Biosynthesis, processing and secretion of von Willebrand factor: biological implications.

von Willebrand factor is a multimeric plasma glycoprotein that is required for normal haemostasis. von Willebrand factor is synthesized by endothelial cells and megakaryocytes, and originates from its precursor pro-von Willebrand factor. The endoproteolytic processing of pro-von Willebrand factor results in mature von Willebrand factor and von Willebrand factor propeptide (also known as von Willebrand Ag II). In endothelial cells, the propeptide controls the polymerization and subsequent targeting of von Willebrand factor to the storage vesicles, the so-called Weibel-Palade bodies. Upon stimulation of the endothelial cells, the Weibel-Palade bodies are translocated to the plasma membrane of the cell, and mature von Willebrand factor and its propeptide are co-secreted. After release, these polypeptides have divergent fates and serve different biological functions. Mature von Willebrand factor both controls platelet adhesion and aggregation at sites of vascular injury and acts as a chaperone protein for coagulation factor VIII. The von Willebrand factor propeptide may serve a role in modulating inflammatory processes. This still growing body of information indicates that the biological function of the von Willebrand factor gene product is more diverse than was previously thought.     

Storage and secretion of von Willebrand factor by endothelial cells

Endothelial cells synthesize and store von Willebrand factor. We have studied the storage and secretion of von Willebrand factor in cultured human umbilical vein endothelial cells. In particular, we were interested in the nature of the storage compartment and the effects of perturbation on the storage and secretion processes. The storage compartment for von Willebrand factor was isolated from homogenates of endothelial cells. By an immunostaining technique the isolated vesicles stained for von Willebrand factor. The staining pattern was similar to that of Weibel-Palade bodies in intact endothelial cells. We concluded that the storage compartment containing von Willebrand factor is identical to the Weibel-Palade body. The von Willebrand factor of the isolated storage vesicles is predominantly constructed of polypeptide chains with a Mr of 220 kD. On the other hand, von Willebrand factor continuously secreted by endothelial cells is constructed of both a 220 kD and a larger precursor (apparent Mr of 275 kD) subunit. The storage vesicles contain von Willebrand factor that supports ristocetin-induced platelet aggregation. Thus, endothelial cells store fully processed, biologically active von Willebrand factor within Weibel-Palade bodies. Short-term (less than 1 h) treatment of endothelial cells with the perturbing phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) results in release of cellular stored von Willebrand factor. 24-48 h after exposure to PMA the endothelial cell distribution of von Willebrand factor is changed distinctly. While the contents of the von Willebrand factor storage sites in the cells are gradually restored within 48 h, enhanced amounts of von Willebrand factor are secreted into the medium. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increase after exposure to phorbol ester, as determined by immunofluorescence microscopy. Phorbol ester treated cells release stored von Willebrand factor 48 h after they have been stimulated. PMA decreases the von Willebrand factor contents of the extracellular matrix; the deposition of von Willebrand factor in the subendothelium is blocked by PMA, whereas the degradation of matrix von Willebrand factor is not affected. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Basal secretion of von Willebrand factor from human endothelial cells

Endothelial cells store the adhesive glycoprotein von Willebrand factor (VWF) in Weibel-Palade bodies (WPBs), distinctively shaped regulated secretory organelles that undergo exocytosis in response to secretagogue. A significant proportion of newly synthesized VWF is also secreted spontaneously from nonstimulated cells, through what is thought to be the constitutive secretory pathway. To learn more about VWF trafficking, we performed kinetic analyses of the storage and nonstimulated secretion of VWF in cultured human endothelial cells. We found that most VWF was secreted through a route that was significantly delayed compared with constitutive secretion, although this pathway was responsible for secretion of a small amount of uncleaved VWF precursor. Disruption of pH-dependent sorting processes with ammonium chloride converted the secretion kinetics of mature VWF to that of its precursor. Conversely, preventing constitutive secretion of nascent protein with brefeldin A had only a modest effect on the spontaneous release of VWF, showing that most VWF secreted by nonstimulated cells was not constitutive secretion but basal release of a post-Golgi storage organelle, presumably the WPB. These data suggest that VWF is sorted to the regulated secretory pathway in endothelial cells much more efficiently than previously reported.     

