PAR-4 Deficiency Rescues The Embryonic Lethal Phenotype Of TFPI Null Mice

Tissue factor pathway inhibitor (TFPI) is a trivalent Kunitz-type serine protease inhibitor that dampens tissue factor (TF) initiated blood coagulation. It is produced by endothelial cells and megakaryocytes. Mice lacking TFPI activity die in utero from yolk sac hemorrhage or a presumptive consumptive coagulopathy. The embryonic lethal phenotype of TFPI null mice can be rescued by TF or factor VII deficiency. Our laboratory has shown that platelet TFPI limits platelet accumulation, but not fibrin formation, in an in vivo vascular injury model. Interestingly, mice lacking PAR4, the major thrombin receptor in mouse platelets, have decreased platelet accumulation but not fibrin formation following vascular injury. Therefore, we hypothesized that PAR-4 deficiency would counterbalance the absence of TFPI in platelets and rescue the embryonic lethal phenotype of TFPI null mice.

TFPI heterozygous mice (TFPI^+/-; C57/Bl6) were bred with PAR4 null mice (PAR4^-/-; C57/Bl6) to generate TFPI^+/-/PAR4^-/- mice. These mice were bred to assess rescue of TFPI lethality. Of 76 pups closely monitored following birth, 12 (15.8%) were TFPI^-/-/PAR4^-/-, with 8 (10.5%) surviving to wean. TFPI^-/-/PAR4^-/- male mice were bred with TFPI^+/-/PAR4^-/- females for subsequent studies. Of 447 pups generated, 101 TFPI^-/-/PAR4^-/- mice (22.6%) were produced. Thus, TFPI^-/-/PAR4^-/- pups survive to wean at approximately one-half the expected frequency. TFPI^-/-/PAR4^-/- mice surviving to wean appeared healthy with no overt signs of thrombosis, and have apparently normal survival to over 12 months of age. Compared to TFPI^+/-/PAR4^-/- littermates, TFPI^-/-/PAR4^-/- have reduced body weight at 8 weeks. Female TFPI^-/-/PAR4^-/- mice can reproduce successfully. Approximately one-third of the TFPI^-/-/PAR4^-/- mice have short or totally absent tails. The short tail phenotype develops in utero and changes in tail:body proportion do not occur following birth. There was no difference in blood loss following tail clip between TFPI^+/+/PAR4^-/-, TFPI^+/-/PAR4^-/- or TFPI^-/-/PAR4^-/- mice. There also was no difference in fibrin or platelet accumulation between TFPI^+/+/PAR4^-/- or TFPI^-/-/PAR4^-/-mice following venous electrolytic injury.

This is the first reported rescue of the TFPI null embryonic lethal phenotype by a platelet activation defect. Almost one-half of the TFPI^-/-/PAR4^-/- mice survive through adulthood despite having normal tissue factor function not regulated by TFPI. This, and our inability, thus far, to identify a procoagulant state in the adult mice, suggests that an essential TFPI anticoagulant function is to modulate PAR-4 mediated platelet function; likely by limiting thrombin generation. TFPI may do this through inhibition of TF-fVIIa, or via inhibition of early forms of prothrombinase on the platelet surface, a very recently recognized inhibitory function of TFPI. The long term survival of these mice also demonstrates that unfettered TF-fVlla-fXa mediated signaling events through PAR-2 are not solely responsible for the embryonic lethality of TFPI null mice. However, the short or absent tails that arise during development of TFPI deficient mice suggests that TF and/or TFPI may have roles in notochord development that is separate from the coagulation system.

Mast:Novo Nordisk: Honoraria, Research Funding.