Von Willebrand Factor Storage Research Articles

Cellular and molecular basis of Von Willebrand disease: Studies on blood outgrowth endothelial cells

Von Willebrand disease (VWD) is a heterogeneous bleeding disorder caused by decrease or dysfunction of von Willebrand factor (VWF). A wide range of mutations in the VWF gene have been characterized; however, their cellular consequences are still poorly understood. Here we have used a recently developed approach to study the molecular and cellular basis of VWD. We isolated blood outgrowth endothelial cells (BOECs) from peripheral blood of 4 type 1 VWD and 4 type 2 VWD patients and 9 healthy controls. We confirmed the endothelial lineage of BOECs, then measured VWF messenger RNA (mRNA) and protein levels (before and after stimulation) and VWF multimers. Decreased mRNA levels were predictive of plasma VWF levels in type 1 VWD, confirming a defect in VWF synthesis. However, BOECs from this group of patients also showed defects in processing, storage, and/or secretion of VWF. Levels of VWF mRNA and protein were normal in BOECs from 3 type 2 VWD patients, supporting the dysfunctional VWF model. However, 1 type 2M patient showed decreased VWF synthesis and storage, indicating a complex cellular defect. These results demonstrate for the first time that isolation of endothelial cells from VWD patients provides novel insight into cellular mechanisms of the disease.

Distinct Functions of pH-Sensing Histidine Residues in the Multimer Assembly and Storage of Von Willebrand Factor in Weibel-Palade Bodies

Abstract 3311The hemostatic function of von Willebrand factor (VWF) depends on the formation of disulfide-linked multimers. We have previously shown that several evolutionarily conserved His residues within the VWF propeptide are critical for pH-dependent VWF multimerization. For example, the charge-neutralization mutations H395A and H460A in the VWF D2 domain prevented multimer assembly, while the charge-stabilization mutation H460K preserved it, suggesting that protonation at these sites is essential. In this study, we investigated the role of these His residues in VWF targeting to storage granules and agonist-induced secretion. Full-length VWF was expressed in transiently-transfected HEK293 cells. If Weibel-Palade body-like storage granules (WPBs) were formed, their morphology was analyzed by immunofluorescence microscopy. A WPB was defined as “long” if length/width was > 2, or “round” if length/width was ≤ 2. To analyze the assembly of VWF multimers, we stimulated VWF secretion by incubating cells with 100 ng/ml of phorbol myristate acetate for 1 hour and collected cell lysates and supernatants for multimer gel electrophoresis. Expression of wild type VWF and VWF H460K induced the formation of approximately 60% long WPBs. Treatment of cells with monensin increases the pH of the Golgi, and as expected monensin reduced the storage of wild type VWF in long WPBs. However, monensin did not impair the storage of VWF H460K or reduce the fraction of long WPBs, indicating that a positive charge at residue 460 can sustain tubular storage of VWF despite neutralization of the late secretory pathway. Both WT VWF and VWF H460K were secreted with a normal distribution of multimers. VWF H395A and VWF H460A formed predominantly round pseudo-WPBs, with only 10–20% long granules, and these mutations also disrupted VWF multimerization. In contrast, VWF H395R and VWF H395A/H460R did not assemble multimers but nevertheless formed long WPBs. Insertion of an extra Gly between the vicinal cysteines in the D1 domain (159CGLC162 to 159CGGLC162) was reported to prevent multimerization but allow storage in long WPBs in AtT-20 cells (Mayadas & Wagner, PNAS 1992; 89:3531). We found that this mutation also inhibited VWF multimerization and resulted in the formation of long WPBs in HEK293 cells. These results indicate that VWF multimerization and packing in elongated WPBs both depend on the acidic pH of the late secretory pathway. However, these processes are independent, with distinct structural requirements, and they can be dissociated by mutations of specific His residues.VWF constructMultimersGranule ShapeGranule Shape (with monensin)Wild typeYesLongRoundH395ANoRoundH395RNoLongH395A/H460RNoLongH460ANoRoundH460KYesLongLong159CGGLC162NoLong Disclosures:No relevant conflicts of interest to declare.

