Variable phenotypic penetrance of thrombosis in adult mice after tissue-selective and temporally controlled Thbd gene inactivation

Abstract

Thrombomodulin (Thbd) exerts pleiotropic effects on blood coagulation, fibrinolysis, and complement system activity by facilitating the thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor and may have additional thrombin- and protein C (pC)-independent functions. In mice, complete Thbd deficiency causes embryonic death due to defective placental development. In this study, we used tissue-selective and temporally controlled Thbd gene ablation to examine the function of Thbd in adult mice. Selective preservation of Thbd function in the extraembryonic ectoderm and primitive endoderm via the Meox2Cre-transgene enabled normal intrauterine development of Thbd-deficient (Thbd^-/-) mice to term. Half of the Thbd^-/- offspring expired perinatally due to thrombohemorrhagic lesions. Surviving Thbd^-/- animals only rarely developed overt thrombotic lesions, exhibited low-grade compensated consumptive coagulopathy, and yet exhibited marked, sudden-onset mortality. A corresponding pathology was seen in mice in which the Thbd gene was ablated after reaching adulthood. Supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen prevented the pathologies of Thbd^-/- mice. However, Thbd^-/- females expressing the PC transgene exhibited pregnancy-induced morbidity and mortality with near-complete penetrance. These findings suggest that Thbd function in nonendothelial embryonic tissues of the placenta and yolk sac affects through as-yet-unknown mechanisms the penetrance and severity of thrombosis after birth and provide novel opportunities to study the role of the natural Thbd-pC pathway in adult mice and during pregnancy.

Graphical abstract

Figure 1.

Tamoxifen-induced systemic thrombomodulin ablation in adult mice. (A-B) Residual expression of Thbd antigen detected in the lungs by western blot analysis of tissue lysate (A) and on histological sections (B). Numbers in panels A and B identify individual animals. (C) Detection of Thbd protein by immunohistochemistry in various tissues of tamoxifen-treated ERCreThbd^loxP mice. Brown staining indicates Thbd protein detected by horseradish peroxidase–coupled anti-Thbd antibody. Hematoxylin counterstain. Red bars indicate 50 μm. In several animals, Thbd antigen was virtually undetectable. (D) Kaplan-Meier survival plot of ERCreThbd^loxP mice after tamoxifen treatment at 8 to 10 weeks. Difference in survival between groups is significant (Mantel-Cox log-rank analysis). Ctrl, non–tamoxifen-treated mice.

Figure 2.

Constitutive Thbd ablation with selective preservation in the placenta in Meox2Cre-Thbd^loxP mice. (A) Thbd-deficient neonates found dead. Arrows indicate hemorrhagic lesions. (B) Photomicrograph of a flash-frozen section prepared from a day 15.5 embryo expressing the Meox2Cre-gene and the mT/mG recombinase reporter gene: red (mT) fluorescence indicates the nonrecombined reporter gene expressed in the placenta, green fluorescence indicates Cre-mediated activation of the mG reporter in the embryo proper, chorionic plate, and fetal blood vessels in the placenta, but lack thereof in all extraembryonic fetal trophoblast cells. (C-D) Immunohistochemical detection of Thbd antigen (brown staining; hematoxylin counterstain) in the day 12 embryo and placenta of Thbd^loxP mice (in panel C, Thbd is detected in the embryo and placenta) and Meox2Cre-Thbd^loxP mice (in panel D, Thbd is present in the placenta, but not the embryo). Scale bars, 1 mm.

Figure 3.

Adult phenotype of Meox2Cre-Thbd-null mice. (A) Smaller body size of Thbd-null mice. (B) Thbd-null mice had significantly lower body weight at all time points measured (n ≥ 16 for each time point), but showed near-normal growth as compared with Thbd-expressing littermates (n ≥ 92 for each time point). (C) Histological immunostaining of different organs at 10 to 16 weeks of age showing persistent absence of Thbd from the endothelium in different vascular beds. (D) Kaplan-Meier survival plot of live-born Meox2Cre-Thbd-null mice (n = 28), as compared with Thbd-expressing littermates of various genotypes. (E) Masson-Trichrome–stained section of affected eye in a Meox2Cre-Thbd-null mouse showing leukocyte infiltration into the anterior eye chamber (left); normal eye histology in a Thbd^loxP/+ (1 loxP-flanked but nondeleted Thbd allele and 1 wild-type Thbd allele) (right); hematoxylin and eosin stain.

Figure 4.

Thrombin generation and vascular permeability in Meox2Cre-Thbd-null mice. (A) Calibrated automated thrombinography of plasma from control mice (Thbd^loxP/+, orange, n = 4) and Thbd-deficient mice (Thbd^−/−; blue, n = 4). (B) Thbd-null mice displayed prolonged lag time and time to peak thrombin generation (ttPeak), reduced endogenous thrombin potential (ETP), and peak thrombin generation (Peak). (C-D) Evans blue extravasation assay showing increased vascular permeability in the lungs of Thbd-null mice compared with controls Thbd^loxP/loxP; n = 5 per group). (D) Data shown in panel C were generated by quantitative scanning of fluorescence (at 720 nm) of whole-organ lung tissue.

Figure 5.

Genetic supplementation of APC by PC transgene expression. (A-B) The D168F/N173K protein C transgene (mAPC^HI) increased plasma levels of PC (A) and activated PC (B) as measured in mice with normal Thbd gene expression. To assess the expression of the mAPC^HI transgene in the absence of endogenous aPC, PC-deficient mice with and without the transgene were added as additional experimental groups. Transgene-derived D168F/N173K PC expression was determined in APC^HI mice lacking endogenous PC (PC^−/−). Heterozygous PC-deficient mice (PC^+/−) were included as controls. (C) The transgene partially restored the body weight of Thbd-null mice (for each time point: Thbd-null, n ≥ 24; mAPC^HI Thbd-null, n ≥ 16; Thbd-expressing littermates, n ≥ 92). (D) D168F/N173K PC supplementation reduced mortality of tamoxifen-induced ERCre-Thbd^loxP mice as well as of constitutively Thbd-deficient Meox2Cre-Thbd-null mice. (E) Pregnant Thbd-null mAPC^HI mice showed pregnancy-induced bleeding, as shown by hemosiderin depositions in the lungs (left; original magnification ×4, hematoxylin and eosin stain) and hemorrhage in the uterus (right).