Proteolytic processing of von Willebrand factor subunit: Heterogeneity in type-IIA von Willebrand disease

Summary Type IIA von Willebrand disease (vWD) is a heterogeneous disorder for which two different pathogenetic mechanisms have been proposed: increased proteolytic susceptibility of von Willebrand factor (vWF), and/or interference of its post-translational processing. Subunit analysis of vWF in type-IIA vWD has revealed an increased relative proportion of the 176- and 140-kDa subunit-derived fragments, suggesting an augmented fragmentation of vWF, even in the resting state. We analyzed the subunit pattern of vWF in plasma from five previously described patients with type-IIA vWD. All of them showed the above-mentioned pattern. In addition, the presence of a new band with an apparent molecular mass of 200 kDa, not described in normal individuals or in patients with vWD, was repeatedly observed in one of these patients. This patient also exhibited an abnormal vWF multimeric structure in platelets and in plasma, before and after desmopressin administration, when the blood was collected either in the presence or in the absence of proteinase inhibitors. We believe that an abnormal primary structure of vWF could be responsible for this abnormal proteolytic fragmentation pattern, as well as for the abnormal multimerization of vWF. Moreover, an abnormal susceptibility to proteolysis appears to be present, as suggested by the increase in the relative proportion of the 176-kDa fragment observed in the same patient. Future sequencing studies and genetic analysis may clarify whether there are one or two different defects related to the vWF of that patient. Our results indicate that the subunit analysis of vWF may reveal additional defects present in type-IIA vWD that may help our understanding of the pathogenesis of such disease.Supported in part by grants 90/3229, 91-92/0372, 94/1509 (FIS, INSALUD, Spain).     

Differential regulation by cytokines of constitutive and stimulated secretion of von Willebrand factor from endothelial cells

We examined the effect of cytokines on basal and agonist-stimulated release of von Willebrand factor (vWf) by human endothelial cells. Treatment of endothelial cells for up to 48 hours with human recombinant or purified interleukin 1 (IL-1) or human recombinant tumor necrosis factor-alpha (TNF-alpha) did not significantly affect constitutive secretion of vWf or intracellular levels of vWf, although basal prostacyclin (PGI2) production was markedly enhanced. In contrast, both IL-1 and TNF-alpha modulated vWf release in response to thrombin or phorbol ester. Pretreatment of endothelial cells for 2 hours with either cytokine enhanced by up to threefold the stimulatory effect of a subsequent 60-minute exposure to thrombin. Addition of cycloheximide (5 micrograms/mL) during the preincubation abolished this enhancement. Moreover, if the cytokine pretreatment time was extended to 24 hours, agonist-stimulated vWf release was significantly suppressed. Cytokine treatment for 2 or 24 hours had no detectable effect on levels of vWf messenger RNA. The effects of cytokines were not the result of contamination with bacterial lipopolysaccharide and were not attributable to endothelial cell injury. These results show that cytokines have little or no direct effect on vWf release from endothelial cells but can significantly modulate its acute release in response to other stimuli in a complex time- and dose-dependent manner.     

Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor

The rapid exocytosis of von Willebrand factor (VWF) in response to vascular injury can be attributed to the fact that VWF is stored in the Weibel-Palade bodies (WPBs) of endothelial cells. We describe a system for examining the ability of VWF to drive both the formation of a storage compartment and the function of that compartment with respect to regulated secretion. Transient transfection of HEK293 cells with wild-type human VWF cDNA leads to the formation of numerous elongated organelles that resemble WPBs. These "pseudo-WPBs" exhibit the internal structure, as well as the ability to recruit membrane proteins including P-selectin, of bona fide WPBs. Finally, VWF was efficiently secreted upon stimulation by phorbol ester. We used this system to examine 3 VWF mutations leading to von Willebrand disease that affect VWF multimerization and constitutive secretion. Surprisingly we find that all 3 mutants can, to some extent, make pseudo-WPBs that recruit appropriate membrane proteins and that are responsive to secretagogues. The most striking defects are a delay in formation and a reduction in the length and number of pseudo-WPBs in proportion to the clinical severity of the mutation. Studies of pseudo-WPB formation in this system thus yield insights into the structure-function relationships underpinning the ability of VWF to form functional WPBs.     

