Expression and release of TM from HUVEC. (A) TM was measured by ELISA in HUVEC and HUVEC₅₃₆ CM and lysates (CM/lysate ratio reflects TM release capacity). (B) TM contained in the CM of HUVEC and HUVEC₅₃₆ was concentrated by Con A or FIIa affinity purification and run on SDS-PAGE alongside sTM produced by HEK293 cells overexpressing TM₅₃₆, then TM was detected by immunoblot. (C) Expression of TM on the surface of HUVEC and HUVEC₅₃₆ was measured using flow cytometry. (D) Immunoblot detection of TM and GAPDH (loading control) in lysates of HUVEC and HUVEC₅₃₆. (E) TM was detected in the lysates of HUVEC and HUVEC₅₃₆ submitted (+) or not (−) to EndoH deglycosylation. Value are mean ± SD expressed as fold change relative to TM_WT or HUVEC. Statistical analyses were performed using an unpaired t test for panel A or Mann-Whitney test for panel C. ∗P < .05; ∗∗∗P < .001. Con A, Concanavalin A; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OE, overexposed blot.