Methods to study the roles of Rho GTPases in platelet function

Rivero, Francisco; Calaminus, Simon

Abstract

Platelets are a critical cell for prevention of bleeding. Part of the response to the formation of the thrombus is the activation of the actin cytoskeleton, with an inability to effectively activate the cytoskeleton linked to thrombus formation defects and instability. The control of this process is linked to activation of the Rho GTPases, Cdc42, Rac1, and RhoA, although additional small GTPases such as Rif and Rap have been shown to play roles in platelet function.

Here we describe the methodology to accurately understand how Rho GTPases are activated in platelets. Due to the technical limitations of working with platelets, such as their lack of ability to be transfected, the majority of work has been carried out either using inhibitors of Rho GTPases or within knockout mouse models. Studies can be conducted both in suspension samples and in spread platelets. In suspension the platelets will undergo a shape change response, but will not be able to spread. In spread platelets it is possible to examine the effects of the matrix environment, such as concentration, type, and stiffness on Rho GTPase function within platelet activation and platelet spreading.

Citation

Rivero, F., & Calaminus, S. (2018). Methods to study the roles of Rho GTPases in platelet function. In F. Rivero (Ed.), Rho GTPases: Methods and Protocols (199-217). Humana Press. https://doi.org/10.1007/978-1-4939-8612-5_14

• Acceptance Date: May 11, 2018
• Online Publication Date: Jul 30, 2018
• Publication Date: Jul 31, 2018
• Deposit Date: Jul 11, 2018
• Publicly Available Date: Jul 31, 2019
• Print ISSN: 1064-3745
• Publisher: Humana Press
• Peer Reviewed: Peer Reviewed
• Volume: 1821
• Pages: 199-217
• Series Title: Methods in Molecular Biology
• Series Number: 1821
• Book Title: Rho GTPases: Methods and Protocols
• ISBN: 9781493986118; 9781493986125
• DOI: https://doi.org/10.1007/978-1-4939-8612-5_14
• Keywords: Actin, RhoA, Rac1, platelets, cAMP, cGMP, pulldown assay, Y27632, Rhosin A
• Public URL: https://hull-repository.worktribe.com/output/922843