Imaging of Von Willebrand Factor Remodeling Upon Secretion From Vascular Endothelial Cells

AbstractAbstract 263In response to vascular injury, endothelial cells rapidly secrete high molecular weight multimers of the coagulation protein Von Willebrand factor (VWF). Once expelled from the cells, VWF unfurls in long strings that bind platelets from the bloodstream to induce primary hemostasis. VWF secreted upon stimulation is released from specialized storage compartments called Weibel Palade bodies (WPB) which have a typical rod or cigar shape. They emerge from the Trans Golgi network in a process driven by the formation of helical tubules consisting of VWF multimers and the VWF propeptide. When WPBs undergo exocytosis and release VWF, rapid structural changes occur which eventually result in platelet capturing VWF strings. It has been postulated that the tubular storage of VWF in WPBs is required for sufficient unfolding of the protein during string formation as agents disrupting the VWF tubules were shown to result in less strings. Recently we described a novel structure involved in VWF exocytosis which is formed only upon stimulation. We refer to this structure as a “secretory pod” as it seemed to derive from multiple WPBs and was identified as a VWF release site where strings seemed to be formed. By transmission electron microscopy (TEM) we identified this structure to be a membrane-delimited organelle containing filamentous material resembling unfurled VWF. The VWF tubules as seen in WPBs are absent in secretory pods suggesting that tubular packaging of VWF is not essential for sufficient release and string formation.To study the formation of secretory pods and the subsequent release and remodeling of VWF, several imaging techniques were used such as live-cell imaging and correlative light and electron microscopy. We expressed propeptide-EGFP in endothelial cells to label the WPBs and stimulated them with PMA. By live-cell imaging we visualized the exocytotic events. We observed, apart from single WPB exocytosis, the formation of secretory pods which occurred by the coalescence of several WPBs. In some cases the individual WPBs rounded up first, before they joined into one round structure while in other cases the coalescence event seemed to happen at once. After coalescence, fusion with the plasma membrane occurred to release the pooled VWF which resulted in the disappearance of the fluorescent signal as the propeptide rapidly diffused into the extracellular medium. How the secreted VWF is remodeled after secretion into VWF strings was studied by correlative light and electron microscopy. We correlated confocal pictures of stimulated endothelial cells, which were stained with VWF specific fluorescent antibodies, to consecutive TEM sections. We found that fluorescently labeled VWF dots that were connected to strings, correlated to secretory pods but also to globular mass of secreted VWF. Interestingly, when we analyzed consecutive EM sections, the globular masses were found to originate from the secretory pods. From the globular masses we also observed deriving strings indicating that once VWF is expelled, remodeling occurs independently from secretion. We hypothesize that fluid flow remodels the secreted globular VWF mass into strings. To study this we stimulated endothelial cells under flow. The intracellular VWF pool in the WPBs was labeled green by transient expression of propeptide-EGFP and the secreted VWF was labeled red with strongly diluted red fluorescent VWF specific antibodies in the perfusate. Using live-cell imaging we observed that upon fusion of EGFP labeled WPBs, the green signal transformed into a red signal revealing dots of labeled secreted VWF. These dots rolled, in the direction of the flow, to the edge of the cell where they aggregated and only then formed strings. In non-transfected cells we performed similar experiments and there we observed the same pattern, confirming even more the VWF aggregation and string formation at the edges of the cell.In conclusion, we demonstrated that several WPBs can fuse with each other to form secretory pods and that VWF is secreted as a globular mass of protein. From these globular masses strings originated indicating that string formation occurs independently from the mechanism of secretion in which the tubular packaging of VWF in WPBs does not seem to be of importance. Disclosures:No relevant conflicts of interest to declare.

Von Willebrand disease biology

*Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; Department of Thrombosis and Hemostasis, C2-R Einthoven Laboratory for Experimental Vascular Medicine, LeidenUniversity Medical Center, Leiden, The Netherlands; Centre for Haematology, Imperial College London, HammersmithHospital Campus, London;§INSERM U770, Cedex, France; and –Department of Pathology and Molecular Medicine, QueensUniversity, Kingston, ON, Canada

Biogenesis of Weibel-Palade bodies in von Willebrand's disease variants with impaired von Willebrand factor intrachain or interchain disulfide bond formation

Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies. The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate. p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser. We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.

