Increased expression of wild-type VWF corrects the biosynthetic and functional defects caused by the M1304R mutation. cDNAs for wild-type and mutant VWF were cotransfected at different ratios into HEK293T cells, and biosynthetic and functional parameters were examined. (A) VWF concentration in the condition medium of transfected cells. As the ratio of wild-type to mutant cDNA increased, VWF concentration in the condition medium also increased. (B) Recombinant VWF multimer patterns. The mutant VWF multimers lack the medium- and high-molecular-weight multimers (lane 1). As the percentage of wild-type cDNA increased, the multimer size of the recombinant VWF also increased (lanes 2-4). (C) Detection of mutant and wild-type VWF in the multimers. The recombinant M1304R VWF (no tag) is detected by antibody AVW-5. The wild-type VWF with myc-His tags was coexpressed with the mutant. The presence of the myc-His tags disrupts the AVW5 epitope and the antibody can no longer recognize the wild-type VWF, which is detected by anti-myc antibody. Mutant VWF (red) was detected in all multimer bands when coexpressed with the wild-type (green) (lanes 2 and 3 in panel C). When the mutant cDNA composed only 25% of the transfected cDNA, the mutant protein was detected only in low-molecular-weight multimers (lane 4 in panel C). (D) Ristocetin-induced platelet binding. (E) Botrocetin-induced platelet binding. In both panels D and E, the M1304R mutation resulted in defective platelet binding, which tended to normalize with an increasing percentage of the wild-type cDNA in cotransfection.