Assessment of the bleeding phenotype in VWF^2N mice using lateral TVT injury model. Male and female mice with ages of 8-20 weeks old of VWF^+/+, VWF^2N/+, VWF^2N/2N, VWF^2N/-, VWF^−/−, and VWF^+/− genotypes were used in this study. Animals were anesthetized with isoflurane, and lateral TVT was performed at the position of 2.5 mm diameter of the prewarmed tail, introducing a 1-mm depth incision of the left lateral tail vein by sliding a scalpel blade through a transverse groove in an aluminum transection template block. The wounded tail was submerged into 14 mL of prewarmed saline and monitored for 15 minutes. The tail was removed from the saline if the bleeding stopped within 15 minutes or at 15 minutes if it did not stop, and clotting was rechallenged a total of 3 times. Primary and rechallenge bleeding times were recorded. Blood loss was quantified 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. Total bleeding times and blood losses during 3 rechallenges were combined. 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 rechallenges. (B) Blood loss during primary challenge and rechallenges. These data demonstrate that 2N VWF can still help to initiate clot formation in vein injury if the subendothelial matrix around the wound remains.