A novel type 1 von Willebrand disease causing mutation (S49R) in the von Willebrand factor pro-peptide that affects von Willebrand factor multimerization and secretion

Abstract number: P1663

Messenger^* S. L., Abuzenadah^* A. M., Gursel† T., Ingerslev‡ J., Peake^* I. R., Daly^* M. E., Goodeve^* A. C.

†Medical School of Gazi University, Turkey; ‡University Hospital of Skejby, Denmark ^*University of Sheffield, UK;

Type 1 von Willebrand disease (VWD) is characterized by a partial quantitative deficiency of plasma von Willebrand factor (VWF), with no evidence of qualitative defects. In a previous study of 25 Turkish families with VWD, we identified four families where type 1 VWD was associated with missense mutations within the DNA encoding the VWF propeptide. One of these, a novel nucleotide transversion 147C > A, was found in the heterozygous form in the index case and father of family XXXIX which predicts a substitution of S49R in the VWF propeptide. The index case and both parents had mild bleeding symptoms and similar levels of VWF:Ag (0.58 IU mL^-1 in the index case, 0.60 and 0.63 IU mL^-1 in father and mother, respectively). This study analyzed the effect of the S49R mutation on the secretion and structure of VWF expressed in vitro. Recombinant VWF encoding the S49R mutation was expressed in COS-7 cells. ELISA analysis of transfected cell lysates and culture media indicated that there was no significant difference in the total amount (secreted + intracellular) of VWF expressed from the wild-type compared to the mutant plasmid. There was however, a significant difference in the proportions of VWF secreted into the culture media. Thus, the rVWFS49R variant was secreted at 139% of the level of wild-type VWF (P = 0.0259). Compared to transfections with just wild-type rVWF, increased secretion of VWF was also observed when rVWFS49R was cotransfected with increasing quantities of wild-type rVWF. Multimer analysis suggests that COS-7 cells transfected with rVWFS49R fail to secrete high-molecular weight multimers with the same efficiency as those transfected with wild-type rVWF. This is in contrast to the in vivo situation where the plasma multimer pattern from the index case and father showed no significant difference from that of normal plasma. It is possible that the S49R mutation affects the trafficking of VWF from the endoplasmic reticulum through the Golgi and trans-Golgi network to the Weibel-Palade bodies of endothelial cells. Repeating transfection experiments in a cell line that mimics the in vivo pathway of VWF synthesis, storage and secretion may give a better indication of how the S49R mutation affects the structure and function of VWF.