879. Effects of Continuous Expression of Activated Protein C (APC) in Novel Murine Thrombosis Models

Abstract

Top of pageAbstract APC mediates anticoagulant functions and signals cellular responses that are anti-inflammatory in nature. To date, APC is the only drug efficacious in human with severe sepsis. Because of its potential role in human diseases other than sepsis, we sought to exploit APC's effects in models of thrombosis. However, assessment of APC's in vivo role has been compromised by its short half-life (15 min). To overcome this limitation, we used adeno-associated viral (AAV) vectors encoding the PC zymogen or APC for liver-specific expression. Initial experiments in mice demonstrated that APC, but not PC, expression (50|[ndash]|400 ng/ml) is effective in preventing in vivo thrombus formation at both macro and microcirculation, with minimal risk of bleeding. Here we described a novel murine model for thrombophilia characterized by fetal loss and spontaneous adult thrombosis. We crossed mice with FVL mutation that renders the protein resistant to APC's proteolytic activity with mice expressing elevated levels of FIX (tFIX). Mice that achieved adulthood developed normally until 7 month of age when 10% of FVL(+/|[minus]|)/tFIX and 5% of FVL(|[minus]|/|[minus]|)/tFIX mice showed spontaneous thrombosis, whereas no other age-matched mice (n=200) developed such complications. Breeding of mice FVL(+/|[minus]|) with mice tFIX generated more than 400 newborns of all genotypes but none of FVL(+/+)/tFIX genotype. Interestingly, the number of reabsorbed embryos increased from 13% (E9.5) to 33% (E16.5, p<0.02), and reached 54% when the mother was tFIX. All FVL(+/+)/tFIX embryos exhibited extensive bruises and hemorrhagic areas which colocalize with marked fibrin deposition, findings consistent with disseminated intravascular coagulation (DIC). Placental analysis (n=20) of FVL(+/+)/tFIX embryos harvested at E12.5 or later demonstrated extensive abnormalities, such as excessive fibrin deposition and necrotic cells (17%) in the labyrinthine trophoblast. These findings were not found in hundreds of embryos of other genotypes or in placental tissues. The maternal genotype also influenced the number of newborns per litter that was significantly lower when the mother carried tFIX compared with father tFIX (4.8 vs. 8.3, P<0.02). Pregnant mice (n=12) received injection of a thrombin inhibitor (hirudin) at doses ranging from 0.3 to 0.6 mg/kg/day throughout the gestation. This strategy resulted in reduced placental fibrin deposition and necrotic areas (6% vs. 17% of untreated mice, p<0.01), but did not rescue FVL(+/+)/tFIX mice. Moreover, the rate of reabsorbed embryos and the litter size were not affected by the anticoagulant. We next injected mice with AAV-APC into female mice FVL(+/|[minus]|)tFIX (n=6) and crossed them with male FVL(+/|[minus]|). The results indicate that APC expression is associated with low numbers of reabsorbed embryos (20%) compared to those gestations without AAV-APC (54%, p<0.02). These data suggest that rescue of embryonic lethality of prothrombotic phenotype models affecting the protein C pathway requires both antithrombotic and anti-inflammatory effects that unlikely can be provide by anticoagulant drugs other than APC.