Rho GTPases are critical for platelet haemostatic functions, but little is known about the roles of Rif and RhoG in platelets. Rho GTPases interact with specific effector proteins to regulate cellular processes. GST -fusion proteins and affinity-purification/ mass-spectrometry (AP-MS) were used to identify interacting proteins of Rif and RhoG in platelets. Rif interacted with cytoskeleton regulators including formins mDia1 and mDia3 in platelets, suggesting RH manipulates the platelet actin cytoskeleton. In platelets, RhoG bound SNARE complex proteins and cytoskeletal regulators including the Rac GEF proteins ELMO and DOCKl, suggesting RhoG regulates both shape change and secretion. The process of platelet filopodia formation is incompletely understood. The canonical pathway involving Cdc42 is not essential in platelets and thus Rif may provide an alternative route to filopodia formation through diaphanous-related formins . A novel knockout mouse line was generated to evaluate the role of Rif in platelets. Rif-/- platelets formed filopodia and spread normally on various agonist surfaces under static and shear conditions. Rif-/- platelets had normal actin dynamics, aggregated and secreted normally from alpha and dense granules. Ultimately, no critical functions for Rif were identified in platelets, likely due to functional overlap with other small GTPases. The role of RhoG in platelets was tested using genetic and pharmacologic approaches. RhoG was activated by both CRP and thrombin in human and mouse platelets. Integrin activation and aggregation were reduced in RhoG-/- platelets in response to CRP, but responses to thrombin were normal. The central defect in RhoG-/- platelets was found to be reduced dense granule secretion, because the integrin activation and aggregation defects could be rescued by ADP costimulation. Defective dense granule secretion by RhoG-/- platelets limited platelet recruitment to growing thrombi under shear and translated into reduced thrombus formation in vivo. The role of RhoG-regulated platelet secretion in health and disease warrants further study.