A role for Rab10 in von Willebrand factor release discovered by an AP‐1 interactor screen in C. elegans

Endothelial von Willebrand factor (VWF) mediates platelet adhesion and acts as a protective chaperone to clotting factor VIII. Rapid release of highly multimerized VWF is particularly effective in promoting hemostasis. To produce this protein, an elaborate biogenesis is required, culminating at the trans-Golgi network (TGN) in storage within secretory granules called Weibel-Palade bodies (WPB). Failure to correctly form these organelles can lead to uncontrolled secretion of low-molecular-weight multimers of VWF. The TGN-associated adaptor AP-1 and its interactors clathrin, aftiphilin and γ-synergin are essential to initial WPB formation at the Golgi apparatus, and thus to VWF storage and secretion. To identify new proteins implicated in VWF storage and/or secretion. A genomewide RNA interference (RNAi) screen was performed in the Nematode C. elegans to identify new AP-1 genetic interactors. The small GTPase Rab10 was found to genetically interact with a partial loss of function of AP-1 in C. elegans. We investigated Rab10 in human primary umbilical vein endothelial cells (HUVECs). We report that Rab10 is enriched at the Golgi apparatus, where WPB are formed, and that in cells where Rab10 expression has been suppressed by siRNA, VWF secretion is altered: the amount of rapidly released VWF was significantly reduced. We also found that Rab8A has a similar function. Rab10 and Rab8A are new cytoplasmic factors implicated in WPB biogenesis that play a role in generating granules that can rapidly respond to secretagogue.

Co-Trafficking of Coagulation Factor VIII with Von Willebrand Factor Alters the Macromolecular Structure Inside Secretory Weibel-Palade Bodies

AbstractAbstract 325The liver is generally recognized as the major site of coagulation factor (F)VIII synthesis. However, there is now increasing evidence that FVIII can also be synthesized in specific endothelial cells where it is stored with its natural carrier protein von Willebrand factor (VWF) in the Weibel-Palade bodies (WPBs). WPBs have a typical cigar-shaped appearance that most likely originates from the macromolecular organization of VWF multimers into tubules. The tubular storage of VWF is thought to be essential for orderly secretion of VWF strings during activation of endothelial cells. Recently we have shown that expression of FVIII with VWF changes the WPB morphology to spherical vesicles. This finding suggests alterations in the biochemical properties of stored VWF. We now studied in detail the effect of FVIII co-expression on the VWF molecule using a combination of innovative techniques, including correlative light-electron microscopy (CLEM), and live-cell fluorescence microscopy under flow conditions. Analysis of human blood outgrowth endothelial cells (BOECs) expressing human B-domain deleted FVIII-GFP by CLEM revealed that FVIII containing WPBs were electron-dense, spherical structures. These structures contained disorganized short VWF tubules, which was confirmed in 3D by electron tomography. Double immunogold labelling with VWF and GFP antibodies showed that the spherical FVIII containing structures were always positive for VWF. These observations imply that FVIII blocks the expansion of VWF tubules, possibly by binding to the N-terminal VWF domains. As the N-terminal domains are also implicated in the formation of multimers, we therefore investigated whether FVIII affects VWF multimer size. Indeed, multimer analysis showed that VWF secreted by FVIII-GFP transduced BOECs was multimerized to a lesser extent when compared to VWF secreted by non-transduced BOECs. The combined absence of high molecular weight (HMW) VWF multimers and long VWF tubules made us question whether these cells could still release ultra-large VWF (UL-VWF) strings. UL-VWF strings play a key role in bleeding arrest, as platelets adhere to the released VWF string which ultimately leads to the formation of a platelet plug. We examined the release of UL-VWF strings under shear stress from BOECs expressing FVIII-GFP employing live-cell confocal imaging. This technique allowed us to follow FVIII release during exocytosis of WPBs in real-time as well. When we stimulated FVIII-transduced BOECs with histamine, these cells were equally able to release VWF strings as non-transduced BOECs. Although spherical WPBs lacked long VWF tubules and did not secrete HMW multimers, released VWF strings were of similar length as strings secreted by non-transduced BOECs. Surprisingly, released VWF strings were completely covered with FVIII which remained attached to the strings throughout the whole experiment. Another remarkable observation was that platelet binding to the FVIII-covered VWF strings was almost completely absent. We hypothesize that FVIII either shields the A1 domain for platelet binding or causes a conformational change in the VWF strings that prevents platelets from binding to the strings. Our results demonstrate that FVIII co-trafficking with VWF has a major impact on properties of VWF as it reduces the degree of multimerization, shortens tubules and prevents platelets from adhering to strings. This leads us to the conclusion that the macromolecular structure of VWF is considerably altered when FVIII is present in WPBs. Disclosures:No relevant conflicts of interest to declare.

