Store vWF Research Articles

Complement promotes endothelial von Willebrand factor and angiopoietin-2 release in obstructive sleep apnea.

Obstructive sleep apnea (OSA) is highly prevalent and triples vascular thromboembolic risk. Intermittent hypoxia (IH) during transient cessation of breathing in OSA impairs endothelial protection against complement. Complement activation stimulates the endothelial release of a pro-thrombotic von Willebrand factor (vWF). We investigated whether increased complement activity in OSA promotes the endothelial release of vWF and pro-inflammatory angiopoietin-2. We further investigated whether improving complement protection with statins reverses these changes. Using endothelial cells (ECs) and blood collected from OSA patients (n = 109) and controls (n = 67), we assessed whether altered cellular localization of complement inhibitor CD59 in OSA modulates exocytosis of Weibel-Palade bodies (WPB), secretory granules that store vWF and angiopoietin-2. These interactions were also assessed in vitro in ECs exposed to normoxia or IH with or without recombinant complement C9 and with or without atorvastatin. Circulating levels of angiopoietin-2 were greater in OSA than controls and levels of vWF cleavage products correlated with OSA severity. In cultured ECs, IH enhanced complement-stimulated angiopoietin-2 and vWF release by reducing EC surface and increasing intracellular expression of complement inhibitor CD59. Intracellular CD59 co-localized with WPB in OSA. IH increased binding of intracellular CD59 to syntaxin-3, which dissociated syntaxin-3 from voltage-sensitive calcium channel Cav1.2, and activated WPB exocytosis in a calcium-dependent manner. Atorvastatin reversed IH-enhanced endothelial release of vWF and angiopoietin-2. IH promotes the complement-mediated release of vWF and angiopoietin-2, which may contribute to pro-thrombotic and pro-inflammatory conditions in OSA. Statin reversed these effects, suggesting a potential approach to reduce cardiovascular risk in OSA.

Creating a DDAVP Releasable Pool of Factor VIII: Its Impact on the Gene Therapy of Hemophilia A † 786

The administration of DDAVP has been know for many years to result in a marked increase in plasma von Willebrand factor (vWF) and Factor VIII (FVIII). This occurs within minutes of intravenous administration and suggests a pre-existing pool of both proteins. The two cells in the body that can synthesize vWF are the endothelial cell and the megakaryocyte which respectively store vWF in the endothelial cell Weibel-Palade Body and the platelet α-granule. Most investigations have suggested that the primary source of the increased plasma vWF is the endothelial cell, but there is no recognized storage source of FVIII. In fact, the cell that produces FVIII is not definitively determined. Transplantation studies suggest that liver, spleen, and lymph nodes may be organ sources of FVIII but the tissue source is not well defined. To further define the nature of FVIII synthesis, storage and release, we studied two patients with severe hemophilia A undergoing prophylactic administration of recombinant FVIII and a type 3 von Willebrand disease (vWD) patient receiving regular infusions of vWF without significant FVIII. These individuals were administered 0.3 μg/kg DDAVP intravenously before and following the administration of the respective therapeutic product to determine if the DDAVP-releasable pool of FVIII was established. Plasma assays of vWF and FVIII demonstrated that there was no measurable release of FVIII into plasma in either disorder. In the vWD patient, the endogenous production of FVIII was at a normal level when the vWF was exogenously administered. Yet, there was no increase of FVIII following DDAVP. These studies suggested that the storage pool of FVIII was vWF dependent and that the storage reservoir of both proteins may need to be in the same cell - presumably the same cell in which they were synthesized. Since we have demonstrated that FVIII co-localizes with vWF when the gene for FVIII is inserted into an endothelial cell, we postulate that the DDAVP-releasable pool of FVIII is in a vWF containing cell. This most likely would occur in a subpopulation of endothelial cells that are not yet defined. Furthermore, gene therapy of hemophilia A may need to be targeted to the vascular endothelium to produce a physiologic storage pool that is protected from the proteolytic activity of plasma proteases.

Morphological alterations in endothelial cells associated with the release of von Willebrand factor after thrombin generation in vivo.

von Willebrand factor (vWF) is synthesized by endothelial cells and stored in endothelium-specific granules, the Weibel-Palade (WP) bodies. The release of vWF from endothelial cells in vitro in response to secretagogues such as thrombin is considered to result in the loss of WP bodies through the fusion of the WP bodies with the plasma membrane. Biochemical and morphological techniques, including transmission (TEM) and scanning (SEM) electron microscopy, were used to examine the plasma profile of vWF in parallel with morphological alterations in endothelial cells associated with the generation of thrombin in vivo. There was a rapid loss of high-molecular-weight multimers of the circulating vWF, with full recovery within 1 hour. Simultaneously, TEM demonstrated that the endothelial cells lost WP bodies and became severely vacuolated; this was associated with the appearance of craters in the endothelial surface on SEM. Release of stored vWF in WP bodies seemed to follow the fusion of multiple rather than individual WP bodies, with the resulting vacuole fusing and rupturing through the plasmatic membrane. Within 1 hour there was increased morphological evidence of metabolic organelle activity associated with replacement of WP bodies, presumably due to de novo synthesis of the basic protomer and its packaging in high-molecular-weight multimeric form in the storage organelles.

