Journal: Redox Biology

Article Title: Inflammation contributes to trauma-induced coagulopathy by oxidation of multiple clotting factors

doi: 10.1016/j.redox.2025.103956

Figure Lengend Snippet: Trauma shows unique features in clotting factors in humans. The activity and protein concentration of each clotting factor, FV ( A ), FVII ( B ), FVIII ( C ), FIX ( D ), FX ( E ), FXI ( F ) and FXII ( G ) was measured using human plasma from trauma patients (n = 22) and healthy controls (n = 10) as described in Methods. Ratio of each clotting factor was calculated by dividing the activity of each factor with protein concentration. Data represent mean ± SEM. ns = not significant. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. control. FV ratio, FVII concentration, FVIII activity, FIX concentration, FXI ratio and FXII ratio panels (Mann-Whitney U test), others (Student's t-test).

Article Snippet: After incubation with the Intercept blocking buffer (LI-COR, #927–6001) for 1 h at room temperature, membranes were incubated with mouse monoclonal antibodies against albumin (Abcam, 15C7, #AB10241, 1:1000), FVII (Invitrogen, AD-1, #MA5-17635, 1:1000), FX (Enzyme Research, 520, #Mab-FX, 1:1000), FXII (Invitrogen, clone 5A6, #MA5-15902, 1:1000), and rabbit polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Millipore, #90451, 1: 150) in case of human samples, or with rabbit antibodies against FVII (Invitrogen, #PA5-115207, 1:1000), FX (Biorbyt, #orb556640, 1:1000), FXII (Proteintech, #12551-1-AP, 1:1000), and Goat polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Bethyl Laboratories, # A150-117A, 1: 1000) in case of rat TIC model, respectively, overnight at 4 °C in Intercept antibody diluent (LI-COR, #927–65001).

Techniques: Coagulation, Activity Assay, Protein Concentration, Clinical Proteomics, Control, Concentration Assay, MANN-WHITNEY

Journal: Redox Biology

Article Title: Inflammation contributes to trauma-induced coagulopathy by oxidation of multiple clotting factors

doi: 10.1016/j.redox.2025.103956

Figure Lengend Snippet: Oxidative stress is increased and correlates with coagulopathy, inflammation, thrombin generation, and FV, FVII and FXII activities in trauma patients. A . Oxidative stress was analyzed by measuring oxidative carbonyl modification in trauma patients (n = 21, closed circle) and healthy controls (n = 10, open circle) as described in Methods. Linear regression relationship was shown between oxidative stress versus coagulopathy (PT. aPTT and fibrinogen, B ), IL6 ( C ), TGA (peak time, peak concentration, slope, lag time and AUC., D ), PGA (peak time, peak concentration, slope, lag time and AUC., E ) or the activity of each clotting factor ( F ) using Pearson correlation coefficient. Data represent mean ± SEM. ∗∗∗p < 0.001 vs. control. Mann-Whitney U test.

Article Snippet: After incubation with the Intercept blocking buffer (LI-COR, #927–6001) for 1 h at room temperature, membranes were incubated with mouse monoclonal antibodies against albumin (Abcam, 15C7, #AB10241, 1:1000), FVII (Invitrogen, AD-1, #MA5-17635, 1:1000), FX (Enzyme Research, 520, #Mab-FX, 1:1000), FXII (Invitrogen, clone 5A6, #MA5-15902, 1:1000), and rabbit polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Millipore, #90451, 1: 150) in case of human samples, or with rabbit antibodies against FVII (Invitrogen, #PA5-115207, 1:1000), FX (Biorbyt, #orb556640, 1:1000), FXII (Proteintech, #12551-1-AP, 1:1000), and Goat polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Bethyl Laboratories, # A150-117A, 1: 1000) in case of rat TIC model, respectively, overnight at 4 °C in Intercept antibody diluent (LI-COR, #927–65001).

Techniques: Modification, Concentration Assay, Activity Assay, Coagulation, Control, MANN-WHITNEY

Journal: Redox Biology

Article Title: Inflammation contributes to trauma-induced coagulopathy by oxidation of multiple clotting factors

doi: 10.1016/j.redox.2025.103956

Figure Lengend Snippet: FVII, FX and FXII are oxidized in trauma patients. FV ( A ), FX ( B ) and FXII ( C ) were immunoprecipitated using monoclonal antibodies against FV, FX and FXII from plasma of trauma patient (n = 5) and healthy controls (n = 4) as described in Methods. Purified FV, FX and FXII was analyzed by OxyBlot and immunoblotting using FV, FX or FXII – specific antibodies. Representative blots are displayed.

Article Snippet: After incubation with the Intercept blocking buffer (LI-COR, #927–6001) for 1 h at room temperature, membranes were incubated with mouse monoclonal antibodies against albumin (Abcam, 15C7, #AB10241, 1:1000), FVII (Invitrogen, AD-1, #MA5-17635, 1:1000), FX (Enzyme Research, 520, #Mab-FX, 1:1000), FXII (Invitrogen, clone 5A6, #MA5-15902, 1:1000), and rabbit polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Millipore, #90451, 1: 150) in case of human samples, or with rabbit antibodies against FVII (Invitrogen, #PA5-115207, 1:1000), FX (Biorbyt, #orb556640, 1:1000), FXII (Proteintech, #12551-1-AP, 1:1000), and Goat polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Bethyl Laboratories, # A150-117A, 1: 1000) in case of rat TIC model, respectively, overnight at 4 °C in Intercept antibody diluent (LI-COR, #927–65001).