The mutation N528S in the von Willebrand factor (VWF) propeptide causes defective multimerization and storage of VWF

AbstractWe characterized a consanguineous Turkish family suffering from von Willebrand disease (VWD) with significant mucocutaneous and joint bleeding. The relative reduction of large plasma von Willebrand factor (VWF) multimers and the absent VWF triplet structure was consistent with type 2A (phenotype IIC) VWD. Surprisingly, platelet VWF was completely deficient of multimers beyond the VWF protomer, suggesting defective α-granular storage of larger multimers. Patients were nearly unresponsive to desmopressin acetate, consistent with a lack of regulated VWF release from endothelial cell Weibel-Palade bodies, suggesting defective storage also in endothelial cells. We identified an N528S homozygous mutation in the VWF propeptide D2 domain, predicting the introduction of an additional N-glycosylation site at amino acid 526 in close vicinity to a “CGLC” disulphide isomerase consensus sequence. Expression studies in mammalian cells demonstrated that N528S-VWF was neither normally multimerized nor trafficked to storage granules. However, propeptide containing the N528S mutation trafficked normally to storage granules. Our data indicate that the patients' phenotype is the result of defective multimerization, storage, and secretion. In addition, we have identified a potentially novel pathogenic mechanism of VWD, namely a transportation and storage defect of mature VWF due to defective interaction with its transporter, the mutant propeptide.

Type 1 von Willebrand disease due to reduced von Willebrand factor synthesis and/or survival: observations from a case series

It may be difficult to diagnose type 1 von Willebrand disease (VWD) because of its heterogeneous and sometimes elusive nature. To evaluate the contribution of a shorter von Willebrand factor (VWF) survival in modulating VWD phenotype, the VWF half-life was assessed in 45 type 1 VWD patients using a 24-h 1-desamino-8-d-arginine vasopressin (DDAVP) test. A shorter VWF survival was observed in patients with C1130F mutations (T(1/2) elimination [T(1/2)el]=4.6+/-1.0h vs normal=15.8+/-2.3h, P<0.0001), in those with other missense mutations investigated (T(1/2)el=9.5+/-0.9h, P<0.02), and in patients not carrying VWF mutations (T(1/2)el=7.0+/-0.7h, P<0.001); the decrease mainly depended on a greater VWF clearance. VWF survival and clearance were normal in patients who carried nonsense mutations. The VWF-propeptide-to-VWF-antigen (VWF:Ag) ratio (VWFpp ratio) was higher in patients with a shorter VWF survival, and the values were inversely correlated with the VWF half-life (P<0.01). The response of VWF to DDAVP administration, which is useful to explore the synthesis and storage of VWF, was normal in patients with no mutations, whereas it decreased in patients with missense and nonsense mutations. Three scenarios, thus, are recognizable in type 1 VWD; one is associated mainly with a shorter survival of VWF, another is associated with its reduced synthesis and release, and a third is characterized by a combination of the two. The shorter VWF half-life found in patients with no VWF mutations suggests that mechanisms other than VWF might be involved in the pathogenesis of type 1 VWD.

Type 3 Von Willebrand Disease: Plasma Versus Platelets.