Vicinal cysteines in the prosequence play a role in von Willebrand factor multimer assembly.

von Willebrand factor (vWf) is synthesized as a large precursor that dimerizes in the endoplasmic reticulum and forms multimers in the trans- and post-Golgi compartments of megakaryocytes and endothelial cells. The disulfide-bonded multimers are stored in alpha granules of platelets and Weibel-Palade bodies of endothelial cells. The prosequence, composed of two homologous D domains, is required for vWf multimerization and storage. Each D domain contains vicinal cysteines (159Cys-Gly-Leu-162Cys and 521Cys-Gly-Leu-524Cys) that are similar to those at the active site of disulfide isomerases that catalyze thiol protein disulfide interchange. As in disulfide isomerases, a positively charged amino acid (lysine) is also found in close proximity to the vicinal cysteines. Although conserved, the lysine present in thioredoxin was shown not to be essential for its redox activity. We investigated the role of the vicinal cysteines and the lysine residue in the vWf propolypeptide by site-directed mutagenesis and expression of the resulting constructs in mammalian cells. Insertion of an extra glycine between the vicinal cysteines in either D domain inhibited multimerization of dimers, whereas alteration of lysine to glycine in both domains (residues 157 and 519) had no effect. This suggests the importance of the vicinal cysteines but not the lysines in vWf multimerization. Expression of the mutant with an additional glycine in the D1 domain in AtT-20 cells, a mouse pituitary cell line that can store vWf, led to the storage of the resulting dimers. This demonstrates that the mutation did not effect the capacity of the propolypeptide to direct vWf storage while its ability to promote interchain disulfide bonding was eliminated.

An ultrastructural and immunohistochemical study of human dental pulp: Identification of Weibel-Palade bodies and von willebrand factor in pulp endothelial cells

Special and specific immunohistochemical techniques as well as routine transmission electron microscopy were used to identify the presence of von Willebrand factor (vWF), a blood clotting factor essential to normal hemostasis, and Weibel-Palade bodies (WPB's), respectively, in the endothelial cells lining the blood vessels from both normal and inflamed human pulpal tissues. In human endothelial cells, WPB's are peculiar and specialized organelles which store vWF. All classes of blood vessels (capillaries, arterioles, arteries, venules, and veins) were vWF positive. The fine structural studies showed similar results with regard to the presence of WPB's. Interestingly, morphometric analyses conducted on the same tissues using either light or transmission electron microscopy showed that significantly more vWF-positive blood vessels were seen in the inflamed tissues. In agreement with the latter observation, transmission electron microscopy showed that more vascular endothelial cells contained WPB's in the inflamed tissues when compared with the normal tissues. From this it appears that during pulpal inflammation, the cascade of events associated with hemostasis may be activated with the increased synthesis and release of vWF by endothelial cells.

Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

γ-Interferon Modulates von Willebrand Factor Release by Cultured Human Endothelial Cells

Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma-interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF-treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

Molecular Studies of von Willebrand Disease: Reduced von Willebrand Factor Biosynthesis, Storage, and Release in Endothelial Cells Derived From Patients With Type I von Willebrand Disease

Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12-myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

Molecular studies of von Willebrand disease: reduced von Willebrand factor biosynthesis, storage, and release in endothelial cells derived from patients with type I von Willebrand disease [published erratum appears in Blood 1990 Nov 1;76(9):1901

Endothelial cells were cultured from the umbilical veins of two neonates with type I von Willebrand disease (vWD) and compared with cells cultured in parallel from normal control umbilical veins. In both cases, cultured vWD endothelial cells contained less messenger RNA (mRNA) encoding von Willebrand factor (vWF), and constitutively secreted two- to fourfold less vWF protein than their matched controls. Regulated secretion of stored vWF induced by thrombin or phorbol-12- myristate-13-acetate (PMA) was also diminished in vWD cells. Both the mRNA and protein produced by each of these type I vWD cells appeared to be of normal size. However, despite the diminished size of the vWF storage pool, electron microscopy of endothelial cells in situ showed normal appearing vWF storage organelles (Weibel-Palade bodies). These studies show that cultured umbilical vein endothelial cells can be used to explore the molecular defects in type I and perhaps other forms of vWD, and suggest that at least some forms of type I vWD are caused by diminished mRNA transcription or subsequent translation due to a defective vWF allele.

Von Willebrand factor and its disorders: An overview of recent molecular studies

von Willebrand factor (vWF) is a plasma protein with multiple functions in haemostasis. The vWF gene, located on chromosome 12p2.1, encodes a primary gene product of 2813 amino acids. Post-translational modification, assembly and secretion of vWF are highly complex. The pro vWF protomer is covalently linked by intermolecular disulphide bonds to form a dimer of MW ∼440 kDa. This then polymerises to form multimers ranging in MW from 1–20 × 10. 6 Simultaneously the pro piece of vWF is cleaved, releasing a 741 amino acid peptide known as vW Ag II from the polymerised protomers. Two distinct secretion pathways are found in the endothelial cell, a regulated pathway with storage in Weibl-Palade bodies and a constitutive pathway. Platelets store vWF in their α-granules. Mature vWF participates in platelet adhesion, spreading and aggregation and is a carrier of factor VIII, protecting the latter from degradation. Disorders of vWF are highly diverse. At least 20 subtypes of von Willebrand's disease have been described to date, based on features of the vWF present in or absent from patients plasma and platelets. Some patients have reduced amounts of apparently normal vWF whilst others have clearly abnormal vWF with aberrant structure and function. Rare patients virtually or completely lack vWF. The genetic and structural basis of some of these abnormalities is just beginning to emerge. This article outlines the molecular biology and physiology of vWF, and reviews some recent progress on the molecular pathology and genetics of von Willebrand's disease.

Fibrin induces release of von Willebrand factor from endothelial cells.

Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.