Cofactor activity of TM. (A) PC activation measured on the surfaces of HUVEC and HUVEC₅₃₆. (B) HEK293 cells expressing EPCR and TM_WT or TM₅₃₉ were treated (+) or not (−) with 10 mM MβCD for 20 minutes before starting the PC activation protocol, and PC activity was measured. (C) Immunoblot detection of the indicated sTM and their CS-devoid counterparts accumulated in the CM. TM_CS+ indicates the position of the CS-modified TM, and Ø indicates the absence of the TM. (D) FIIa-dependent PC activation measured in vitro in the presence of the same amount of the indicated sTM. (E) Immunoblot detection of soluble CS-TM₅₃₆ and CS-TM₅₁₄ and their efficacy as cofactors in PC activation. Relative PC activities (mean ± SD) obtained after 30 to 50 minutes of PC substrate conversion are shown. Statistical analyses were performed using unpaired t test. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. CS-TM, CS-devoid TM; MβCD, methyl-β-cyclodextrin.