Abstract 3059Poster Board II-1035Type 3 von Willebrand disease (VWD) is characterized by the virtual absence of von Willebrand factor (VWF) in plasma. Affected individuals are usually either homozygous or compound heterozygous for mutations that have been identified throughout the 52 exon VWF gene (listed in the ISTH SSC VWF database, http://www.sheffield.ac.uk/vwf/index.html). Most are null mutations associated with deletions, insertions, frameshifts, splicing defects and premature stop codons, however, a number of missense mutations have also been identified. VWF is normally stored in the Weibel-Palade bodies of vascular endothelial cells and within platelet alpha granules. Thus, the absence of plasma VWF in type 3 VWD patients carrying non-null mutations could be explained by one or more of: 1) premature mRNA degradation; 2) improper folding or assembly of VWF within cells resulting in proteolytic degradation; 3) rapid clearance of defective VWF molecules from plasma; 4) inability of synthesized VWF to be properly stored and/or released.We set out to detect type 3 VWD patients with possible VWF plasma clearance, storage or release defects by measuring the relative amounts of VWF in platelets and plasma, reasoning that such defects may be associated with the accumulation of VWF in platelets. Blood was prospectively collected from 21 previously diagnosed cases. Laboratory evaluation confirmed type 3 VWD, if the VWF:Ag and VWF:RCo levels were '5 IU per dL or if the VWF:Ag or VWF:RCo levels were 6-10 IU per dL accompanied by FVIII levels of '10 IU per dL. From this cohort, 16 unrelated type 3 VWD index cases were identified. Equivalent amounts of plasma and platelet lysates were analyzed for VWF by immunoblotting. As expected, platelet and plasma VWF were observed to be very low or absent in most cases. Interestingly, 5 patients were found to have relatively more (∼10 fold) platelet than plasma VWF using semiquantitative immunoblotting and equivalent sample loading. Type 3 VWD index cases together with available type 1 VWD siblings were subjected to VWF gene sequencing analyses. VWF exons 1-52 as well as ∼1500 bp of the promoter and intron/exon boundaries were sequenced. Strategies to identify partial gene deletions are ongoing. VWF gene mutations were identified in 12/16 patients. Surprisingly, in 4 out of the 5 patients with discrepant VWF content in plasma and platelets, no mutation in the VWF gene was identified by sequencing, suggesting either that an in-frame partial deletion or intronic VWF mutation may be present or that other genes such as those regulating the assembly, storage or release of VWF could be affected. A novel homozygous frameshift mutation at position c.8418_8419 resulting from a TCCC insertion was identified in one case. The presence of platelet VWF in this patient suggests that despite this frameshift mutation, VWF is expressed and packaged into platelets. Two siblings with the same homozygous mutation had an identical pattern (no plasma VWF but platelet VWF present) whereas two heterozygous siblings had 10-30% plasma VWF:Ag with normal amounts of platelet VWF. Studies are underway to determine whether increased clearance or defects in storage and/or release from endothelial cells or platelets are the cause of absent plasma VWF. Our studies also suggest that type 3 VWD can be subcategorized into subtype 0 (no plasma no platelet VWF) versus subtype P (no plasma but platelet VWF present) by measuring plasma and platelet VWF. We hypothesize that patients with type 3 VWD with platelet-harbouring VWF may have milder clinical manifestations compared to the subtype 0, however further studies are needed to test this proposal. DisclosuresNo relevant conflicts of interest to declare.

Genetic alteration of the D2 domain abolishes von Willebrand factor multimerization and trafficking into storage.

The large von Willebrand factor (VWF) propeptide (VWFpp) plays a critical role in the multimerization and regulated storage of the mature VWF protein. Although our laboratory and others have identified mutations in von Willebrand disease patients that disrupt VWF multimerization, little is known about the affect of mutations on the regulated storage of VWF. We identified a heterozygous 18 base pair, in-frame deletion in exon 12 of the VWF gene in a patient with an unusual, dimer-intense multimer pattern. This deletion results in loss of amino acids 436-442 of VWFpp, which include one cysteine. Through expression studies, we demonstrate reduced secretion, loss of VWF multimerization, and defective regulated storage of the variant VWF. The loss of VWF storage is secondary to loss of propeptide storage resulting from an apparently defective sorting signal on VWFpp. Suprisingly, coexpressed wild-type VWF or VWFpp functioned in trans to partially restore multimerization of VWF from the variant allele. The deletion of six amino acids in VWFpp results in defects in VWF processing, regulated storage, and function. Although VWFpp may usually function in a homotypic fashion, acting on its own mature VWF subunit, VWFpp may retain the ability to function in trans on VWF expressed from the variant allele.

