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Wdr-1, an actin interacting protein, enhances cofilin's capacity to accelerate depolymerization of F-actin filaments. Wdr-1-deficient mice have impaired hemostasis due to defective inside-out integrin signaling in platelets. Here, we studied the role of Wdr-1 on outside-in signaling necessary for retraction of the clot and platelet spreading. Outside-in signaling was assessed by fibrin clot retraction assay and by adhesion and spreading of unstimulated platelets on fibrinogen substrate. The spatial distribution of actin, cofilin-1 and Wdr-1 were determined by immunofluorescence microscopy. Interaction of F-actin with focal adhesion kinase was assessed in dual-color confocal images and by immunoblotting of F-actin filaments. Clot retraction is markedly impaired in Wdr-1-deficient platelets. Wdr-1-deficient platelets adhere and spread poorly on fibrinogen substrate compared with wild-type controls. In resting platelets, Wdr-1 is colocalized with cofilin-1 in cortical actin. Following platelets spreading on fibrinogen substrate, Wdr-1 translocates to the cytoskeleton in association with cofilin-1. In Wdr-1-deficient platelets, cofilin-1 is aberrantly localized throughout the cytoplasm and there is no significant change following adhesion to fibrinogen substrate. The actin filaments formed upon spreading on fibrinogen are mostly in the periphery of the platelets and does not traverse the cytoplasm. Furthermore, there is diminished colocalization of actin filaments with focal adhesion kinase. These studies show that Wdr-1 is essential for the localization of cofilin-1 to the platelet membrane skeleton. F-actin fails to attach to focal adhesions resulting in defective reorganization of actin filaments necessary for platelet spreading and clot retraction.