Recombinant Mouse Anti-TF Antibody (D3)

(a) Amidolytic activity of sTF:F.VIIa towards the small synthetic substrate Chromozym t-PA. Antibodies were incubated together with sTF (10 nM) and F.VIIa (10 nM) in HBS buffer, 5 mM CaCl₂ for 15 min. Chromozym t-PA (0.5 mM) was added and the rates of substrate cleavage were measured. The results were expressed as fractional activities (vᵢ/v_o) (average of 3 experiments ± SD). (b) Prolongation of human plasma clotting by anti-TF antibodies. The antibodies (D3 Fab and control Ab were not tested) were incubated with TF reagent (Innovin) for 15 min, then added to human citrated plasma. The increase in clotting times is reported as the ratio of clotting times in the presence of antibody and baseline values. The results are the average of two independent experiments. (filled circle) D3, (open circle) D3 Fab, (x) 5G6, (filled square) 7G11, (open square) 6B4, (filled triangle) HTF1, (open triangle) isotype-matched control Ab

Kirchhofer, D., Moran, P., Chiang, N., Kim, J., Riederer, M. A., Eigenbrot, C., & Kelley, R. F. (2000). Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thrombosis and haemostasis, 84(12), 1072-1081.

Relipidated TF₁₋₂₄₃ (0.4 nM) and CaCl₂ was used to initiate the reaction in citrated human plasma that was supplemented with increasing concentrations of anti-TF antibodies. A specific thrombin inhibitor was added to plasma to block thrombin activity. Timed aliquots of the reaction mixture were quenched in EDTA and F.Xa activity measured by adding S2765 and monitoring increase in absorbance at 405 nm on a kinetic microplate reader. The inhibited rates of F.Xa generation during the initial phase of 45 sec were calculated and expressed as fractional activity (vᵢ/v_o). (filled circle) D3, (x) 5G6, (filled square) 7G11, (open square) 6B4, (filled triangle) HTF1, (open triangle) isotype-matched control Ab

Kirchhofer, D., Moran, P., Chiang, N., Kim, J., Riederer, M. A., Eigenbrot, C., & Kelley, R. F. (2000). Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thrombosis and haemostasis, 84(12), 1072-1081.

Antibodies were infused to blood flowing from the antecubital vein of human donors and were mixed in a mixing device without interruption of flow. The blood then entered plexiglass chambers containing coverslips with monolayers of TF-expressing human J82 cells. The shear rate on the cell layer was 65 s⁻¹ representing venous blood flow conditions. Coagulation initiated on the cell layer resulted in the generation of FPA which was quantified from blood taken at the exit of the chambers. The FPA concentrations in plasma were expressed in percent of control values. Each value is the average of 3-8 experiments except for the highest concentrations of D3 Fab and 5G6 (n = 1 and n = 2, resp.) and for two 6B4 concentrations (n = 2 for 1.5 μg/ml and 15 μg/ml). The average of all control values (n = 36) was 1348.6 ± 46.1 ng/ml (± SEM). (filled circle) D3, (open circle) D3 Fab, (x) 5G6, (open square) 6B4, (filled triangle) HTF1

Kirchhofer, D., Moran, P., Chiang, N., Kim, J., Riederer, M. A., Eigenbrot, C., & Kelley, R. F. (2000). Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thrombosis and haemostasis, 84(12), 1072-1081.

Antibodies were added to citrated human plasma for 5 min. Clotting was initiated by addition of relipidated human tissue factor reagent containing CaCl₂ (Innovin) and clotting times measured on an ACL 300. Prolongation of clotting times are reported as the ratio of clotting times in the presence of antibody and baseline values. The results are the average of two independent experiments. (filled circle) D3, (x) 5G6, (filled square) 7G11, (open square) 6B4, (filled triangle) HTF1

Kirchhofer, D., Moran, P., Chiang, N., Kim, J., Riederer, M. A., Eigenbrot, C., & Kelley, R. F. (2000). Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thrombosis and haemostasis, 84(12), 1072-1081.

Kirchhofer, D., Moran, P., Chiang, N., Kim, J., Riederer, M. A., Eigenbrot, C., & Kelley, R. F. (2000). Epitope location on tissue factor determines the anticoagulant potency of monoclonal anti-tissue factor antibodies. Thrombosis and haemostasis, 84(12), 1072-1081.