Annexin A2 promotes liver fibrosis by mediating von Willebrand factor secretion

BackgroundLiver fibrosis can lead to cirrhosis and hepatocellular carcinoma if not treated in the early stages. The molecular mechanisms of the pathogenesis of hepatic fibrosis remain unclear.AimTo identify the molecules involved in the pathogenesis of liver fibrosis and to investigate the potential effect and mechanism of Annexin A2 up-regulation during liver fibrosis progression.MethodsTwenty Sprague-Dawley rats were divided into two groups: the carbon tetrachloride (CCl₄)-induced liver fibrosis group and the normal control group. Hematoxylin and eosin staining or Masson Trichrome staining and enzyme-linked immunosorbent assay were applied to assess the degree of liver damage and fibrosis in rats with CCl₄-induced liver fibrosis. Liver tissue protein profiles were analyzed using iTRAQ and mass spectrometry. RT-PCR and western blotting analyses were employed to validate differentially expressed proteins. Small interfering RNA-based silencing was performed to study the function of Annexin A2.ResultsTwelve weeks after CCl₄ injection, significant body weight changes and liver injury and liver fibrosis were observed in rats. In addition, 130 proteins were differentially expressed in the liver fibrosis group. Overexpression of Annexin A2 was confirmed by RT-PCR and Western blotting analysis. Silencing of Annexin A2 expression in HepG2 and LX-2 cells significantly reduced the secretion of von Willebrand factor (vWF).ConclusionAnnexin A2 promotes liver fibrosis by mediating vWF secretion, which can be used to mitigate the progression of liver fibrosis.     

Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.     

Alloantibody from a patient with severe von Willebrand disease inhibits von Willebrand factor-FVIII interaction

 Older patients with RAEB-T or AML are extremely difficult to treat. They are at high risk of infection and/or bleeding complications and have a low probability of cure and short overall survival with conventional treatments. We treated 12 patients with an outpatient low-dose chemotherapy regimen consisting of Ara-C 100 mg subcutaneously on day 1, and 6-thioguanine 80 mg orally on days 2–5, repeated every week. Nine patients had MDS, six RAEB-T, and three RAEB (median age 57 years) and three had de novo AML (median age 73 years). All patients were transfusion dependent. The mean peripheral blast count at the beginning of treatment was 29% (4–51%). The median follow-up is 13 months (2–34 months) for all the patients and 14 months (2–34 months) for those with RAEB-T. Nine of the 12 patients are alive, including seven RAEB-T patients with a median of 18 months (range 6–34+ months). During treatment, the peripheral blast count was markedly reduced to a mean of 5% (0–23%). The mean pre-therapy platelet count, with transfusion support, was 24.0×10⁹/l, while the mean post-therapy platelet count without transfusion support is 95.0×10⁹/l. All patients except two became transfusion independent at some time. Treatment for 6–10 weeks was required to show reduction of blast number and increase in hemoglobin, platelet, and WBC counts. Initial cytopenias were the only side effects of this regimen. One patient had granulocytopenic fever. In conclusion, this low-dose regimen is effective and well tolerated for outpatient palliation in high-risk or elderly patients with RAEB-T or AML. Received: 11 September 1996 / Accepted: 8 January 1997     

A monoclonal antibody to factor VIII inhibits von Willebrand factor binding and thrombin cleavage

