Kozak sequence polymorphism in the GP Ib gene and in vitro expression of the polymorphic variants. (A) The GP Ib sequence surrounding the start codon and the location of the polymorphism aligned with the consensus sequence determined for these regions by Kozak.15 (B) Expression of GP Ib in CHO cells transfected with GP Ib cDNAs containing either C or T at position −5. The cells were transfected with either plasmid alone or with the same quantity of an equal mixture of the 2 plasmids. Expression of GP Ib on the cell surface was evaluated by flow cytometry after staining the cells with monoclonal antibody WM23 and a FITC-conjugated secondary antibody. Expression levels were determined by measuring the mean fluorescence of the whole cell population and are expressed as percentages of the expression obtained for the more common −5T variant. The increased surface levels of GP Ib in the cells transfected with the −5C plasmid alone or with a combination of the −5C and −5T plasmids as compared with the cells transfected only with the −5T plasmid were both statistically significant (P= .05 and P < .003, respectively, Student’s two-tail t-test, n = 5). (C) Coexpression of GP Ib variants with green fluorescent protein. Plasmids encoding the GP Ib T and C variants were cotransfected with a plasmid containing the GFP cDNA. GP Ib was detected with WM23 followed by a phycoerythrin-conjugated secondary antibody. Values are expressed as the ratio of mean fluorescence in FL-2 (PE) to the mean fluorescence in FL-1 (GFP). The expression of the C variant was again significantly higher than that of the T variant (P = .02, n = 4, Student’s two-tail t-test).