Biogenesis and Exocytosis of Weibel-Palade Bodies Is Affected by Naturally Occurring Von Willebrand Disease Variants within the A1-A3 Domains of VWF
| Autor: | Karine M. Valentijn, Jan Voorberg, Dafna J. Groeneveld, Jeroen Eikenboom, Marjon J. Mourik, Richard J. Dirven, Jiong-Wei Wang, Pieter H. Reitsma |
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| Rok vydání: | 2012 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities medicine.medical_specialty biology Chemistry Immunology HEK 293 cells ER retention Cell Biology Hematology Transfection medicine.disease Biochemistry Cell biology Endocrinology Von Willebrand factor hemic and lymphatic diseases Internal medicine Von Willebrand disease medicine biology.protein Weibel–Palade body Secretion Platelet circulatory and respiratory physiology |
| Zdroj: | Blood. 120:1072-1072 |
| ISSN: | 1528-0020 0006-4971 |
| Popis: | Abstract 1072 Background: Von Willebrand Factor (VWF) is synthesized in endothelial cells and megakaryocytes and is either secreted constitutively into plasma or stored in specific organelles; Weibel-Palade bodies (WPB) in endothelial cells or α -granules in megakaryocytes and platelets. Release of von Willebrand factor from WPB in response to desmopressin (DDAVP), an agonist of WPB exocytosis, is clinically applied to raise VWF plasma levels in patients with von Willebrand disease (VWD). A subset of patients with VWD type 1, a quantitative defect, shows a reduced response to DDAVP. This reduced response suggests the absence of recruitable WPB. The VWF propeptide (D1-D2 domains) together with the D'D3 domains are necessary for the tubular assembly of VWF. The assembly of VWF into tubules drives WPB formation. Some variants in the VWF A1 to A3 domains have been linked to reduced DDAVP responsiveness. This suggests that determinants for WPB formation may reside outside the D1-D2-D'-D3 domains. We hypothesize that a reduced tendency to assemble into VWF tubules underlies the defective DDAVP response in VWD type 1 patients with mutations in the A domains of VWF. Methods: Human Embryonic Kidney (HEK) 293 cells were transiently transfected with plasmids containing full-length wild-type VWF or 6 naturally occurring VWF variants located throughout the A domains of VWF. The following mutations, originally identified in type 1 VWD patients, were studied: p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His (A1 domain), p.Tyr1584Cys, p.Arg1583Trp (A2 domain) and p.Val1822Gly (A3 domain). Medium and lysates of transfected cells were collected to measure basal VWF secretion. Transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the regulated VWF secretion. Confocal- and Transmission electron microscopy (TEM) were used to study the formation of WPB. Results: Cells transfected with WT-VWF or VWF p.Arg1583Trp formed numerous elongated pseudo-WPB as evidenced by confocal microscopy. p.Ser1285Pro, p.Leu1307Pro, p.Arg1374His, p.Tyr1584Cys and p.Val1822Gly were able to form these organelles, but in most cases the pseudo-WPB were shorter and more round compared to those formed by WT-VWF. Retention of VWF in the ER was present in about 50% of the cells expressing p.Leu1307Pro and 25–35% of the cells expressing p.Ser1285Pro and p.Val1822Gly as compared to 10% in WT-VWF. The ER retention in p.Arg1374His, p.Tyr1584Cys and p.Arg1583Trp variants was comparable with WT-VWF. Upon co-transfection with WT-VWF the defective elongation of pseudo-WPB was partly corrected. WPB formation was further studied using TEM. In WT-VWF transfected cells, elongated and electron dense pseudo-WPB were observed. p.Arg1583Trp showed cigar-shaped pseudo-WPB with typical VWF striations. Shorter and more round pseudo-WPB were found in cells expressing the VWF variants p.Ser1285Pro, p.Arg1374His, p.Tyr1584Cys. The p.Val1822Gly variant showed some elongated pseudo-WPB, although most of the structures were round. The round organelles are, however, recognizable as pseudo-WPB as they contain tubular structures indicating storage of VWF tubules. The p.Leu1307Pro and p.Val1822Gly showed reduced VWF basal secretion, even in the heterozygous state. Both mutations, together with p.Ser1285Pro, also showed impaired regulated secretion of VWF in both single and co-transfections with wt-VWF. Conclusion: Our data shows that naturally occurring VWD variants within the A domains of VWF can cause defects in both WPB formation and (regulated) secretion of VWF. This is in contrast with previous data which suggests that only the propeptide and D1'-A1 domain of VWF are essential for normal WPB formation. In our study however, we found that two mutations located within the A2 and A3 domain (p.Tyr1584Cys and p.Val1822Gly) also interfere with the formation of elongated WPB as evidenced by round storage organelles containing VWF tubules. Defects in WPB formation and regulated secretion of VWF may be the underlying cause of the poor response to DDAVP infusion seen in a subset of VWD type 1 patients. Disclosures: No relevant conflicts of interest to declare. |
| Databáze: | OpenAIRE |
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