Techniques: Immunoprecipitation, Bioprocessing, Clinical Proteomics, Purification, Western Blot

Journal: Redox Biology

Article Title: Inflammation contributes to trauma-induced coagulopathy by oxidation of multiple clotting factors

doi: 10.1016/j.redox.2025.103956

Figure Lengend Snippet: IL6-activated leukocytes mimic oxidation and reduce the activity of clotting factors shown in trauma patients while the anti-inflammatory protein, AQB-565 and antioxidant, vitamin C prevent these phenomena. IL6-stimulated RBC-lysed leukocytes from healthy donors (n = 5) were incubated with FVII, FX and FXII, or AQB-565 and vitamin C as described in Methods for 4 h. After that, FVII, FX and FXII were isolated by immunoprecipitation as described in Methods. Purified FV ( A ), FX ( B ) and FXII ( C ) was analyzed by OxyBlot and immunoblotting using FV, FX or FXII – specific antibodies (left panel). Representative blots are displayed. Also, each activity of these coagulation factors was measured in isolated FVII, FX and FXII as described in methods (right panel). Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. IL6. ANOVA and Tukey post-hoc test.

Article Snippet: After incubation with the Intercept blocking buffer (LI-COR, #927–6001) for 1 h at room temperature, membranes were incubated with mouse monoclonal antibodies against albumin (Abcam, 15C7, #AB10241, 1:1000), FVII (Invitrogen, AD-1, #MA5-17635, 1:1000), FX (Enzyme Research, 520, #Mab-FX, 1:1000), FXII (Invitrogen, clone 5A6, #MA5-15902, 1:1000), and rabbit polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Millipore, #90451, 1: 150) in case of human samples, or with rabbit antibodies against FVII (Invitrogen, #PA5-115207, 1:1000), FX (Biorbyt, #orb556640, 1:1000), FXII (Proteintech, #12551-1-AP, 1:1000), and Goat polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Bethyl Laboratories, # A150-117A, 1: 1000) in case of rat TIC model, respectively, overnight at 4 °C in Intercept antibody diluent (LI-COR, #927–65001).

Techniques: Activity Assay, Coagulation, Incubation, Isolation, Immunoprecipitation, Purification, Western Blot

Journal: Redox Biology

Article Title: Inflammation contributes to trauma-induced coagulopathy by oxidation of multiple clotting factors

doi: 10.1016/j.redox.2025.103956

Figure Lengend Snippet: Each clotting factor shows distinct and unique roles in thrombin generation and ROTEM. TGA ( A , C , E , G , and I ) and ROTEM ( B , D , F , H , J ) were performed using each clotting factor-specific DP (n = 3) as described in Methods. A and B . control amount (control) of FV which we found in healthy controls or reduced amount (treated) of FV which we found in trauma patients were added in FV DP. Variables from TGA, lag time, peak time, slope, peak concentration and AUC, or variables from ROTEM, clotting time, a-angle, maximum clot firmness were obtained in each experiment as described in Methods. C and D . control FVII (control) and oxidized FVII (treated) which were obtained by incubation with leukocytes as described in Methods were added to FVII DP. E and F . control amount (control) or excess amount (treated)of FVIII were added to FVIII DP. G and H . control FX (control) and oxidized FX (treated) which were obtained by incubation with leukocytes as described in Methods were added to FX DP. I and J . control FXII (control) and oxidized FXII (treated) which were obtained by incubation with leukocytes as described in Methods were added to FXII DP. Data represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 vs. control. Student's t-test.

Article Snippet: After incubation with the Intercept blocking buffer (LI-COR, #927–6001) for 1 h at room temperature, membranes were incubated with mouse monoclonal antibodies against albumin (Abcam, 15C7, #AB10241, 1:1000), FVII (Invitrogen, AD-1, #MA5-17635, 1:1000), FX (Enzyme Research, 520, #Mab-FX, 1:1000), FXII (Invitrogen, clone 5A6, #MA5-15902, 1:1000), and rabbit polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Millipore, #90451, 1: 150) in case of human samples, or with rabbit antibodies against FVII (Invitrogen, #PA5-115207, 1:1000), FX (Biorbyt, #orb556640, 1:1000), FXII (Proteintech, #12551-1-AP, 1:1000), and Goat polyclonal antibody against 2,4-dinitrophenylhydrazone (anti-DNP, Bethyl Laboratories, # A150-117A, 1: 1000) in case of rat TIC model, respectively, overnight at 4 °C in Intercept antibody diluent (LI-COR, #927–65001).

