Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading

Background: During platelet spreading, the actin cytoskeleton undergoes marked changes, forming filopodia, lamellipodia and stress fibres. In the present study, we report the identification of a novel actin-rich structure, termed an actin nodule, which appears prior to lamellipodia and stress fibre formation. Methods: Platelet spreading was monitored using human platelets and mouse GFP-actin platelets using real-time and end-point DIC, and fluorescent and electron microscopy (EM). Results: We identified a small, novel actin structure, the actin nodule, in the early stages of adhesion and spreading, which we hypothesize to be a precursor of lamellipodia and stress fibres. Nodule formation shows an inverse correlation to Rho kinase and myosin-II activity, is independent of PI3-kinase, but dependent on Src kinase activity. Actin nodules contain multiple proteins, including Arp2/3, Fyn, Rac, and β1- and β3- integrins, but not Src. EM analysis revealed that actin filaments extend in all directions from the nodules. Actin nodules are present on multiple matrices, including fibrinogen, laminin and VWF+botrocetin. Conclusion: This work identifies a novel platelet actin structure, which we propose is a precursor to both lamellipodia and stress fibres and acts to drive platelet spreading.