Intersection of Mechanisms of Type 2A Von Willebrand Disease through Defects in Von Willebrand Factor Multimerization, Secretion, ADAMTS-13 Susceptibility, and Regulated Storage

Type 2A von Willebrand disease (VWD) is characterized by the absence of large von Willebrand factor (VWF) multimers and decreased platelet binding function. These variants are classified as either group 1 (2A-1) with impaired assembly and secretion of VWF multimers, or group 2 (2A-2) with increased susceptibility to proteolysis by ADAMTS13. However, laboratory parameters in individual patients may not discriminate between 2A-1 and 2A-2; this must be established through expression studies. Type 2A VWD patients recruited through the TS Zimmerman Program For The Molecular And Clinical Biology Of VWD (ZPMCB VWD) were phenotypically identified based upon laboratory parameters. By sequencing genomic DNA we identified 13 potentially causative sequence variations in the VWF D2, D3, A1, and A2 domains of type 2A VWD patients. Expression studies were performed to examine the effect of these mutations onVWF processing and proteolysis, andVWF regulated storage. The VWF variants L1503R, S1506L, and V1607D demonstrated severely impaired secretion and lacked mid- and high-molecular weight multimers, consistent with a group 1 classification (2A – 1).Some variants including I1568N, G1579R, and G1631D were normally multimerized and secreted, but had an increased susceptibility to cleavage by ADAMTS13 cleavage, indicative of group 2 classification (2A – 2). Five variants involving cysteine residues (C1190S, C1099P, C1272R/S/Y) had multimer abnormalities, a modest reduction in secretion, and variably enhanced ADAMTS-13 cleavage. These variants did not easily fit the criteria for either 2A subgroup. We also identified sequence variations (M740I and I1380V) that had no effect on VWF secretion, structure, or proteolysis that are likely innocuous polymorphisms but are present in patients with a type 2A phenotype. VWF is stored for regulated release in endothelial cell Weibel-Palade bodies and in platelet alpha-granules. The effect of mutations on VWF regulated storage has been documented for few VWF variants. The 2A variants were expressed in HEK293 cells, immunostained, and examined by confocal microscopy. Several variants including C1272S/Y, L1503R, S1506L, and V1607D did not form storage granules. A loss or reduction in endothelial cell Weibel-Palade bodies may explain the diminished desmopressin response observed in many type 2A VWD patients. In sum, our data indicate that 2A-2 variants are associated with VWF A2 domain mutations and normal regulated storage, while 2A-1 mutations are not restricted to a particular domain and result in loss of regulated VWF storage. Mutations involving cysteines may not affect VWF secretion, but are likely to cause abnormalities in multimer structure. In summary, type 2A VWD appears to result from at least three intersecting mechanisms: intracellular retention, defective multimerization, or increased plasma proteolysis.

Basal secretion of von Willebrand factor from human endothelial cells

AbstractEndothelial 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.

Characterization of Recessive Severe Type 1 and 3 von Willebrand Disease (VWD), Asymptomatic Heterozygous Carriers Versus Bloodgroup O-Related von Willebrand Factor Deficiency, and Dominant Type 1 VWD

Recessive type 3 von Willebrand disease (VWD) is caused by homozygosity or double heterozygosity for two non-sense mutations (null alleles). Type 3 VWD is easy to diagnose by the combination of a strongly prolonged bleeding time (BT), absence of ristocetine-induced platelet aggregation (RIPA), absence of von Willebrand factor (VWF) protein, and prolonged activated partial thromboplastin time (aPTT) due to factor VIII:coagulant (FVIII:C) deficiency. VWD type 3 is associated with a pronounced tendency to mucocutaneous and musculoskeletal bleedings since early childhood. Carriers of one null allele are usually asymptomatic at VWF levels of 50% of normal. Recessive severe type 1 VWD is caused by homozygosity or double heterozygosity for a missense mutation. Recessive type 1 VWD differs from type 3 VWD by the presence of detectable von Willebrand factor: antigen VWF:Ag and FVIII:C levels between 0.09 and 0.40 U/mL. Patients with recessive type 1 VWD show an abnormal VWF multimeric pattern in plasma and/or platelets consistent with severe type 2 VWD. Carriers of a missense mutation may have mild bleeding and mild VWF deficiency and can be diagnosed by a double VWF peak on cross immunoelectrophoresis (CIE). There will be cases of mild and moderate recessive type 1 VWD due to double heterozygosity of two missense mutations, or with the combination of one missense mutation with a non-sense or bloodgroup O. Mild deficiency of VWF in the range of 0.20 to 0.60 U/mL, with normal ratios of von Willebrand factor: ristocetine cofactor/antigen VWF:RCo/Ag and VWF:collagen binding/antigen (VWF:CB/Ag), normal VWF multimers, and a completely normal response to desmopressin acetate (DDAVP) with VWF level rising from below to above 1.00 U/mL are very likely cases of so-called pseudo-VWF deficiency in individuals with normal VWF protein and gene. Autosomal dominant type 1 VWD variants are in fact type 2 variants caused by a heterozygous missense mutation in the VWF gene that produces a mutant VWF protein that has a dominant effect on normal VWF protein produced by the normal VWF allele with regard to the synthesis, processing, storage, secretion, and/or proteolysis of VWF in endothelial cells. A DDAVP challenge test clearly differentiates between dominant type 1 VWD phenotype and dominant type 2 M VWD.

