Pull-down assay for analysis of integrin-mediated activation of Rap proteins in adherent platelets.
Rap1 GTPases operate as molecular switches by cycling between a GDP-bound inactive state and a GTP-bound active state and regulate several cellular pathways in response to different stimuli. Circulating blood platelets express high levels of Rap1 proteins, mainly Rap1b, which plays a critical role in platelet adhesion and activation. Rap1 is a key element in the inside-out signaling pathway leading to the conversion of integrins into the high-affinity state for their ligands. In platelets, Rap1b regulates inside-out activation of both integrin αIIbβ3 and α2β1. In addition, Rap1b is also involved in integrin outside-in signaling. Integrin-mediated platelet adhesion leads to accumulation of GTP-bound Rap1b, which promotes integrin-mediated processes such as spreading and clot retraction. Rap1b is thus a bidirectional regulator of platelet integrin function. Here we describe a method to analyze Rap1b activation induced by platelet adhesion via integrin α2β1.
Department of Biology and Biotechnology, University of Pavia, Pavia, Italy.
Gianni Francesco
GF
Guidetti
Mauro
M
Torti
D001792:Blood Platelets / Q000166:cytology / Q000502:physiology*; D015153:Blotting, Western; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D047468:Immunoprecipitation / Q000379:methods*; D038982:Integrin alpha2beta1 / Q000378:metabolism*; D010973:Platelet Adhesiveness; D020827:rap1 GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D038982:Integrin alpha2beta1; D020827:rap1 GTP-Binding Proteins