To study the interaction of human factor VIII (FVIII) with its various ligands, select regions of cDNA encoding FVIII light chain were cloned into the plasmid expression vector pET3B to overproduce FVIII protein fragments in the bacterium Escherichia coli. Partially purified FVIII protein fragments were used to produce monoclonal antibodies. One monoclonal antibody, 60-B, bound both an FVIII protein fragment (amino acid residues 1563 through 1909) and recombinant human FVIII, but not porcine FVIII. This antibody prevented FVIII-vWF binding and acted as an inhibitor in both the activated partial thromboplastin time (APTT) assay and a chromogenic substrate assay that measured factor Xa generation. The ability of the antibody to inhibit FVIII activity was diminished in a dose-dependent fashion by von Willebrand factor. This anti-FVIII monoclonal antibody bound to a synthetic peptide, K E D F D I Y D E D E, equivalent to FVIII amino acid residues 1674 through 1684. The 60-B antibody did not react with a peptide in which the aspartic acid residue at 1681 (underlined) was changed to a glycine, which is the amino acid present at this position in porcine FVIII. Gel electrophoretic analysis of thrombin cleavage patterns of human FVIII showed that the 60-B antibody prevented thrombin cleavage at light chain residue 1689. The coagulant inhibitory activity of the 60-B antibody may be due, in part, to the prevention of thrombin activation of FVIII light chain.     

Syntaxin 5 determines Weibel-Palade body size and von Willebrand factor secretion by controlling Golgi architecture

von Willebrand factor (VWF) is a multimeric hemostatic protein primarily synthesized in endothelial cells. VWF is stored in endothelial storage organelles, the Weibel-Palade bodies (WPB), whose biogenesis strongly depends on VWF anterograde trafficking and Golgi architecture. Elongated WPB morphology is correlated to longer VWF strings with better adhesive properties. We previously identified the SNARE SEC22B, which is involved in anterograde endoplasmic reticulum-to-Golgi transport, as a novel regulator of WPB elongation. To elucidate novel determinants of WPB morphology we explored endothelial SEC22B interaction partners in a mass spectrometry-based approach, identifying the Golgi SNARE Syntaxin 5 (STX5). We established STX5 knockdown in endothelial cells using shRNA-dependent silencing and analyzed WPB and Golgi morphology, using confocal and electron microscopy. STX5-depleted endothelial cells exhibited extensive Golgi fragmentation and decreased WPB length, which was associated with reduced intracellular VWF levels, and impaired stimulated VWF secretion. However, the secretion-incompetent organelles in shSTX5 cells maintained WPB markers such as Angiopoietin 2, P-selectin, Rab27A, and CD63. In brief, we identified SNARE protein STX5 as a novel regulator of WPB biogenesis.     

von Willebrand factor proteolytic processing and multimerization precede the formation of Weibel-Palade bodies

We investigated the intracellular site of pro-von Willebrand factor (pro-vWF) cleavage and multimerization, as well as the fate of the propolypeptide (von Willebrand antigen II) after cleavage. Analysis of subcellular fractions of endothelial cells metabolically labeled with sulfate showed that both cleavage and covalent multimerization occur after sulfation and precede the formation of Weibel-Palade bodies. Because sulfation is a processing step localized to the trans-Golgi network (TGN), our results indicate that multimerization and prosequence cleavage also occur in this organelle. After cleavage, the propolypeptide remains noncovalently associated with the mature vWF subunit. This association is promoted by a high calcium concentration and an acidic pH (conditions thought to prevail in the TGN) and explains the 1:1 stoichiometry of the propolypeptide and mature vWF found in Weibel-Palade bodies. The propolypeptide remains an integral part of the large multimeric vWF aggregates in the Weibel-Palade body until secretion. When secretion occurs under slightly acidic conditions, such as may be found in poorly perfused wounds, the propolypeptide remains associated with the endothelial surface-bound vWF, and may thus participate in the wound healing process.     

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.     

