Hyperactivatable Protein C Expression Partially Rescues the Embryonic Lethality of TFPI Null Mice but Surviving Mice Develop Severe Hydrocephalus

Abstract

Introduction: Tissue factor pathway inhibitor (TFPI) and thrombomodulin (TM) are the two anticoagulant proteins directly bound to endothelium. TFPI inhibits the initiation of coagulation by inhibiting TF-FVIIa and early forms of prothrombinase. TM slows the propagation of coagulation by promoting the activation of protein C (APC), which inactivates FVa and FVIIIa. An in vitro model system of purified coagulation proteins has shown that TFPI and thrombomodulin act synergistically to quench tissue factor mediated thrombin generation via neutralization of prothrombinase activity. However, it is unclear how these two proteins cooperate within different vascular beds in vivo, particularly in the brain that has large amounts of tissue factor procoagulant activity. We have previously shown that mice with decreased thrombomodulin function (TMpro) do not have cerebral fibrin deposition even following LPS injection, while partial TFPI deficiency does induce intravascular fibrin deposition in the brain of the TMpro mice, suggesting that these two anticoagulant proteins have distinct functions within the brain vasculature. TFPI null mice die in utero with brain fibrin deposition. A hyperactivatable murine protein C (hMPC) can be produced by altering two acidic amino acids near the thrombin cleavage site. hMPC is activated 30-fold more efficiently by thrombin than wild type protein C and its activation does not require TM. We have used murine model systems to investigate how hMPC expression alters the (patho)physiology of mice with TFPI deficiency.Methods: Transgenic mice producing hMPC under control of the transthyretin promoter were produced. hMPC was bred into TFPI+/- mice, which were then characterized to define the anticoagulant activity of the transgene. Offspring from these initial matings were interbred to determine if hMPC expression would rescue the embryonic lethality of TFPI null mice.Results: hMPC expression elevated plasma protein C 2-fold and plasma APC 3-fold producing a potent anticoagulant effect. In thrombin generation assays, plasma from TFPI+/-/hMPC+ mice had peak thrombin generation of 44.9±7.5 nM vs. 63.0±3.5 nM in TFPI+/-/hMPC- mice (p<0.001). In a TF-induced pulmonary embolism model 5 of 14 TFPI+/-/hMPC+ mice survived over 5 minutes, while only 1 of 10 TFPI+/-/hMPC- did (p=0.02). When TFPI+/-/hMPC+ mice were mated with TFPI+/-/hMPC- mice, TFPI-/-/hMPC+ pups were born at approximately 30% of the expected frequency regardless of whether the transgene was expressed by the mother or the father. About 35% of the surviving TFPI-/-/hMPC+ mice developed a domed shaped head and succumbed to severe hydrocephalus by 8 weeks of age. Those surviving beyond 8 weeks did not develop severe hydrocephalus and were fertile. One TFPI-/-/hMPC+ mouse with a domed shaped head survived long enough to be examined by MRI, which documented severe hydrocephalus. In addition, two lesions (1mm and 0.5 mm diameter) were present in remaining brain tissue. These lesions contained iron suggesting they were areas of blood clot or hemorrhage. MRI exam of 12 week old TFPI-/-/hMPC+ mice identified subclinical hydrocephalus in 1 of 4 mice. Hydrocephalus did not occur in TFPI+/+/hMPC+ or TFPI+/-/hMPC+ mice. Histological examination of brain tissue from TFPI-/-/hMPC+ mice confirmed hydrocephalus with little remaining normal brain tissue. Mice with hydrocephalus had hemorrhage in the ventricles and brain parenchyma that was associated with areas of fibrin(ogen) deposition. There was also congestion of pial vessels and hemorrhage within the subarachnoid space. These findings were not observed in TFPI+/-/hMPC+ mice.Conclusions: Expression of the hMPC transgene produces a potent anticoagulant effect that partially rescues TFPI null embryonic lethality. Surviving TFPI-/-/hMPC+ pups are susceptible to death from severe hydrocephalus associated with hemorrhage and vascular abnormalities in the brain as they mature. These findings demonstrate that TFPI produces physiologically important anticoagulant activity within murine brain vasculature that is not fully compensated by over-expression of activated protein C. DisclosuresMast:Novo Nordisk: Honoraria, Research Funding.