Characterization of hGPIbα transgenic mice. (A) hGPIbα was immunoprecipitated from human platelets or hGPIbα^WT (WT) and hGPIbα^FW (FW) transgenic mouse platelet lysates using 10 μg/mL ALMA.12 mAb (hGPIbα) or an isotype-matched control mAb (immunoglobulin G control). The presence of filamin A was detected using pAb H-300 (Ai), and human GPIbα was immunoblotted with WM23 mAb (Ai-ii). Data shown are from 1 experiment representative of 3 performed. Substitution of Phe568 and Trp570 to alanine completely disrupts the interaction between GPIbα and filamin A but has no negative impact on 14.3.3ζ binding to GPIb (Aii). (B) Flow cytometric analysis of hGPIbα expression on the surface of hGPIbα^WT (black line) and hGPIbα^FW (gray line) platelets in whole blood samples (1:10 dilution) and stained with anti–human CD42b-PE mAb demonstrating that loss of filamin A binding has no effect on receptor surface expression. (C) Alexa Fluor 488-VWF binding to hGPIbα expressed on transgenic platelets was measured in the presence of 5 μg/mL botrocetin and 20 μg/mL Xia.B2 murine GPIbα blocking antibody. No differences were measured between hGPIbα^WT and hGPIbα^FW platelets over all concentrations of VWF (1-20 μg/mL).