Atherosclerosis and von Willebrand factor

S ummary. The prevalence of von Willebrand's disease (VWD) in Western European countries and Israel was assessed. Possible patients were identified initially by questionnaire and those with plasma levels of von Willebrand factor antigen (WFAg; factor VIII related antigen) less than 1 u/dl (<1%) were confirmed by immunoradiometric assay performed at four reference centres. The prevalences of severe VWD in Scandinavian countries were similar to each other and about 10 times greater than those in other Western European countries. The prevalence in Israel was intermediate. The Registry will form the basis for future non-invasive epidemiological studies of atherosclerosis in these individuals.     

beta2-Glycoprotein I inhibits von Willebrand factor dependent platelet adhesion and aggregation

Patients with antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity and the presence of antiphospholipid autoantibodies. Particularly, anti-beta(2)-glycoprotein (beta(2) GPI) autoantibodies correlate with thrombosis, suggesting an antibody-induced gain of prothrombotic function and/or an antibody-induced loss of antithrombotic function of beta(2) GPI. In the search for potential antithrombotic properties of beta(2) GPI, we found that beta(2) GPI inhibits von Willebrand factor (VWF)-induced platelet aggregation. In addition, platelet adhesion to a VWF-coated surface was decreased by 50% in the presence of beta(2) GPI (P < .03). beta(2) GPI binds to the A1 domain of VWF but preferably when the A1 domain is in its active glycoprotein Ibalpha-binding conformation. Anti-beta(2) GPI antibodies isolated from a subset of antiphospholipid syndrome patients neutralized the beta(2) GPI-VWF interactions and thus the inhibitory activity of beta(2) GPI. In comparison to healthy individuals, the amounts of active VWF in circulation were increased 1.5-fold (P < .001) in patients positive for lupus anticoagulant (LAC) due to anti-beta(2) GPI antibodies. Thus, beta(2) GPI is a biologically relevant inhibitor of VWF function by interfering with VWF-dependent platelet adhesion. Anti-beta(2) GPI autoantibodies neutralize this inhibitory function and are associated with increased levels of active VWF. This mode of action could contribute to the thrombosis and consumptive thrombocytopenia observed in patients with anti-beta(2) GPI antibodies.     

Antioxidants reduce endoplasmic reticulum stress and improve protein secretion

Protein misfolding in the endoplasmic reticulum (ER) contributes to the pathogenesis of many diseases. Although oxidative stress can disrupt protein folding, how protein misfolding and oxidative stress impact each other has not been explored. We have analyzed expression of coagulation factor VIII (FVIII), the protein deficient in hemophilia A, to elucidate the relationship between protein misfolding and oxidative stress. Newly synthesized FVIII misfolds in the ER lumen, activates the unfolded protein response (UPR), causes oxidative stress, and induces apoptosis in vitro and in vivo in mice. Strikingly, antioxidant treatment reduces UPR activation, oxidative stress, and apoptosis, and increases FVIII secretion in vitro and in vivo. The findings indicate that reactive oxygen species are a signal generated by misfolded protein in the ER that cause UPR activation and cell death. Genetic or chemical intervention to reduce reactive oxygen species improves protein folding and cell survival and may provide an avenue to treat and/or prevent diseases of protein misfolding.     

N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I-deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher K(m) but similar k(cat) value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.     

A monomeric von Willebrand factor fragment, Leu-504--Lys-728, inhibits von Willebrand factor interaction with glycoprotein Ib-IX [corrected]

von Willebrand factor interaction with glycoprotein Ib alpha (GPIb alpha) plays a critical role in the initial phase of platelet adhesion at high shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474--Pro-488 (peptide 153) and Ser-692--Pro-708 (peptide 154), inhibit von Willebrand factor--GPIb alpha interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504--Lys-728 [corrected], with a single intrachain disulfide bond linking residues Cys-509--Cys-695 and examined its ability to inhibit von Willebrand factor--GPIb alpha interactions and platelet adhesion at high shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetin-induced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an IC50 = 0.011-0.260 microM, significantly lower than the IC50 of peptide 153 or 154, IC50 = 86-700 microM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (IC50 greater than 500 microM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 microM and at 7.6 microM inhibited greater than 80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.