FIX therapy to support wound healing following hemarthrosis in the FIX^−/−mouse joint. Joint hemorrhage was induced by needle puncture of the left knee joint capsule in all treatment groups, followed by tail vein infusion of first treatment or placebo (NS) at 20 minutes after wounding. Some treatment groups received repeated doses of rFIX at later time points as indicated. Treatment groups (6-8 mice/group) consisted of FIX^−/− mice treated with a single dose of NS on day 0 (NS, ●); FIX^−/− mice treated with a single dose of N9-GP on day 0 (N9-GP d0, ▪); FIX^−/− mice treated with a single dose of rFIX on day 0 (rFIX d0, ▲); FIX^−/− mice treated with rFIX on day 0, 1 and 3 (rFIX d0,1,3, ▼); FIX^−/− mice treated with rFIX on day 0, 1,3,5,7,9,11,13 (rFIX d0-13, ♦); and WT mice treated with NS on day 0 (WT, ○). Points that are significantly different from “WT” are indicated by “*.” Points that are significantly different from “NS” are indicated by “#.” Markers of significance are used as follows: *P < .05, **P < .01, ***P < .001, ****P < .0001, ^#P < .05, ^##P < .01, ^####P < .0001; ^fP < 0.05, ^ffP < 0.01. (A) Synovitis grade. As reported previously,⁸  untreated FIX^−/− mice always demonstrate synovitis pathology graded greater than two-tenths on the Valentino scale at 2 weeks after this injury, whereas hemostatically normal mice never score greater than two-tenths. The dashed line indicates the historical threshold value of 2. FIX^−/− mice that received N9-GP but not unmodified rFIX demonstrated mean synovitis score below this threshold of 2 out of 10. Apoptotic (TUNEL stain positive) chondrocytes (B) and total chondrocytes (C) were counted from femoral and tibial cartilage using a 20× objective lens, and the mean and standard deviation for the entire cohort of animals at each time point are plotted. (D) Iron released from degraded erythrocytes was observed as the intensity of Prussian blue staining ferric iron at ×40 magnification and was graded in the heaviest areas of synovial and subsynovial staining as described in the supplemental Methods. (E) Macrophage infiltration was identified by immunostaining for CD68 observed at ×40 magnification and graded in the most heavily infiltrated portion of synovium as described in the supplemental Methods. (F) Vascular endothelial cells were identified by staining for VWF observed at ×40 magnification and counted in the staining areas of the synovium and subsynovium.