stimulated platelets, representing the operationally defined actin assembly [20]. We therefore further investigated the association of mDia1 with the actin cytoskeleton in thrombin-stimulated platelets, by determining if mDia1 translocated to the Triton X-100-insoluble pellets in platelet aggregates. As shown by anti-mDia1 immunoblotting analysis, mDia1 was found mostly in the Triton X-100-soluble fraction in resting platelets. After stimulation of platelets with thrombin, however, mDia1 translocated to the actin-rich Triton X-100-insoluble fraction in 2.5 min (Fig. 1A). This translocation was significantly blocked by pretreatment with the PI 3-kinase inhibitors, wortmannin or LY294002 (Fig. 1B). These findings suggest that mDia1 is involved in the dynamic reorganization of the
actin cytoskeleton following thrombin stimulation of platelets, and that the effect of thrombin stimulation on the association of mDia1 with the actin cytoskeleton is probably exerted through PI 3-kinase. This is consistent with recent data showing that the stimulation of platelets with thrombin resulted in the activation of PI 3-kinase and synthesis of the PI 3-kinase lipid products, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate [17]. Both of these are involved in the regulation of platelet spreading [18]. Given the crucial roles of mDia1 and PI 3-kinase products in actin-filament rearrangement [10] and [18], our data imply that PI 3-kinase mediates thrombin-induced mDia1 translocation to the cytoskeleton fraction, where mDia1 then modulates actin remodeling.
