Purification and Characterization of Human vWF

Human vWF was purified as described previously (Booth et al., 1984 (link)). Human α-thrombin was purchased from Enzyme Research Laboratories. RGDS peptide was from Bachem, and ristocetin was from Sigma-Aldrich. Monoclonal antibodies against integrin α_vβ₃ (anti-VNR1) (O'Toole et al., 1990 (link)), against integrin β₃ (Mab15) (Frelinger et al., 1990 (link)), and against the integrin α_IIbβ₃ complex (D57 and 2G12) (Frojmovic et al., 1991 (link)) were provided by M. Ginsberg (The Scripps Research Institute, La Jolla, CA) and V. Woods (University of California, San Diego, CA). Monoclonal antibodies against GPIb (SZ2) (Ruan et al., 1987 (link)) were a gift from C. Ruan (Jiangsu Institute of Hematology, Suzhou, China). Calpain cleavage site–specific antibodies were generated by immunizing rabbits with pentapeptides (AKWDT for Ab741, NNPLY for Ab747, ATSTF for Ab754, TNITY for Ab759, and TYRGT for Ab762) as described previously (Du et al., 1995 (link)). Oregon green 488–conjugated human fibrinogen, Alexa Fluor 488–conjugated goat anti–mouse IgG, and Alexa Fluor 546–conjugated goat anti–rabbit IgG were from Molecular Probes. Integrin α_IIb and β₃ cDNA clones in pCDM8 vector were provided by M. Ginsberg, and a mutant β₃ cDNA clone bearing the Y⁷⁵⁹A substitution was a gift from J. Ylänne (University of Helsinki, Helsinki, Finland). Cell lines expressing GPIb-IX complex (1b9), integrin α_IIbβ₃ (2b3a), or both GPIb-IX and α_IIbβ₃ (123) were described previously (Gu et al., 1999 (link)).