Functional domains of Desmolaris. (A) Domain representation for Desmolaris. All truncated forms were designed based on sites of introns determined for TFPI,¹²  as indicated in Figure 1B. (B) SDS-PAGE of the mutated and truncated forms of Desmolaris. Gels were Coomassie Blue-stained. Lane 1, R32L; lane 2, K1K2; lane 3, K1; lane 4, K2Long; lane 5, K2Short. (C) Effects on the catalytic activity of FXa. FXa (1 nM) was incubated for 30 minutes with the indicated forms of Desmolaris followed by addition of S2222 (250 μM). The ratio Vs/Vo obtained was plotted against Desmolaris concentration and data fitted (when appropriate) with the Morrison equation to calculate the IC₅₀ for each form. (D) Effects on the catalytic activity of FXIa. FXIa (1 nM) was incubated for 30 minutes with the indicated forms of Desmolaris followed by addition of S2366 (250 μM). The ratio Vs/Vo obtained was plotted against Desmolaris concentration and data fitted (when appropriate) with the Morrison equation to calculate the IC₅₀ for each form. (E) Effects on the PT. Desmolaris forms were incubated with plasma followed by addition of PT reagent and Ca²⁺. Clotting was estimated with a coagulometer. Reference time is 14.6 seconds. (F) Effects on the aPTT. Desmolaris forms were incubated with plasma followed by addition of aPTT reagent and Ca²⁺. Clotting was estimated with a coagulometer. Reference time was 35.9 seconds. (G) Effects of heparin on FXa inhibition by K1 and K1K2. Comparison with Desmolaris. Experiments were performed as in panel C in the absence or presence of heparin (1 μg/mL) and the indicated concentrations of Desmolaris forms. FXa, 0.5 nM. Data points were fitted with Morrison equation. (H) Effects of heparin on FXIa inhibition by K1 and K1K2. Comparison with Desmolaris. Experiments were performed as in panel D in the absence or presence of heparin (1 μg/mL) and the indicated concentrations of Desmolaris forms. FXIa, 0.5 nM. Data points were fitted with Morrison equation. (I) Interaction of Desmolaris forms with a heparin-agarose column. Desmolaris, R32L, K1K2, K1, K2Long, or K2Short were loaded in the heparin column and equilibrated in TBS, pH 7.4. A total of 200 μL of each form at ∼20 μM were applied. After washing the column, a gradient (NaCl, 0-2 M) was applied for 40 minutes. The peak corresponding to Desmolaris eluted at ∼1 M NaCl. (J) Diagram of the putative interactions of Desmolaris domains with FXa, FXIa, and heparin. ?, putative interactions with exosites of FXIa or FXa.