Dasgupta Swapan K, Thiagarajan Perumal
Department of Pathology, Center for Translational Research on Inflammatory Diseases, Michael E. DeBakey Veterans Affairs Medical Center.
Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.
Blood Coagul Fibrinolysis. 2018 Sep;29(6):540-545. doi: 10.1097/MBC.0000000000000756.
: 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.
Wdr-1是一种与肌动蛋白相互作用的蛋白，可增强丝切蛋白加速F-肌动蛋白丝解聚的能力。Wdr-1基因缺陷小鼠由于血小板中由内向外的整合素信号传导缺陷而导致止血功能受损。在此，我们研究了Wdr-1在凝块回缩和血小板铺展所需的由外向内信号传导中的作用。通过纤维蛋白凝块回缩试验以及未刺激血小板在纤维蛋白原底物上的黏附与铺展来评估由外向内信号传导。通过免疫荧光显微镜确定肌动蛋白、丝切蛋白-1和Wdr-1的空间分布。在双色共聚焦图像中以及通过对F-肌动蛋白丝进行免疫印迹来评估F-肌动蛋白与粘着斑激酶的相互作用。Wdr-1基因缺陷的血小板中凝块回缩明显受损。与野生型对照相比，Wdr-1基因缺陷的血小板在纤维蛋白原底物上的黏附与铺展较差。在静息血小板中，Wdr-1与丝切蛋白-1在皮质肌动蛋白中共定位。血小板在纤维蛋白原底物上铺展后，Wdr-1与丝切蛋白-1一起转位至细胞骨架。在Wdr-1基因缺陷的血小板中，丝切蛋白-1异常定位于整个细胞质中，并且在黏附于纤维蛋白原底物后无明显变化。在纤维蛋白原上铺展时形成的肌动蛋白丝大多位于血小板周边，未穿过细胞质。此外，肌动蛋白丝与粘着斑激酶的共定位减少。这些研究表明，Wdr-1对于丝切蛋白-1定位于血小板膜骨架至关重要。F-肌动蛋白无法附着于粘着斑，导致血小板铺展和凝块回缩所需的肌动蛋白丝重组缺陷。
