1. Two novel mutations in the αIIb calcium-binding domains identify hydrophobic regions essential for αIIbβ3 biogenesis
by W. Beau Mitchell, Ji Hong Li, Fiza Singh, Alan D. Michelson, James Bussel, Barry S. Coller, and Deborah L. French
Blood
Volume 101(6):
March 15, 2003
©2003 by American Society of Hematology

2. Immunoblots of platelet αIIb and β3 for patients M and C
Immunoblots of platelet αIIb and β3 for patients M and C.Platelets were solubilized in SDS, electrophoresed into a 7% polyacrylamide gel under reduced (αIIb) and nonreduced (β3) conditions, and electrotransferred to PVDF membranes.
Immunoblots of platelet αIIb and β3 for patients M and C.Platelets were solubilized in SDS, electrophoresed into a 7% polyacrylamide gel under reduced (αIIb) and nonreduced (β3) conditions, and electrotransferred to PVDF membranes. Equal amounts of protein were electrophoresed as judged by the staining of the gel after electrophoresis for platelet myosin heavy chain (Mr about 200 000). Membranes analyzed for αIIb were incubated with the mAb PMI-1, and membranes analyzed for β3 were incubated with the mAb 7H2.28 (A) Samples from patient M and his mother and father. (B) Samples from patient C and his 2 siblings.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

3. Ribbon diagram of the αV subunit showing the relative locations of the αIIb mutations of patients M and C.The αIIb and αV subunits share 40% sequence homology.
Ribbon diagram of the αV subunit showing the relative locations of the αIIb mutations of patients M and C.The αIIb and αV subunits share 40% sequence homology. This ribbon diagram of αV shows the relative positions of the 4 αIIb mutations, which are indicated by spheres (missense mutation) or a block (nonsense mutation) centered on the amino acid α-carbons (red and underlined for patient M, black for patient C). The β-propeller blades are designated W1-W7; the calcium atoms are shown in yellow. Three mutations lie within the β-propeller domain, blades W5 and W6, and the fourth is within the calf-1 domain. This image was generated with MOLMOL36 from the PDB file 1JV2.13
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Immunoblot for αIIb of 293T cells transfected with mutant αIIbβ3 receptors.Cells were solubilized in Triton X-100, electrophoresed into a 7% polyacrylamide gel under reduced conditions, and electrotransferred to PVDF membranes.
Immunoblot for αIIb of 293T cells transfected with mutant αIIbβ3 receptors.Cells were solubilized in Triton X-100, electrophoresed into a 7% polyacrylamide gel under reduced conditions, and electrotransferred to PVDF membranes. Equal amounts of protein (150 μg), as determined by the BCA assay, were loaded per lane. The membrane was analyzed for αIIb using the mAb PMI-1. (A) Schematic diagram showing the processing and molecular weights of pro-αIIb in the endoplasmic reticulum and of mature αIIb in the Golgi. (B) Immunoblot of αIIb showing pro-αIIb and mature forms of control and mutant subunits.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Pulse-chase analysis of transfected cells expressing control and mutant αIIbβ3 receptors.Cells were cotransfected with cDNA constructs expressing control or mutant (Val298Phe and Ile374Thr) αIIb and control β3 subunits for 36 hours.
Pulse-chase analysis of transfected cells expressing control and mutant αIIbβ3 receptors.Cells were cotransfected with cDNA constructs expressing control or mutant (Val298Phe and Ile374Thr) αIIb and control β3 subunits for 36 hours. Following preincubation in methionine/cysteine-free medium, cells were labeled with 35S-methionine/cysteine (300 μCi/well [11.1 MBq]) containing medium for 15 minutes and chased in medium containing methionine and cysteine (1 mg/mL each). Cells were harvested at the indicated time points. Using equivalent TCA precipitable counts (about 1.5 × 106 counts/sample), cell lysates were immunoprecipitated using a combination of αIIb-specific mAbs, B1B and M-148 (Santa Cruz Biotechnology; 4 μg each/sample) that recognize pro-αIIb and mature αIIb subunits. Samples were electrophoresed under reduced conditions and dried gels were exposed to film. Bands representing pro-αIIb, mature αIIb, and β3 are shown by arrows.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Effect of replacing residues Val298 and Ile374 with different amino acids on the maturation and processing of pro-αIIb to mature αIIb.Cells were cotransfected with cDNA constructs expressing control or mutant αIIb and control β3 subunits for 36 hours.
Effect of replacing residues Val298 and Ile374 with different amino acids on the maturation and processing of pro-αIIb to mature αIIb.Cells were cotransfected with cDNA constructs expressing control or mutant αIIb and control β3 subunits for 36 hours. Whole cell lysates were prepared and immunoprecipitated with antibodies to αIIb (B1B5 and M-148). Immunoblots were performed with another antibody to αIIb (PMI-1). Bands representing pro-αIIb and mature αIIb are shown by arrows. (A) Schematic model of a calcium-binding loop showing residues numbered −3 to −1 and The amino acid sequences of the second and third calcium-binding domains of αIIb are shown. The Val298 and Ile374 residues are underlined and the amino acids in the putative N-linked glycosylation site created by the Ile374Thr mutation are bracketed. The amino acid substitutions for residues Val298, Ile374, and Asn372 are shown in the boxes. (B) Immunoblot of cells expressing amino acid substitutions for residue Val298. (C) Immunoblot of cells expressing amino acid substitutions for residue Ile374. (D) Immunoblot of cells expressing amino acid substitutions for Ile374 and Asn372.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

