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. 2019 Feb 12;3(3):489-498.
doi: 10.1182/bloodadvances.2018018853.

Maintaining extraembryonic expression allows generation of mice with severe tissue factor pathway inhibitor deficiency

Michelle M Castillo  1   2 , Qiuhui Yang  1 , Min Zhan  1 , Amy Y Pan  3 , Michael W Lawlor  1 , Alan E Mast  2   4 , Rashmi Sood  1   2
Affiliations

Maintaining extraembryonic expression allows generation of mice with severe tissue factor pathway inhibitor deficiency

Michelle M Castillo et al. Blood Adv. .

Abstract

Tissue factor pathway inhibitor (TFPI) is a serine protease with multiple anticoagulant activities. The Kunitz1 (K1) domain of TFPI binds the active site of factor VIIa and is required for inhibition of tissue factor (TF)/factor VIIa catalytic activity. Mice lacking TFPI K1 domain die in utero. TFPI is highly expressed on trophoblast cells of the placenta. We used genetic strategies to selectively ablate exon 4 encoding TFPI K1 domain in the embryo, while maintaining expression in trophoblast cells. This approach resulted in expected Mendelian frequency of TFPI K1 domain-deficient mice. Real-time polymerase chain reaction confirmed 95% to 99% genetic deletion and a similar reduction in transcript expression. Western blotting confirmed the presence of a truncated protein instead of full-length TFPI. Mice with severe TFPI K1 deficiency exhibited elevated thrombin-antithrombin (TAT) levels, frequent fibrin deposition in renal medulla, and increased susceptibility to TF-induced pulmonary embolism. They were fertile, and most lived normal life spans without any overt thrombotic events. Of 43 mice observed, 2 displayed extensive brain ischemia and infarction. We conclude that in contrast to complete absence of TFPI K1 domain, severe deficiency is compatible with in utero development, adult survival, and reproductive functions in mice. Inhibition of TFPI activity is being evaluated as a means of boosting thrombin generation in hemophilia patients. Our results show that in mice severe reduction of TFPI K1 activity is associated with a prothrombotic state without overt developmental outcomes. We note fibrin deposits in the kidney and rare cases of brain ischemia.

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Conflict of interest statement

