SMIFH2 inhibition of platelets demonstrates a critical role for formin proteins in platelet cytoskeletal dynamics

Background: Reorganisation of the actin cytoskeleton is required for proper functioning of platelets following activation in response to vascular damage. Formins are a family of proteins which regulate actin polymerisation and cytoskeletal organisation via number of domains including the FH2 domain. However, the role of formins in platelet spreading has not been studied in detail. Objectives: Several formin proteins are expressed in platelets and so we used an inhibitor of FH2 domains (SMIFH2) to uncover the role of these proteins in platelet spreading and in maintenance of resting platelet shape. Methods: Washed human and mouse platelets were treated with various concentrations of SMIFH2 and the effect on platelet spreading, platelet size, platelet cytoskeletal dynamics and organisation and were analysed using fluorescence and electron microscopy. Results: Pre-treatment with SMIFH2 completely blocks platelet spreading in both mouse and human platelets through effects on the organisation and dynamics of actin and microtubules. However, platelet aggregation and secretion are unaffected. SMIFH2 also caused a decrease in resting platelet size and disrupted the balance of tubulin post-translational modification. Conclusions: These data therefore demonstrated an important role for formin mediated actin polymerisation in platelet spreading and highlighted the importance of formins in cross talk between the actin and tubulin cytoskeletons.