7. Effect of replacing αIIb residues Val298 and Ile374 with different amino acids on the surface expression of αIIbβ3 receptors.Flow cytometry was performed on 293T cells cotransfected with αIIb constructs containing control, Val298 and Ile374, or substituted ...
Effect of replacing αIIb residues Val298 and Ile374 with different amino acids on the surface expression of αIIbβ3 receptors.Flow cytometry was performed on 293T cells cotransfected with αIIb constructs containing control, Val298 and Ile374, or substituted (Val298Phe, Val298Leu, Ile374Thr, Ile374Leu, Ile374Val) amino acid residues and a control β3 construct. Cells were incubated with the complex-dependent mAb, 10E5, then with FITC-labeled secondary antibody (shaded) and flow cytometry was performed. Background controls were cells incubated with secondary antibody alone (unshaded). Percentages indicate percent positive of gated cells.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

8. Effect of αIIb mutations on electrostatic potential
Effect of αIIb mutations on electrostatic potential.The electrostatic potential was calculated at each of the 4 calcium positions in the αIIb β-propeller model.
Effect of αIIb mutations on electrostatic potential.The electrostatic potential was calculated at each of the 4 calcium positions in the αIIb β-propeller model. The Glanzmann thrombasthenia mutations Val298Phe and Ile374Thr, and the experimental mutations Val298Ala, Val298Leu, Ile374Leu, and Ile374Val were then inserted into the model and the electrostatic potentials were calculated. The relative change in electrostatic potential resulting from each mutation is depicted for each calcium position as a positive or negative bar. The normal electrostatic potential at each calcium position is arbitrarily set to 0. A positive change in electrostatic potential of more than 2 (the charge of calcium) would be expected to decrease the ability of calcium to bind, whereas a negative change in electrostatic potential would increase the ability of calcium to bind.
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

9. Mutations within the αIIb calcium-binding domains
Mutations within the αIIb calcium-binding domains.(Left) Schematic of the β-propeller domain of αIIb and αv showing the central cage motif.
Mutations within the αIIb calcium-binding domains.(Left) Schematic of the β-propeller domain of αIIb and αv showing the central cage motif. The blades are labeled W1 through W7. The cage motif comprises 2 concentric rings of predominantly aromatic residues, which line the upper, inner rim of the propeller core. Each blade contributes 2 residues to the cage structure, represented here by hexagons. (Right) Schematic of one blade of the αIIb β-propeller derived from molecular modeling, showing the relative locations of 7 Glanzmann thrombasthenia mutations that lie within blades W4-W7. The blade is viewed from the side, with the 2 cage residues indicated and the calcium-binding loop at the bottom. Starting from the cage residues, the 4 antiparallel β-strands of the blade form the legs of a “W.” Seven mutations reported to be in or near the calcium-binding domains (including the 2 from this study, underlined) are shown in their relative positions: Gly273Asp from W4; Val298Phe, Glu324Lys, and Arg327His from W5; Ile374Thr from W6; and Gly418Asp and deletion Val425/Asp426 from W7.7-11
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology

10. Model of the αIIb β-propeller showing calcium-binding mutations and cage residues.A model of the αIIb β-propeller was generated as described in “Patients, materials, and methods.” (A) Top view of the propeller showing the 7 blades (W1-W7), the 4 calcium ion...
Model of the αIIb β-propeller showing calcium-binding mutations and cage residues.A model of the αIIb β-propeller was generated as described in “Patients, materials, and methods.” (A) Top view of the propeller showing the 7 blades (W1-W7), the 4 calcium ions (yellow), and the locations of 7 reported calcium-binding domain mutations (black and cyan spheres; mutations identified in this study are cyan). (B) Side view of the propeller with the mutations labeled. (C) Blades W4-W7 are viewed from the side. The 2 critical cage residues are displayed. A sphere centered on the amino acid α-carbon identifies the positions of each calcium-binding domain mutation. This figure was generated with MOLMOL.36
W. Beau Mitchell et al. Blood 2003;101:
©2003 by American Society of Hematology