Figure 1.
Figure 1. TF-induced coagulation is rate-limiting for VT that follows inhibition of antithrombin and protein C. (A) Thrombin generation initiated by TF-positive cell extract (mouse smooth muscle cells). In a parallel group of mice (n = 3), control antibody or 1H1 was injected intraperitoneal (no siRNA treatment). Forty-one hours after antibody treatment, mice were euthanized, and citrated blood was collected via the vena cava. Platelet-poor plasma was used for thrombin generation analysis. Curves represent the average of 3 mice treated with either control antibody (–1H1) or 1H1 antibody (+1H1). (B) Clinical phenotype in mice treated with siRNAs targeting Serpinc1 and Proc, treated with control antibody or 1H1. Mice exhibiting characteristic clinical signs (blue bars) and mice unaffected (red bars) 2 days after siRNA treatment (end of experiment). P = .001, Fisher’s exact test. Of note, results from experiment 1 (–1H1: 3 of 3 mice affected vs +1H1: 1 of 3 mice affected) and experiment 2 (–1H1: 8 of 10 mice affected vs +1H1: 2 of 10 mice affected; P = .02) were pooled. (C) Representative cross-sections of the whole head of mice (formalin-fixed and decalcified) from the –1H1 and the +1H1 groups (n = 5). Cross-sections were taken behind the eye and show the presence of macroscopically visible coagulopathy in and around the mandibular area (white asterisks). In this area, coagulopathy was visible for 11 of 13 −1H1 mice and 3 of 13 +1H1 mice. All mice were treated with siSerpinc1/siProc. The black bar represents 1 cm. (D) Descriptive scoring for the type of detected thrombi. 0: no thrombi found; I + II: thrombi categories were based on structure and layering (see Methods section and supplemental Figure 4). Blue bars: −1H1 (n = 26); red bars: +1H1 (n = 26). Of note, results from experiment 1 (n = 6) and experiment 2 (n = 20) were pooled. (E) Levels of fibrin deposition in the liver in −1H1 (open circles) and +1H1 (filled circles) mice. P = .001. Of note, for the second 1H1 experiment (n = 10) liver fibrin deposition: −1H1: 37.4 ng/mg (16.1, 289.8) and +1H1: 15.6 ng/mg (12.5, 25.9); P = .003. Mann-Whitney rank sum test. Dashed line indicates the detection limit of 12.5 ng/mg. Fibrin deposition levels for C57BL/6J female mice (n = 6), treated with 1H1 or control antibody only, were below the detection limit (<12.5 ng/mg).

TF-induced coagulation is rate-limiting for VT that follows inhibition of antithrombin and protein C. (A) Thrombin generation initiated by TF-positive cell extract (mouse smooth muscle cells). In a parallel group of mice (n = 3), control antibody or 1H1 was injected intraperitoneal (no siRNA treatment). Forty-one hours after antibody treatment, mice were euthanized, and citrated blood was collected via the vena cava. Platelet-poor plasma was used for thrombin generation analysis. Curves represent the average of 3 mice treated with either control antibody (–1H1) or 1H1 antibody (+1H1). (B) Clinical phenotype in mice treated with siRNAs targeting Serpinc1 and Proc, treated with control antibody or 1H1. Mice exhibiting characteristic clinical signs (blue bars) and mice unaffected (red bars) 2 days after siRNA treatment (end of experiment). P = .001, Fisher’s exact test. Of note, results from experiment 1 (–1H1: 3 of 3 mice affected vs +1H1: 1 of 3 mice affected) and experiment 2 (–1H1: 8 of 10 mice affected vs +1H1: 2 of 10 mice affected; P = .02) were pooled. (C) Representative cross-sections of the whole head of mice (formalin-fixed and decalcified) from the –1H1 and the +1H1 groups (n = 5). Cross-sections were taken behind the eye and show the presence of macroscopically visible coagulopathy in and around the mandibular area (white asterisks). In this area, coagulopathy was visible for 11 of 13 −1H1 mice and 3 of 13 +1H1 mice. All mice were treated with siSerpinc1/siProc. The black bar represents 1 cm. (D) Descriptive scoring for the type of detected thrombi. 0: no thrombi found; I + II: thrombi categories were based on structure and layering (see Methods section and supplemental Figure 4). Blue bars: −1H1 (n = 26); red bars: +1H1 (n = 26). Of note, results from experiment 1 (n = 6) and experiment 2 (n = 20) were pooled. (E) Levels of fibrin deposition in the liver in −1H1 (open circles) and +1H1 (filled circles) mice. P = .001. Of note, for the second 1H1 experiment (n = 10) liver fibrin deposition: −1H1: 37.4 ng/mg (16.1, 289.8) and +1H1: 15.6 ng/mg (12.5, 25.9); P = .003. Mann-Whitney rank sum test. Dashed line indicates the detection limit of 12.5 ng/mg. Fibrin deposition levels for C57BL/6J female mice (n = 6), treated with 1H1 or control antibody only, were below the detection limit (<12.5 ng/mg).

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