Mouse Anti-Anti-tissue Factor Antibody D3H44 Recombinant Antibody (clone 6A6) (CAT#: PABZ-009)

Figure 1 Analysis of FcγRIV Function In Vivo

(A–C) For analysis of FcγRIV function in vivo, experimental immune thrombocytopenia was induced in wt (A), FcγRI^−/− (B), and FcγRIII^−/− (C) mice by intravenous injection of 3.5 μg of an IgG2b variant of the 6A6 anti-platelet antibody (red lines). As a control, platelet depletion in γ^−/− mice (g^−/−) was included to demonstrate FcR dependence (blue lines). (D) To block IC binding to FcγRIV in vivo, mice were pretreated with 200 μg of an FcγRIV-specific monoclonal antibody (2C6) that interferes with IC binding to this Fc receptor or an isotype-matched control antibody, followed by injection with the platelet depleting 6A6-IgG2b antibody. *p < 0.001; **p < 0.01. (E) The percentage of platelet depletion 4 hr after injection of a 6A6-IgG1, 6A6-IgG2a, or 6A6-IgG2b isotype variant with or without (mock) previous injection of the 2C6- or 9E9-blocking antibodies or isotype-matched control antibodies (Iso) is shown. (F) Platelet depletion mediated by the 6A6-IgG1 antibody is FcγRIII dependent. C57BL/6 wt, common γ^−/−, and FcγRIII^−/− mice were injected with the 6A6-IgG1 isotype variant, and platelet counts were followed over time. Additionally, mice were injected with 200 μg of the FcγRIV-blocking antibody 2C6 or an isotype control antibody before injection with 6A6-IgG1. One out of three independent experiments with five mice per group is shown. For each group, the mean ± SD was calculated, and Student's t test was used for statistical analysis.

Nimmerjahn, F., Bruhns, P., Horiuchi, K., & Ravetch, J. V. (2005). FcγRIV: a novel FcR with distinct IgG subclass specificity. Immunity, 23(1), 41-51.

Figure 2 Neutralization of D3H44 anticoagulant activity by 6A6 antibody in whole blood and plasma.

(a) Endotoxin-mediated whole blood clotting assay. Endotoxin exposure for three hours reduced clotting time by about 50%. Addition of 10 nM D3H44 (F(ab0)2, Fab or IgG4b) prolonged clotting time to control values. 6A6 antibody (200 nM) alone neither prolonged nor reduced clotting times, but effectively neutralized D3H44. Unpaired two-tailed t-test: *p , 0.05; p , 0.01. (b) Prothrombin time (PT). Addition of 300 nM D3H44- F(ab')2, 300 nM D3H44-IgG4b, or 600 nM D3H44-Fab resulted in prolongation of the PT in citrated human plasma. 6A6 antibody at a concentration of 1.7 mM fully restored normal PT values. Dark bars for no 6A6, light bars for 6A6 present.

Eigenbrot, C., Meng, Y. G., Krishnamurthy, R., Lipari, M. T., Presta, L., Devaux, B., ... & Kirchhofer, D. (2003). Structural insight into how an anti-idiotypic antibody against D3H44 (anti-tissue factor antibody) restores normal coagulation. Journal of molecular biology, 331(2), 433-446.