In vitro transfection of full-length Vwf cDNA constructs and mutagenized variants. (A) Reference full-length Vwf cDNA is shown with the Myc-tag, relevant restriction enzyme sites, and SNPs noted (lines below construct). Larger representations of the NotI/EcoRI fragment and the region flanking R2657Q are shown below in CASA/RkJ and A/J VWF cDNA full-length constructs (no. 1 and no. 2, respectively) and mutagenized variants (nos. 3-10). In the individual constructs, black boxes represent CASA/RkJ sequence and gray boxes represent A/J sequence. The various chimeras are depicted as mixed gray and black, with corresponding amino acid designations below. VWF levels in conditioned media from COS-1 transfections are shown at right, represented as a percentage of CASA/RkJ transfection levels ± SD. (B) VWF levels in conditioned media following transfection of COS-1 cells with full-length A/J (pA) and CASA/RkJ (pC) cDNA. A strain-specific increase (1.43-fold) is noted. Error bars represent ± 95% confidence interval. *P < .001. (C) Expression of Vwf plasmids are summarized by pooling the results for all transfections by the sequence differences at each amino acid position. For example, in the T20A columns, the gray bar summarizes VWF expression in the 5 constructs which have the A/J-specific amino acid at position 20 (T), and the black bar summarizes VWF levels in the 5 constructs which have the CASA/RkJ amino acid (A) at the equivalent location. A similar pattern is repeated for each amino acid. An amino acid–specific increase (1.32-fold) is noted. Error bars represent ± 95% confidence interval. †P < .001.