Shielding the front-strand β3 of the von Willebrand factor A1 domain inhibits its binding to platelet glycoprotein Ibα by Arnaud Bonnefoy, Hiroshi Yamamoto, Chantal Thys, Morikazu Kito, Jos Vermylen, and Marc F. Hoylaerts Blood Volume 101(4):1375-1383 February 15, 2003 ©2003 by American Society of Hematology

Purification of the wt-A1 fusion protein Purification of the wt-A1 fusion protein.Wild-type A1, prepurified with glutathione-Sepharose beads, was dialyzed (24 hours, 4°C) against 20 mM Tris-HCl, pH 8.0 buffer containing 50 mM NaCl and loaded onto a Q-Sepharose column. Purification of the wt-A1 fusion protein.Wild-type A1, prepurified with glutathione-Sepharose beads, was dialyzed (24 hours, 4°C) against 20 mM Tris-HCl, pH 8.0 buffer containing 50 mM NaCl and loaded onto a Q-Sepharose column. (A) wt-A1 was eluted with a 50- to 500-mM NaCl gradient and collected as indicated. (B) SDS-PAGE and silver staining analysis of the collected wt-A1. Lane 1: prepurified wt-A1 after elution from the glutathione-Sepharose beads; lane 2: wt-A1 eluted from the Q-Sepharose. The fusion protein exhibits an apparent Mr of 49 kDa, which increases to 56 kDa after reduction with 100 mM DTE (lane 3). Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Functional evaluation of the wt-A1 in flow-dependent platelet tethering.(A) Glass coverslips coated with purified wt-A1 (300 μg/mL) were untreated (control) or treated for 30 minutes at 37°C with 1mM CuSO4 or 10 mM DTE as indicated and were perfused with pl... Functional evaluation of the wt-A1 in flow-dependent platelet tethering.(A) Glass coverslips coated with purified wt-A1 (300 μg/mL) were untreated (control) or treated for 30 minutes at 37°C with 1mM CuSO4 or 10 mM DTE as indicated and were perfused with platelets in reconstituted blood at a wall shear rate of 1500 seconds−1. After 3 minutes, interacting platelets were counted. Results are expressed as percentages of interacting platelets relative to the nontreated wt-A1 (*P < .01). (B) Images (205 × 330 μm) show the flow-dependent tethering of fluorescently labeled platelets on glass coverslips coated with wt-A1 (300 μg/mL) or native VWF (50 μg/mL) at 200 seconds−1(i,vi), 1000 seconds−1 (ii,vii), and 1500 seconds−1 (iii,viii) and the inhibitory effect of 30 μg/mL G19H10 (iv,ix) and 20 μg/mL AJvW-2 (v,x) at 1500 seconds−1. Corresponding platelet counts ± SDs are provided in subpanels. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Ribbon representation of the human VWF A1 domain and location of the mutated residues.The tridimensional structure front view of the A1 domain, based on the 3-dimensional coordinates by Celikel et al,12 is shown with the spatial distribution of all 9 mutate... Ribbon representation of the human VWF A1 domain and location of the mutated residues.The tridimensional structure front view of the A1 domain, based on the 3-dimensional coordinates by Celikel et al,12 is shown with the spatial distribution of all 9 mutated residues, displayed in ball-and-stick representation. Critical secondary structural elements were colored for clarity. For space considerations, single-letter codes were used for amino acids. The helix α3 is shown in red; strands β3 and β4 are shown in yellow; β-turn sequences are displayed in violet. Pictures were created with RasMol v2.6 (Glaxo Research and Development, London, United Kingdom). Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Binding of AJvW-2 to wt-A1 and mutants Binding of AJvW-2 to wt-A1 and mutants.Plateau values for the binding of saturating concentrations of AJvW-2 (A) and the conformation-dependent anti-VWF A1 antibodies, poly–anti-VWF, A21H3, and A7C6 (B), to microtiter plate-coated A1 mutants (2 μg/mL), were... Binding of AJvW-2 to wt-A1 and mutants.Plateau values for the binding of saturating concentrations of AJvW-2 (A) and the conformation-dependent anti-VWF A1 antibodies, poly–anti-VWF, A21H3, and A7C6 (B), to microtiter plate-coated A1 mutants (2 μg/mL), were determined by ELISA. Results are expressed as the mean percentages ± SDs of triplicate determinations (*P < .01), relative to binding to wt-A1. For space considerations, single-letter codes were used for amino acids. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Heparin binding.(A) Binding of wt-A1 and mutants to heparin. Heparin binding.