Formation of pro-VWF heterodimers.

(A) Schematic structure of VWF constructs Cys1149Arg and dA13. Selected structural domains of VWF are labeled (A1, A2, A3, D3). The hatched and labeled segment of Cys1149Arg represents the substitution of Cys1149 to Arg in the D3 domain. Numbers below the structures indicate the positions of amino acid residues numbered from the initiation codon of pre-pro-VWF. In dA13, Gly-Ala replaces amino acid residues 1353-1458, and Ala-Gly replaces amino acid residues 1686-1872. (B) Analysis of VWF dimers by SDS-PAGE; 293T cells were transfected with plasmids encoding Cys1149Arg and dA13 as indicated. Cell lysates (L) and conditioned medium (S) were collected and immunoprecipitated with polyclonal anti-VWF antibody 082 (anti-VWF) or monoclonal anti-A1 domain antibody B710 (anti-A1). Samples were analyzed by SDS-PAGE on a 5% polyacrylamide gel under reducing conditions, and VWF was detected by Western blotting. The positions of intracellular pro-VWF and mature VWF subunits are indicated by brackets. The masses of the subunits are about 350 kd (pro-VWF Cys1149Arg), about 310 kd (pro-VWF dA13), about 250 kd (mature Cys1149Arg), and about 210 kd (mature dA13).