Conflict-of-interest disclosure: M.Z. is a current employee of MPP Group LLC. M.W.L. receives research support from and is a member of Scientific Advisory Boards for Audentes Therapeutics, Solid Biosciences, and Ichorion Therapeutics and is a consultant for Wave Life Sciences. A.E.M. receives research grant from Novo Nordisk. The remaining authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
TFPI K1 domain–deficient embryos and placentas from epiblast-specific deletion appear grossly normal with no overt signs of hemorrhage. Representative images of littermate TFPI_K1δ/+ (A) and TFPI_K1δ/δ (B) 15.5 dpc embryos and corresponding placentas among progeny of Meox2Cretg/+ TFPI_K1δ/+ male mice mated to TFPI_K1Lox/Lox female mice are shown. (C-D) Hematoxylin and eosin–stained sections of placentas shown in panels A and B, respectively. (E-F) Enlarged images of boxed regions in panels C and D, respectively. Maintaining expression of full-length TFPI in trophoblast cells corrects the abnormal vascularization observed in K1 null placentas.
Figure 2.
Figure 2.
Intracranial hemorrhage and reduced placental vascularization in global TFPI K1 null embryos generated by TFPI_K1+/−intercrosses. To replicate previously published observations with global deletion of TFPI K1 domain, we examined pregnancies from TFPI_K1+/− intercrosses. Whole-mount images of 11.5 dpc TFPI_K1+/+ (A) and TFPI_K1−/− (B) embryos and placentas are shown. The arrow points to intracranial hemorrhage of the TFPI_K1−/− embryo. Labyrinth regions of placentas are highlighted with dotted circles; the placenta of the TFPI_K1−/− embryo shows reduced labyrinth region. Carstairs’ stained histological sections of both placentas are shown in panels C and D, and boxed regions are enlarged in panels E and F, respectively. Arrows point to large fetal vessels that may not have been optimally branched.
Figure 3.
Figure 3.
RNA and protein expression in TFPI_K1δ/δmice. (A) Expression of exon 4 containing RNA in TFPI_K1δ/+ and TFPI_K1δ/δ embryos relative to TFPI_K1Lox/+ litter mates is shown. TFPI_K1δ/δ embryos show 95% to 99% reduction in exon 4 containing TFPI RNA. RNA was isolated from whole embryos. Primers specific for exon 4 were used for real-time qPCR analysis. (B) TFPI_K1δ/δ mice express a truncated protein corresponding to K1-deleted TFPI. FXa-conjugated beads were used to pull down TFPI, and western immunoblotting was performed to evaluate the level of full-length protein in the organs of adult TFPI_K1δ/δ mice (lanes marked “Lo”) and WT C57Bl/6 controls (lanes marked “+”). No full-length TFPI protein could be detected in TFPI_K1δ/δ mice. A truncated protein at reduced level of expression was readily detected.
Figure 4.
Figure 4.
Evidence of prothrombotic tendency of TFPI_K1δ/δmice. (A) Plasma from TFPI_K1δ/δ mice supports significantly enhanced thrombin generation as compared with plasma from WT C57Bl/6 mice. Thrombin generation assays conducted using TF as a trigger are shown for WT (blue solid line) and TFPI_K1–deficient plasma (red solid line). Background thrombin generation was observed in the absence of any added trigger in TFPI_K1–deficient (red dashed line), but not in WT plasma (blue dashed line). (B) Plasma TAT complex is elevated in TFPI_K1δ/δ mice compared with WT controls. TAT levels in TFPI K1–deficient and WT plasma were measured to be 3.8 ± 0.5 ng/mL vs 6.7 ± 1.9 ng/mL; mean ± standard deviation; P = .027. (C) Percent of surviving TFPI_K1δ/δ mice (solid line) and WT (TFPI_K1+/+) controls (dotted line) over time after IV injection of TF is shown. TFPI_K1δ/δ mice are more susceptible to TF-induced pulmonary embolism as compared with controls (P = .006 at 30 minutes). (D) Lung perfusion scores (arbitrary units) with Evan’s blue for TFPI_K1δ/δ and control mice following TF injection are shown. TFPI_K1δ/δ mice have significantly increased scores indicating increased perfusion defect (P = .003).
Figure 5.
Figure 5.
Frequent fibrin deposits in renal medulla of TFPI_K1δ/δmice. Histological sections of kidneys from TFPI_K1δ/δ (A) and WT C57Bl/6 control mice (B) with Carstairs’ staining are shown. Bright orange/red stains in panel A (yellow arrows) are fibrin deposits seen in 5 out of 7 TFPI_K1δ/δ mice examined, but in none of the 5 controls.
Figure 6.
Figure 6.
In rare instances, TFPI_ K1δ/δmice exhibited large infarcted lesions in the brain. Hematoxylin and eosin–stained coronal sections and enlarged views of brains from a TFPI-deficient (≤1% residual full-length TFPI) (A,C) and WT control (B,D) mice are shown. Arrow points to an old lesion in the cerebral cortex replaced by scar tissue.
Figure 7.
Figure 7.
Kaplan-Meier disease-free survival curves for TFPI_K1δ/δand TFPI_K1δ/+mice. TFPI_K1–deficient mice (red line) exhibit a probability of adverse event-free survival comparable to heterozygous controls (black line) (P = .37). Censored observations (animals removed from the study) are indicated as cross lines. Two out of 43 TFPI_K1δ/δ mice were found dead, and 2 others showed acute disease during the 14th and 16th weeks, but the probability of adverse event-free survival was not found to be significantly different from 73 controls of which 3 were found dead.
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