Structure and function of the von Willebrand factor A1 domain: analysis with monoclonal antibodies reveals distinct binding sites involved in recognition of the platelet membrane glycoprotein Ib-IX-V complex and ristocetin-dependent activation by Mariagrazia De Luca, David A. Facey, Emmanuel J. Favaloro, Mark S. Hertzberg, James C. Whisstock, Tracy McNally, Robert K. Andrews, and Michael C. Berndt Blood Volume 95(1):164-172 January 1, 2000 ©2000 by American Society of Hematology

Mapping the epitope for the anti-vWf monoclonal antibody, 6G1 Mapping the epitope for the anti-vWf monoclonal antibody, 6G1.Inhibition of binding of 125I-labeled monoclonal antibody 6G1 (final concentration, 1 μg/mL) to immobilized vWf by synthetic peptides (final concentration, 100 μM) in 30 minutes at 22°C. Mapping the epitope for the anti-vWf monoclonal antibody, 6G1.Inhibition of binding of 125I-labeled monoclonal antibody 6G1 (final concentration, 1 μg/mL) to immobilized vWf by synthetic peptides (final concentration, 100 μM) in 30 minutes at 22°C. Inhibition is expressed relative to maximal specific binding in the absence of peptide. Results are the mean of quadruplicate determinations, with standard deviation from the mean of ≤ 5%. Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology

Epitopes for anti-vWf monoclonal antibodies Epitopes for anti-vWf monoclonal antibodies.Structure of the von Willebrand factor A1 domain based on the x-ray crystal coordinates25 with highlighted sequences representing epitopes for the anti-39/34-kd vWf fragment monoclonal antibodies determined using ... Epitopes for anti-vWf monoclonal antibodies.Structure of the von Willebrand factor A1 domain based on the x-ray crystal coordinates25 with highlighted sequences representing epitopes for the anti-39/34-kd vWf fragment monoclonal antibodies determined using synthetic peptides. Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology

Cross blocking of anti-vWf monoclonal antibodies Cross blocking of anti-vWf monoclonal antibodies.Binding of 125I-labeled monoclonal antibodies (final concentration, 1 μg/mL) to immobilized native vWf (A-D) or 39/34-kd vWf fragment (E-F) in 30 minutes at 22°C. Cross blocking of anti-vWf monoclonal antibodies.Binding of 125I-labeled monoclonal antibodies (final concentration, 1 μg/mL) to immobilized native vWf (A-D) or 39/34-kd vWf fragment (E-F) in 30 minutes at 22°C. Unlabeled antibodies were at a final concentration of 100 μg/mL. Specific binding in the presence of blocking antibodies was expressed as a percentage of maximal specific binding measured in the absence of blocking antibody. Panel, monoclonal antibody: A, 2C9; B, 5D2; C, 6G1; D, CR1; E, 6G1; F, CR11. Results are the mean of triplicate determinations, with standard deviation from the mean of ≤ 5%. Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology

Effect of anti-vWf monoclonal antibodies on vWf binding to platelets Effect of anti-vWf monoclonal antibodies on vWf binding to platelets.Antibodies (final concentration, 50 μg/mL) were included in assays measuring binding of 125I-labeled vWf (1 μg/mL) to GP Ib-IX-V on washed platelets induced by (A) botrocetin (25 μg/mL), (... Effect of anti-vWf monoclonal antibodies on vWf binding to platelets.Antibodies (final concentration, 50 μg/mL) were included in assays measuring binding of 125I-labeled vWf (1 μg/mL) to GP Ib-IX-V on washed platelets induced by (A) botrocetin (25 μg/mL), (B) jaracetin (25 μg/mL), or (C) ristocetin (1 mg/mL). Antibodies were preincubated with vWf for 5 minutes at 22°C before the addition of washed platelets (final concentration, 5 × 108/mL). Specific binding was expressed as a percentage of maximal specific binding measured in the absence of antibody. Nonspecific binding was assessed in the presence of 50 μg/mL of the inhibitory anti-GP Ibα monoclonal antibody, AK2. Results are the mean of quadruplicate determinations, with standard deviation from the mean of ≤ 5%. Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology

Effect of anti-vWf monoclonal antibodies on vWf binding to botrocetin or jaracetin.Antibodies (final concentration, 50 μg/mL) were included in assays measuring binding of 125I-labeled vWf (1 μg/mL) to (A) botrocetin-coated beads or (B) jaracetin-coated bead... Effect of anti-vWf monoclonal antibodies on vWf binding to botrocetin or jaracetin.Antibodies (final concentration, 50 μg/mL) were included in assays measuring binding of 125I-labeled vWf (1 μg/mL) to (A) botrocetin-coated beads or (B) jaracetin-coated beads for 30 minutes at 22°C. Specific binding was expressed as a percentage of maximal specific binding measured in the absence of antibody. Nonspecific binding was assessed in the presence of a 100-fold excess of unlabeled vWf. Results are the mean of triplicate determinations, with standard deviation from the mean of ≤ 5%. Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology

Effect of the anti-vWf monoclonal antibody 6G1 on binding of the 39/34-kd vWf fragment to platelets.(A) Specific binding of 125I-labeled 39/34-kd vWf fragment (final concentration, 1 μg/mL) to washed platelets (final concentration, 5 × 108/mL) in the presen... Effect of the anti-vWf monoclonal antibody 6G1 on binding of the 39/34-kd vWf fragment to platelets.(A) Specific binding of 125I-labeled 39/34-kd vWf fragment (final concentration, 1 μg/mL) to washed platelets (final concentration, 5 × 108/mL) in the presence of 6G1 in 30 minutes at 22°C. Nonspecific binding was determined in the absence of 6G1 (see Materials and Methods). (B) Specific binding of125I-labeled 39/34-kd vWf fragment (final concentration, 1 μg/mL) to platelets in the presence of botrocetin and either the absence (squares) or presence (circles) of 6G1 (final concentration, 50 μg/mL). Mariagrazia De Luca et al. Blood 2000;95:164-172 ©2000 by American Society of Hematology