In the current study, mDia1 was shown to translocate to the platelet cytoskeleton following thrombin stimulation, in a PI 3-kinase-dependent manner. Anti-mDia1 loading or pretreatment with PI 3-kinase inhibitors essentially abrogated thrombin-elicited AM095 stress fiber formation, with a corresponding decrease in the proportion of platelets exhibiting a fully spread morphology. We also investigated the mechanisms underlying mDia1 effects on thrombin-induced actin remodeling and platelet spreading, and found that these involved PI 3-kinase-mediated induction of mDia1 interaction with RhoA.
Materials and methods
Platelet isolation. Platelets were isolated from venous blood collected from healthy volunteer donors, as previously described, with minor modifications [2] and [5]. Platelet-rich plasma (PRP) was prepared by centrifugation of whole blood at 150g for 15 min. The platelets were then isolated from PRP by centrifugation at 750g for 15 min in the presence of 1 μM prostaglandin E1 (PGE1) (Cayman Chemical, Ann Arbor, MI, USA). Finally, platelets were suspended in modified HEPES/Tyrode buffer [129 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 1 mM MgCl2, 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 5 mM glucose; pH 7.4] at a concentration of 4–5 × 108 platelets/ml, without stirring, before stimulation with 1 IU/ml human thrombin (Sigma–Aldrich, St. Louis, MO, USA), 100 nM wortmannin (Sigma–Aldrich) or 30 μM LY294002 (Sigma–Aldrich).
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