(A) Binding of wt-A1 and mutants to heparin. Fusion proteins (50 μg/mL) were incubated with heparin-Sepharose beads for 2 hours at 37°C. The bound fraction of the proteins was calculated from the concentration of unbound material measured after centrifugation of the beads. Results are expressed as mean percentages ± SDs of protein bound to the beads for triplicate determinations (*P < .01). A GST/human VWF A3 domain fusion protein was tested as a control for nonspecific binding to heparin-Sepharose beads. For space considerations, single-letter codes were used for amino acids. (B) Inhibition of wt-A1 binding to immobilized AJvW-2 by soluble unfractionated heparin during perfusions in a BIACore 1000 instrument. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Inhibition of mediator-induced GPIb-VWF interaction by wt-A1 and mutants.(A) Botrocetin assay. Inhibition of mediator-induced GPIb-VWF interaction by wt-A1 and mutants.(A) Botrocetin assay. Reciprocal IC50 values for the competitive binding of the indicated A1 mutants, in comparison with wt-A1, during the binding of 0.5 μg/mL VWF to microtiter plate-coated glycocalicin, mediated by 2 μg/mL botrocetin. Results are expressed as relative inhibition (1/IC50). (B) Inhibition of RIPA by wt-A1 and mutants conducted in PRP. Means ± SDs of triplicate determinations (*P < .05; **P < .01). For space considerations, single-letter codes were used for amino acids. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Shear-dependent interaction of platelets with immobilized wt-A1 and mutants.Reconstituted blood containing fluorescently labeled platelets (10 000/μL) was perfused over wt-A1 or mutant fusion proteins, immobilized onto glass coverslips coated at 300 μg/mL i... Shear-dependent interaction of platelets with immobilized wt-A1 and mutants.Reconstituted blood containing fluorescently labeled platelets (10 000/μL) was perfused over wt-A1 or mutant fusion proteins, immobilized onto glass coverslips coated at 300 μg/mL in a flow chamber (see “Materials and methods”). (A) Number of platelets translocating over the A1 domain mutants per field (205 × 330 μm), after 3 minutes of perfusion, at 200 seconds−1 (■), 1000 seconds−1 (▨), and 1500 seconds−1 (▪). Data are expressed as the means ± SDs of at least 3 separate assays (*P < .05). (B) Median velocities of platelet translocation on immobilized wt-A1 and mutants were determined from real-time movies by measuring the distance traveled by single platelets during 1 second of perfusion at 1500 seconds−1. For each mutant, the velocity of 50 individual platelets was measured in one typical experiment. Ratios of the platelet count to the median velocity of platelets interacting with the mutants are represented as black bars. Median platelet velocity (μm/sec) is indicated in parentheses. ♦ indicates a contact time that was too short to allow velocity measurement. For space considerations, single-letter codes were used for amino acids. (C) Platelets in reconstituted blood were perfused at 1500 seconds−1 over wt-A1 (○) and over Ala587Thr-A1 (●) in the presence of increasing concentrations of AJvW-2. After 3 minutes of perfusion, interacting platelets were counted. Results are expressed as the percentages of platelet numbers in the absence of AJvW-2. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology

Location of amino acid mutations impairing GPIbα, heparin, and AJvW-2 binding to VWF A1.Space-filling, 3-dimensional representation of the human VWF A1 domain. Location of amino acid mutations impairing GPIbα, heparin, and AJvW-2 binding to VWF A1.Space-filling, 3-dimensional representation of the human VWF A1 domain. (A) Front (left) and back (right) view of the A1 domain. Surface residues of mutations previously shown to abolish shear-dependent platelet interactions with the recombinant VWF A1 domain16 19 20 or to cause VWD type 2M (ISTH SSC VWF database: www.shef.ac.uk/vwf/) are indicated in orange. Location of buried residues is indicated by dotted lines. Amino acid substitutions detected in type 2M VWD patients are underlined. For space considerations, single-letter codes were used for amino acids. (B) Surface residues of mutations in the present study that impaired the shear-dependent interaction of the VWF A1 domain with platelet GPIbα are indicated in red. The location of the buried residue Ala618 is indicated by a dotted line. (C) Residues of mutations in the present study that impaired heparin and AJvW-2 recognition are indicated in blue. Amino acid Ala587, whose mutation only impairs AJvW-2 binding, is indicated in cyan. Pictures were created with RasMol v2.6. Arnaud Bonnefoy et al. Blood 2003;101:1375-1383 ©2003 by American Society of Hematology