Assembly of Multimeric von Willebrand Factor Directs Sorting of P-Selectin

Abstract

Abstract-We designed a model system to study the role of von Willebrand factor (vWF) in the sorting of P-selectin and the biogenesis of Weibel-Palade body (WPB)-like organelles. For that purpose, a human epithelial cell line (T24) that synthesizes P-selectin mRNA, but which is devoid of vWF mRNA synthesis and storage organelles, was transfected with full-length vWF cDNA or a deletion mutant thereof. Stable transfectants of T24 with full-length vWF cDNA revealed the generation of WPB-like organelles as demonstrated by colocalization of vWF and P-selectin with double-labeling immunofluorescence. In contrast, T24 cells transfected with vWF delD'D3 cDNA, encoding a mutant that is unable to form vWF multimers, displayed only perinuclear vWF staining, whereas no indication was found for the presence of WPB-like organelles. The contents of the organelles in full-length vWF cDNA-transfected T24 cells were released on activation of the protein kinase C pathway, similar to the situation with genuine endothelial cells. The expression of vWF did not affect the biosynthesis of P-selectin, as deduced from the observation that untransfected and vWF cDNA-transfected T24 cells contained the same amount of P-selectin mRNA. We propose that the biosynthesis of multimeric vWF directs the generation of WPB-like organelles, as evidenced by the sequestering and anchoring of P-selectin into these storage granules. Key Words: von Willebrand factor Ⅲ P-selectin Ⅲ Weibel-Palade bodies Ⅲ protein sorting M ultimeric von Willebrand factor (vWF) is an obligatory glycoprotein for the formation of a hemostatic plug. It connects the platelet to the subendothelium that is exposed on injury of the vessel wall. vWF is synthesized by megakaryocytes and vascular endothelial cells. Endothelial cells display 2 secretory routes for newly synthesized vWF, namely, a constitutive and a regulated pathway. 1 Constitutively secreted vWF is rapidly transported in secretory vesicles toward the plasma membrane. This material consists of vWF dimers and low-molecular-weight multimers. Another portion of the newly synthesized vWF is encountered in specific storage organelles, denoted Weibel-Palade bodies (WPBs). These organelles fuse with the plasma membrane on activation of the protein kinase C signal transduction pathway, followed by release of their contents (regulated secretion). 2,3 vWF routed by this pathway consists of high-molecular-weight multimers that are Ϸ2 orders of magnitude more effective in mediating adhesion of platelets to the injured vessel wall than are vWF dimers or low-molecular-weight multimers. 4 The WPBs are considered to be endothelial cell-specific, rod-shaped, electron-dense storage organelles. 5 Apart from vWF multimers and the vWF propolypeptide, 6 -8 the WPB contains a number of other proteins, namely CD63, a transmembrane glycoprotein that is also present in lysosomal membranes of many cell types 9,10 ; interleukin-8 11 ; tissue plasminogen activator 12 ; and the transmembrane receptor P-selectin (also called GMP-140 or PADGEM). 13,14...

The role of the D1 domain of the von Willebrand factor propeptide in multimerization of VWF

“…Hence, even though the mutation in the propeptide rendered the region incapable of its key chaperone function, it does not preclude interactions necessary for granular storage. While other studies have demonstrated multimerization of VWF to be necessary for granular storage, 37,38 our results demonstrate that VWF multimerization is not a prerequisite for storage. Rather, our model of VWF storage requires intact propeptide and structurally competent VWF that can associate noncovalently with VWFpp to cotraffick to storage granules.…”

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

“…In contrast, transfection of P-selectin cDNA leads to its storage in the endogenous granules of the transfected cell lines (28,29). Even though this result indicates that P-selectin contains an independent sorting signal now identified in the cytoplasmic tail (30), it also indicates that P-selectin needs some pre-existing storage granules to be directed to, as recently confirmed in transfected cells (31). Immunolocalization of P-selectin with the use of confocal microscopy showed that in resting vWf Ϫ͞Ϫ endothelial cells, P-selectin is found in a reduced number of small vesicles, compared with vWf ϩ͞ϩ cells (Fig.…”

Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

“…25,36 A similar degradation of P-selectin was demonstrated in the T24 human epithelial cell line that does not synthesize VWF. 29 We were unable to definitively identify the P-selectincontaining granules as lysosomes due to lack of availability of antibodies cross-reacting with canine lysosomal proteins. However, the degradation of P-selectin in the absence of a regulated secretory pathway is well-defined as previously described.…”