Detailed Biosynthethic Analysis of the Y1584C and R924Q Von Willebrand Factor Variants.

Von Willebrand factor (VWF) is a multimeric that has a major role in hemostasis by serving as a carrier for FVIII and by mediating platelet adhesion to subendothelium in response to vascular injury. Vascular endothelium is the major site of VWF biosynthesis where it is synthesized as a 2813 amino acid pre-pro-VWF. Following translation, the signal sequence is removed and pro-VWF is dimerized and glycosylated in the endoplasmic reticulum (ER). Multimerization occurs in the Golgi apparatus (GA), and the VWF molecule is either secreted constitutively or stored in the newly formed Weibel-Palade bodies (WPB) and released following a defined stimulus. Quantitative or qualitative defects in the biosynthesis of the VWF molecule are associated with Type 1/Type 3 or Type 2 Von Willebrand disease (VWD), respectively. Most of the missense mutations identified in the VWF gene of patients with Type 1 or Type 3 VWD gene are associated with the ER retention and degradation. In order to elucidate the molecular pathology of Type 1 VWD, a Canadian Type 1 study has been initiated. Y1584C and R924Q have been identified as the most common alterations in the Canadian Type 1 VWD population with frequencies of 13 % (25/194) and 8.5 % (10/117), respectively. Previous in vitro expression studies with recombinant VWF revealed that Y1584C and R924Q result in significant intracellular retention. In this study we report the effect of Y1584C and R924Q on the biosynthesis and function of WP bodies. In order to analyze ER retention of recombinant VWF possessing one or the other missense changes, pulse chase experiments were performed in transfected Cos-7 cells and intracellular location of the recombinant VWF molecules was analyzed by immunofluorescence antibody staining in AtT-20 cells. Pulse chase experiments revealed that both types of recombinant VWF molecules are successfully transported to the GA from the ER. Immunofluorescence antibody staining revealed that both mutant forms of recombinant VWF have a similar subcellular localization to that of wild-type recombinant VWF and that they appear to be stored in pseudo-WPB. In addition to the utilization of the AtT-20 heterologous expression system, the intracellular location of native mutant VWF molecules was analyzed in blood outgrowth endothelial cells (BOECs) and human umbilical vein endothelial cells (HUVECs) with immunofluorescence antibody staining. BOECs were isolated from a patient who is a compound heterozygote for R924Q and R816W and HUVECs were prepared from a patient who is heterozygous for R924Q. In the R924Q heterozygous HUVECs, normal localization of VWF in WPBs was observed. In the compound heterozygous R924Q/R816W BOECs, both normal and some abnormal larger WPB structures were observed. In addition to these experiments, the ability of the intracellular pseudo-WPB in transfected AtT-20 cells to release recombinant 1584C or 924Q VWF upon treatment with a stimulant (BaCl₂) was quantified by ELISA. Pseudo-WPB formed by both types of recombinant VWF molecules were responsive to stimulation and there was no significant difference in the fold-increase in the amount of the secreted VWF between wild-type and mutant VWF constructs. In summary, although molecular basis of the abnormal WPB structures in the heterozygous R924Q/R816W BOECs is still unknown, these studies have demonstrated that neither Y1584C nor R924Q result in major defects in VWF biosynthesis, and that both mutant proteins are associated with the development of pseudo-WPBs that are normally responsive to secretagogues in a heterologous cell system.