Restoring Initial Thrombin Generation Does Not Normalize Cutaneous Wound Healing in Hemophilia B.

We recently described abnormal wound healing in a mouse model of hemophilia B (Hoffman et al, Blood 2006; DOI 10.1182/blood-2006-05-020495). Specifically: epithelial closure was delayed in hemophilia B mice; hemophilic animals, unlike wild type, developed subcutaneous hematomas; macrophage influx was delayed compared to wild-type mice; and, surprisingly, angiogenesis was enhanced in the hemophilia B mice. We hypothesized that restoring the initial hemostatic burst of thrombin generation following wounding by administration of a single dose of factor IX (FIX) replacement or factor VIIa (FVIIa) bypassing therapy would not only prevent bleeding, but correct the subsequent wound healing process. One dose of therapy was given thirty minutes prior to placement of a single three mm punch biopsy wound on the dorsal skin of each wild type, untreated hemophilia B, and treated hemophilia B mouse. The size of the wounds was measured daily until full epithelial closure. The time course of epithelial closure in treated hemophilia B was intermediate between wild type and untreated hemophilia B. FVIIa-treated hemophilia B began to heal earlier than FIX-treated. Skin from the wound site was collected at different days and examined histologically. Macrophage influx was earlier in treated hemophilia B mice compared to untreated hemophilia B, likely a due to the increased thrombin and fibrin acting as chemotactic agents. The macrophage influx in FVIIa-treated HB was significantly greater at certain time points than in FIX-treated mice, possibly reflecting some signaling effect of TF/FVIIa in addition to its effects on thrombin generation. With the earlier influx in macrophages, hemoglobin was degraded to storage iron at earlier time points. However, tissue iron continued to persist in treated hemophilia B mice similar to untreated hemophilia B, suggesting continued rebleeding. FIX treatment led to significantly more angiogenesis than FVIIa. The reasons for this difference remain to be determined. Untreated and some treated hemophilia B mice developed subcutaneous hematomas both before and after wound closure. The early hematomas are likely caused by a combination of the initial wounding trauma and vulnerability to bleeding related to the high level of vascularity within the granulation tissue. We propose the late hematomas are due to a cycle of bleeding, leading to more inflammation with production of more pro-angiogenic cytokines, leading to greater angiogenesis with its attendant risk of bleeding. In conclusion, restoring initial hemostatic thrombin generation did not normalize cutaneous wound healing in a hemophilia B mouse model. While the time frame of healing may not be the same in human and murine hemophiliacs, our findings suggest that this model could be helpful in rational determination of treatment schedules for replacement or bypassing therapy following injury or surgery.