Klotho counteracts IS-induced platelet hyperactivation via inhibiting ROS/p38MAPK signaling. (A-C) Platelets were pretreated with indicated concentrations of Klotho and then stimulated with 500 μM IS. (A) Flow cytometric analysis of P-selectin expression in platelets in response to 2 μg/mL collagen after Klotho pretreatment (n = 6). (B) Flow cytometric analysis of the percentage of platelet-monocyte aggregates in response to 2 μg/mL collagen after Klotho pretreatment in vitro (n = 6). (C) Representative images of thrombus formation on a collagen-coated flow chamber under shear-flow conditions (i). Scale bar, 50 μm. Quantification of the area of platelet adhesion for each group (n = 6) (ii). (A-C) Data are mean ± SD. *P < .05, **P < .01 vs IS group, ANOVA. (D-F) Platelets were pretreated with 400 pM Klotho and then stimulated with 500 μM IS. (D) Flow cytometric analysis of ROS levels in platelets (n = 6). (E) Flow cytometric analysis of the expression of P-p38MAPK in platelets (n = 6). (F) Western blot analysis of the expression of P-p38MAPK and p38MAPK in platelets (i). Quantification of the ratio of P-p38MAPK/p38MAPK for each group (n = 3) (ii). (D-F) Data are mean ± SD. **P < .01 vs control group, ^##P < .01 vs IS 500 μM group, ANOVA. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.