Assessment of the bleeding phenotype in VWF2N mice by a TTT injury model. Male and female mice age 8 to 20 weeks of VWF^+/+, VWF^2N/+, VWF^2N/2N, VWF^2N/−, VWF^−/−, and VWF^+/− genotypes were assessed for their bleeding phenotype. Animals were anesthetized with isoflurane, and 4 mm of the tail tip was clipped. The wounded tail was submerged in 14 mL of prewarmed saline and monitored for 10 minutes. The tail was removed if the bleeding stopped within 10 minutes or at 10 minutes if it did not stop and rechallenged once. Bleeding times were recorded. Blood loss was measured by lysing red cells in 10 mL of distilled H₂O and measuring hemoglobin at OD₅₇₅ nm, and blood loss was calculated according to a standard curve generated from known amounts of pooled blood from wild-type C57BL/6J mice. Mice from our wild-type C57BL/6J colony and VWF^+/+ (VWF2N littermates, also on a C57BL6 background) served as controls. (A) Bleeding times during primary challenge and rechallenge. (B) Blood loss during primary challenge and rechallenge. These data demonstrate that both primary and secondary hemostasis are impaired in VWF^2N/2N mice in a severed tail tip injury model.