Secreted ATX interacts with integrin β3 and mediates Hcy-induced platelet integrin αIIbβ3 activation. Washed platelets were purified from C57BL/6J mice. (A-B) Supernatant ATX levels were measured by enzyme-linked immunosorbent assay (ELISA). All data are expressed as the mean ± SEM (n = 4). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. (C-D) Platelet aggregation after HA130 (0.6 μM) stimulation with collagen (1.5 μg/mL) (C) or thrombin (0.01 U/mL) (D) in the presence/absence of Hcy (100 μM) was monitored with a turbidimetric aggregometer. (E) Washed platelets from C57BL/6J mice were left to adhere and spread on fibrinogen-coated wells with 100 μM Hcy or vehicle in the presence of HA130 (0.6 μM). The spreading area of individual platelets (stainted with phalloidin, original magnification, 200×) was measured with ImageJ software. All data are expressed as the mean ± SEM (n = 3-5). *P < .05 compared with ctrl; #P < .05 compared with Hcy. (F-G) p-AKT^Ser473 and p-SRC^Tyr416 protein levels in platelets with or without Hcy incubation were analyzed after treatment with 0.6 μM HA130 for 10 minutes with collagen (1.5 μg/mL) (F) or thrombin (0.01 U/mL) (G) by western blot analysis. All data are expressed as the mean ± SEM (n = 3). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists. (H) Coimmunoprecipitation of ATX with integrin β3 in platelets from C57BL/6J mice. (I) p-AKT^Ser473 and p-SRC^Tyr416 protein levels in platelets from C57BL/6J mice incubated with ATX (+, 10 nM; ++, 20 nM; +++, 50 nM) were analyzed by western blot after incubation with mP6 (20 μM) for 10 minutes. All data are expressed as the mean ± SEM (n = 3). *P < .05 compared with agonists; #P < .05 compared with Hcy+agonists or Th-ATX+++.