Techniques: Coagulation, Control, Concentration Assay, Incubation

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Sustained high expression of human FVII following AAV8-mediated gene delivery in mice

doi: 10.1016/j.omtm.2025.101523

Figure Lengend Snippet: Evaluation of AAV5- versus AAV8-mediated FVII expression (A) rAAV5 was packaged with either hFVIIwt or cohFVII(-22) under the control of the hAAT promoter and injected at a dose of 1.6 × 10 13 gc/kg; while rAAV8 was packaged with the same constructs and injected at a dose of 1.6 × 10 12 gc/kg instead; (B) hFVIIwt levels in plasma of male C57BL/6 mice delivered by either rAAV5 or rAAV8; (C) human cohFVII(-22) levels in plasma of male C57BL/6 mice delivered by either rAAV5 or rAAV8. In both rAAV5- and rAAV8-mediated expression of hFVII recombinant protein, the hFVIIwt was outperformed by the shorter hFVII(-22) transcript. Data are presented as means (SD). See also . Data were analyzed by Student’s t test. ∗∗∗∗ p < 0.0001.

Article Snippet: FVII activity in mouse plasma was assayed using the BIOPHEN FVII chromogenic assay (Ref 221304, Hyphen BioMed) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Injection, Construct, Clinical Proteomics, Recombinant

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Sustained high expression of human FVII following AAV8-mediated gene delivery in mice

doi: 10.1016/j.omtm.2025.101523

Figure Lengend Snippet: Long-term expression using AAV8 capsid (A) AAV8 capsid was packaged with either FVIIwt and coFVII(-22) or two alternative codon-optimized versions, RBD-FVIIwt and RBD-FVII(-22), under the control of the hAAT promoter. Injection was performed at a dose of 0.8 × 10 12 gc/kg in wild-type C57BL/6 mice ( n = 6/group). (B) Plasma levels of FVII after blood collection up to 48 weeks post-injection. No difference was observed between FVIIwt and the correspondent codon-optimized version RBD-FVIIwt as well as between coFVII(-22) and its codon-optimized version RBD-FVII(-22); however, both RBD-FVII(-22) and coFVII(-22) overperformed FVII(wt) and RBD-FVII(wt). For extended dataset, see also . (C) FVII activity assay is used to test the same samples used for plasma antigen level. FVII activity assay is directly proportional to the amount of active protein present in the plasma of the injected mice. Dotted black line corresponds to mouse FVII threshold level (1 U/mL). Transcripts derived from FVII(-22) variants were confirmed to overperform FVIIwt variants. For extended dataset, see also . Graphs show group means (SD); data were analyzed by two-way ANOVA with Tuckey’s multiple comparisons test. ns, non-significant; ∗∗∗∗ p < 0.0001.

Article Snippet: FVII activity in mouse plasma was assayed using the BIOPHEN FVII chromogenic assay (Ref 221304, Hyphen BioMed) according to the manufacturer’s instructions.

Techniques: Expressing, Control, Injection, Clinical Proteomics, Activity Assay, Derivative Assay

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Sustained high expression of human FVII following AAV8-mediated gene delivery in mice

doi: 10.1016/j.omtm.2025.101523

Figure Lengend Snippet: In vivo dosing study (A) To identify the optimal dosage, hAAT-RBD-FVII(-22) was packaged into AAV8. Injections were performed at a concentration of 0.3 × 10 11 , 0.8 × 10 11 , 1.6 × 10 11 , or 0.8 × 10 12 gc/kg in wild-type C57BL/6 mice ( n = 5–6/group); (B) FVII antigen level detected after injection at 0.3 × 10 11 , 0.8 × 10 11 , 1.6 × 10 11 , or 0.8 × 10 12 gc/kg or 1.6 × 10 11 , 0.8 × 10 11 , and 0.3 × 10 11 gc/kg. Graphs show group means (SD); data were analyzed by two-way ANOVA with Tuckey’s multiple comparisons test. ns, non-significant; ∗∗∗∗ p < 0.0001.

Article Snippet: FVII activity in mouse plasma was assayed using the BIOPHEN FVII chromogenic assay (Ref 221304, Hyphen BioMed) according to the manufacturer’s instructions.

Techniques: In Vivo, Concentration Assay, Injection

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Sustained high expression of human FVII following AAV8-mediated gene delivery in mice

doi: 10.1016/j.omtm.2025.101523

Figure Lengend Snippet: Anti-AAV8 antibodies in treated samples The bar graph showed plasma levels of anti-AAV8 antibodies in mice injected with rAAV8 capsids encoding for FVIIwt, coFVII(-22) RBD-FVIIwt, and RBD-FVII(-22), under the control of the hAAT promoter, after 6, 12, and 48 weeks post-injection as well as PBS-injected animals ( n = 5–6/group). Graphs show group means (SD); data were analyzed by one-way ANOVA with Tuckey’s multiple comparisons test; ∗ p < 0.05.

Article Snippet: FVII activity in mouse plasma was assayed using the BIOPHEN FVII chromogenic assay (Ref 221304, Hyphen BioMed) according to the manufacturer’s instructions.

Techniques: Clinical Proteomics, Injection, Control