Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality

_T ^issue extracellular _{TF shows}^factor_high^(TF) allosteric _evolutionary^{is highly} disulfide ^expressed_conservation bond ⁱⁿ Cys186-Cys209 ^{sub-endothelial}_and in vitro _evidence^tissue. of human _sug-^The gests that it significantly contributes to TF procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant Tf mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine Tf. A bleeding phenotype was prominent in homozygous C213G Tf mice. Pre-natal lethality of one third of homozygous offspring was observed between embryonic (E) day E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G Tf male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (at least 100-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G Tf males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the TF allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine TF. Although C213G Tf mice do not display the severe embryonic lethality of Tf knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional TF for hemostasis.