Abstract
Tissue factor (TF) triggers blood coagulation and is translated from two mRNA splice isoforms, encoding membrane-anchored full-length TF (flTF) and soluble alternatively-spliced TF (asTF). The complete knockout of TF in mice causes embryonic lethality associated with failure of the yolk sac vasculature. Although asTF plays roles in postnatal angiogenesis, it is unknown whether it activates coagulation sufficiently or makes previously unrecognized contributions to sustaining integrity of embryonic yolk sac vessels. Using gene knock-in into the mouse TF locus, homozygous asTF knock-in (asTFKI) mice, which express murine asTF in the absence of flTF, exhibited embryonic lethality between day 9.5 and 10.5. Day 9.5 homozygous asTFKI embryos expressed asTF protein, but no procoagulant activity was detectable in a plasma clotting assay. Although the α-smooth-muscle-actin positive mesodermal layer as well as blood islands developed similarly in day 8.5 wild-type or homozygous asTFKI embryos, erythrocytes were progressively lost from disintegrating yolk sac vessels of asTFKI embryos by day 10.5. These data show that in the absence of flTF, asTF expressed during embryonic development has no measurable procoagulant activity, does not support embryonic vessel stability by non-coagulant mechanisms, and fails to maintain a functional vasculature and embryonic survival.
Highlights
Tissue factor (TF) triggers blood coagulation and supports nonhemostatic processes through cell signaling
Generation of alternatively-spliced TF (asTF) Knock-in Mice To study the function of asTF in the absence of full-length TF (flTF) in vivo, an asTF knock-in mouse was generated by replacing the TF gene with the murine asTF coding sequence under the control of the murine TF(F3) promoter (Fig. 1A)
To determine whether homozygous asTF knock-in (asTFKI) embryos develop to this stage, 48 E9.5 embryos were genotyped. 13 homozygous asTFKI (27%), 25 heterozygous asTFKI/wtTF (52%), and 10 wt (21%) embryos were identified, demonstrating normal Mendelian segregation significantly different from the genotype distribution found at birth (p = 0.0001) (Fig. 2)
Summary
Tissue factor (TF) triggers blood coagulation and supports nonhemostatic processes through cell signaling. The TF transcript can undergo alternative splicing of exon 4 to 6, which creates a soluble protein isoform termed alternatively-spliced TF (asTF) with a unique C-terminus that lacks the transmembrane and intracellular domains of full-length TF (flTF) [1]. The C-termini of human and mouse asTF show appreciable sequence homology within the first 40 amino acids [2]. It is controversial whether asTF exhibits procoagulant activity [1,3,4,5]. Recombinant murine asTF exhibits minimal, phospholipiddependent cofactor activity [2]; procoagulant activity of murine asTF has not been characterized in in vivo in the absence of flTF
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