Extracellular platelet TMX1 inhibits aggregation of human platelets. (A) TMX1 expression on the platelet surface increases with platelet activation. TMX1 expression on nonactivated (NA) and thrombin-activated platelets. Representative line graph (left panel) and combined results (right panel). Data are mean ± standard error of the mean (SEM), n = 5 for each group. Human platelets were incubated with 1 U/mL thrombin for 5 minutes, followed by flow cytometry measurement using the anti-TMX1 antibody B01P. The anti-TMX1 antibody B01P increases platelet aggregation induced by SFLLRN (B), convulxin (C), and thrombin (D). In these studies, human platelets were incubated with IV.3 (10 μg/mL) for 5 minutes and then with B01P (20 μg/mL) for 5 minutes, after which they were activated. Representative tracings for aggregation and ATP release (left panels) and the combined results (right panels) are shown; data are mean ± SEM, n = 3 (B) and n = 4 (C-D). rTMX1 containing the CPAC-motif (rTMX1) inhibits convulxin-induced platelet aggregation (E) and thrombin-induced platelet aggregation and ATP release (F). Human platelets were incubated with rTMX1 for 5 minutes at the indicated concentrations before stimulation. Representative aggregation and ATP release (left panels) and combined results (right panels); data are mean ± SEM, n = 4 for each group. (G) Inactivated rTMX1 [TMX1(oo)] increases convulxin-induced platelet aggregation. Representative aggregation (left panel) and combined results (right panel); data are mean ± SEM, n = 6 for each group. *P < .05, **P < .01, ***P < .001, Student t test. IgG, normal mouse immunoglobulin G; MFI, mean fluorescence intensity.