Table 1.
Fig 1.
Bleeding phenotype of the F8-/y mice.
(A) Patella diameter before and 24h after joint bleeding induction to the right knee (n = 7,6) of C57BL/6J and F8-/y with the left knee (n = 6,6) set as a control. (B) Representative rotational thromboelastometry (ROTEM) graphs of a C57BL/6J and a F8-/y, illustrating clotting time (green), clot formation time (pink) and clot firmness (blue). (C) Clotting times and (D) clot formation times of C57BL/6J, F8-/y, and F8-/y blood samples reconstituted with 2.5 U/ml of human recombinant FVIII (n = 3). All data were expressed as means ± SEM. Statistical comparisons were performed using one-way ANOVA or two-way ANOVA * p< 0.05, ** p<0.01, ***p<0.001.
Fig 2.
Impaired thrombin generation by platelets from F8-/y mice.
(A), (B) and (C) Representative thrombograms of a F8+/y (WT) vs a F8-/y mouse. Thrombin generation in unstimulated platelet-rich plasma (PRP) (A), in PRP induced by 1 pM tissue factor (TF) (B), and in PRP induced by 0.1 U/ml thrombin (thr) (C), respectively. (D) Thrombin peak in unstimulated PRP (n = 5,2), PRP plus 1 pM TF (n = 5,2), and 0.1 U/ml thr-stimulated PRP (n = 5,2). (E) Endogenous thrombin potential in unstimulated PRP (n = 5,2), PRP triggered by 1 pM TF (n = 5,2), and 0.1U/ml thr-stimulated platelets in PRP (n = 5,3), analyzed by calibrated automated thrombography of F8+/y vs F8-/y mice. All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test, * p< 0.05, ** p<0.01, ***p<0.001.
Fig 3.
Low-grade inflammatory phenotype in the liver of F8-/y mice.
(A) FVIII (n = 5,4), (B) TNFα (n = 6,7), (C) CD45 (n = 7), and (D) TLR4 (n = 6,4) hepatic transcript levels of F8+/y (WT) vs F8-/y mice. (E) Representative hepatic tissue sections of F8+/y vs F8-/y stained with anti-mouse F4/80 antibody (200x magnification). (F) Visual scoring of the F4/80 stained hepatic tissue sections (n = 5). (G) Hepatic transcript levels of the macrophage marker F4/80 in F8+/y (WT) vs F8-/y mice (n = 4,7). (H) Hepatic SAA3 transcript levels in F8+/y (WT) vs F8-/y mice (n = 5,5). (I) Serum glutamate-pyruvate-transaminase levels in F8+/y (WT) vs F8-/y mice (n = 5,5). (J) Serum glutamat-oxalacetat-transaminase levels in F8+/y (WT) vs F8-/y mice (n = 6,5). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test, * p< 0.05, ***p<0.001, ****p<0.0001.
Fig 4.
Increased VWF staining of the hepatic endothelium of F8-/y mice.
(A) Representative hepatic tissue sections of F8+/y (WT) vs F8-/y mice stained with anti-human VWF antibody (100x and 400x magnification). (B) Visual scoring of the VWF stained hepatic tissue sections of F8+/y (WT) vs F8-/y mice (n = 7,6). (C) Hepatic VWF transcript levels in F8+/y (WT) vs F8-/y mice (n = 4,7). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test, ** p<0.01.
Fig 5.
Increased VWF levels in the plasma of F8-/y mice.
(A) VWF antigen levels in plasma of F8+/y (WT) vs F8-/y mice (n = 9). (B) VWF multimer analysis of plasma samples from F8+/y vs F8-/y mice (n = 5). (C) ADAMTS13 levels in plasma of F8+/y vs F8-/y mice (n = 7). (D) VWF antigen levels in plasma of F8-/y mice, 2h after tail vein injection of recombinant FVIII (Kogenate) at a dose of 1.5 U per 30g body weight or with 0.9% NaCl solution as a vehicle control (n = 11,10,12). All data were expressed as means ± SEM. Statistical comparisons were performed using the Student’s t-test or one-way ANOVA, * p< 0.05, ** p<0.01.