In Vivo Evidence of Modulation of the Hemophilia Phenotype by the Factor V Leiden.

The amelioration of hemophilia phenotype or delayed onset of the bleeding episodes in subjects with severe hemophilia A (FVIII deficiency) has been associated with inherited resistance to activated protein C due to factor V Leiden (R506Q), FVL. These observations were confirmed by in vitro systems in which homozygous phenotype of FVL increased thrombin formation in presence of < 1% FVIII by nearly 5-fold (Blood 90:3067). Here we provide in vivo evidence of the beneficial interaction of FVL on the hemophilia phenotype in mice. Animals with severe deficiency of factor VIII due to deletion of intron 16 of factor VIII gene (HA) or large gene deletion of factor IX (HB) were crossed with FVL homozygous mice [+/+] on C57Bl6 strain [Cui et al. (Blood 96:4222)] We used a modified activated partial thromboplastin time (aPTT) assay to compared clotting times among male HA (n=30), HA/FVL [+/+] (n=7), FVL [+/+] (n=5), and litermate WT mice (n=6) with age ranging from 6–12 weeks. Blood samples were collected by tail vein transection into 3.8% sodium citrate. Values for the aPTT in male HA were 67.3 ± 4 sec, and among WT or FVL [+/+] values were 37 ± 4 sec or 31± 2 sec, respectively. Whereas intermediate aPTT values of 53 ± 3.7 sec were determined in HA/FVL [+/+], which differs from HA mice (p<0.0001) but also from WT or FVL (p<0.0001). A similar shortening of aPTT was also determined among HB/FVL[+/+] which were compared to HB, 55 ± 7 sec vs. 64 ± 0.9. Hemostatic challenge by tail clipping assay failed to revealed differences in bleeding times/blood loss among hemophilia animals with or without FVL mutation. To test whether a more sensitive technique would provide further evidence of the improved hemostasis in HA/FVL mice, we assessed real-time in vivo thrombus formation by confocal and widefield microscopy. Mice were anesthetized and the cremaster muscle was exposed for intravital microscopy. Infusion of fluorescently labeled antibody to murine platelet glycoprotein IIb/IIIa complex via the jugular vein allowed monitoring of platelet deposition upon laser-mediated endothelial injury at several sites of the arterial vessel wall. No thrombus formation was observed in severe HA mice following successive vascular injuries, a finding also common in severe HB mice. However, infusion of factor VIII concentrated clearly induced the thrombi formation upon vascular injury. HA/FVL mice tested presented thrombus formation in a comparable fashion of HA-FVIII transfused mice. These in vivo data provide support to the hypothesis that the FVL mutation has the potential to improve the phenotype of severe hemophilia and may offer a novel therapeutic target for hemophilia.