WT and VWF variant expression and functionality from transfected HEK293T cells. (A) Constitutive expression was derived from unstimulated cells and release of VWF from pseudo-WPB was measured after stimulation with PMA. 100% indicates homozygous transfections; 50% indicates transfections in the mock heterozygous state where HEK293T cells were transfected with 50% WT vector and 50% mutant vector. Black significance stars without a bar below represent a significant difference in constitutive expression of the mutants compared with WT. Significance stars with a bar below indicate a significant increase in secretion between constitutive and stimulated HEK293T cells (N = 3; *P < .05; **P < .01; ***P < .001) The absence of VWF from the 100% mock, and restoration of normal VWF levels in the 50% mock, show that expression reductions across the other variables are in fact due to the respective mutant VWF. (B) Functionality of the VWF splice variants from expression in transiently transfected HEK293Tcells measured by VWF:RCo, FVIII:B ELISA, and collagen-binding ELISA. 100% indicates homozygous transfections; 50% indicates transfections in the mock heterozygous state where HEK293T cells were transfected with 50% WT vector and 50% mutant vector (N = 3). Significance stars compare the binding properties of the splicing mutants to WT VWF (*P < .05; **P < .01).