The Role of Thrombin Deficiency on Skeletal Health

Introduction Persons with hemophilia have decreased bone mineral density (BMD) and increased risk of fracture. Our group has demonstrated that mice with factor VIII deficiency have decreased BMD and decreased biomechanical strength. The underlying mechanism for the decreased skeletal health remains undetermined. This study uses small interfering(si) RNA to knockdown prothrombin gene expression to test the hypothesis that thrombin deficiency results in decreased skeletal health. Method Seventeen 3-week old male C57BL/6J wild-type mice were given subcutaneous injections of siRNA against α-prothrombin (1 mg/Kg) and compared to an equal number of mice injected with saline. Mice were injected at 3, 4, 5, 7, 9, 11, 13, 15, 17, and 19 weeks of age with a volume of 200 ul and measurements taken at peak bone mass at 20 weeks of age. In initial dose response studies, this regimen results in >85% knockdown of prothrombin gene expression (data not shown). Studies included whole body and femoral BMD, thromboelastography (TEG) of plasma, micro computed tomography (µCT), and three-point breaking. Results TEG studies show significant differences between mice treated with siRNA (n=17) and mice treated with normal saline (n=6) with R, K and α-angle being p < 0.02, p < 0.014, and p < 0.005 respectively. These results confirm that the thrombin is knocked down by the siRNA against α-prothrombin. Comparative analysis of the whole body and femoral BMD between the treatment and saline groups demonstrate no significant differences (p = 0.64 and p = 0.74). There was no significant difference seen in µCT scans of the right femur, including femoral cortical area (p=0.19), cortical thickness (p= 0.63), and cancellus area (p= 0.25). Three-point break data show no significant difference in ultimate force (p = 0.268), strength (p = 0.546), or modulus (p = 0.07). Conclusion This study seeks to understand the underlying mechanism for bone disease in hemophilia. The results indicate that >85% prothrombin knockdown in wild-type mice starting at 3 weeks of age does not significantly alter skeletal health. This suggests that either 1) thrombin deficiency is not involved in the mechanism of decreased skeletal health in hemophilia or; 2) the role of thrombin deficiency occurs early in bone development (prior to 3 weeks of age). To evaluate these two potential explanations, thrombin deficiency needs to be evaluated earlier in development. Thrombin deficient mice are embryonically lethal. Therefor