Mutations C1157F and C1234W of von Willebrand factor cause intracellular retention with defective multimerization and secretion.

The D3 domain of von Willebrand factor (VWF) is involved in the multimerization process of the protein through the formation of disulfide bridges. We identified heterozygous substitutions, C1157F and C1234W, in the VWF D3 domain in two unrelated families with unclassified and type 2A von Willebrand disease, respectively. VWF was characterized by a low plasmatic level, an abnormal binding to platelet GPIb and a high capacity of secretion from endothelial cells following DDAVP infusion. Using site-directed mutagenesis and expression in mammalian cells, we have investigated the impact of these mutations upon the multimerization, secretion and storage of VWF. Using COS-7 cells both mutated recombinant VWF (rVWF) displayed only lower molecular weight multimers. Pulse-chase analysis and endoglycosidase H digestion experiments showed the intracellular retention of mutated rVWF in pre-Golgi compartments. Study of hybrid rVWF obtained with a constant amount of wild-type (WT) DNA and increasing proportions of mutated plasmids established that both substitutions reduced the release of WT VWF in a dose-dependent manner and failed to form high molecular weight multimers. Using transfected AtT-20 stable cell lines, we observed similar granular storage of the two mutants and WT rVWF. Our data suggest that cysteines 1157 and 1234 play a crucial role in the early step of the folding of the molecule required for a normal transport pathway, maturation and constitutive secretion. In contrast, their substitution does not prevent the storage and inducible secretion of VWF.

D Dimer Levels Are Markedly Raised in HIV-Related TTP.

Background Thrombotic Thrombocytopenic Purpura (TTP) is now being seen with increasing frequency in association with HIV infection. Successful treatment depends on early institution of plasma infusion or plasmapheresis and it is often necessary to make the diagnosis in the presence of a microangiopathic haemolytic anaemia (MIAHA) and thrombocytopenia alone before the full diagnostic pentad of features has emerged. This requires exclusion of other possible causes for the clinical picture including Disseminated Intravascular Coagulopathy (DIC). The absence of abnormalities on a coagulation (DIC) screen is therefore often quoted as an important diagnostic criterion. However, although other coagulation parameters appear normal, we have frequently observed extremely high D-Dimer levels in patients with HIV related TTP. This study was done to analyze the results of coagulation screening in patients with HIV related TTP and to compare these with the findings in HIV negative patients with TTP. Materials and Methods The HIV status and results of DIC screens done on consecutive patients diagnosed with TTP at the Johannesburg Hospital between August 2002 and January 2005 were reviewed. Patients were identified from records of haematology consults received during this period. The diagnosis of TTP required at least the presence of MIAHA and thrombocytopaenia in the absence of other possible causes. Laboratory data was retrieved from the hospital computer system. HIV status was confirmed on ELISA testingResults Fifteen patients with HIV related TTP and 3 patients with Idiopathic TTP were identified. Two patients with pregnancy related TTP who suffered spontaneous abortions prior to assessment were excluded as alterations in coagulation parameters in these patients were not solely dependent on the TTP process. The median haemoglobin and platelet levels at diagnosis were 5.4g/dl and 11x10^9/l respectively for the patients with HIV related TTP and 8.0g/dl and 19X10^9/l respectively for the patients with idiopathic TTP. Median INR, PTT and Antithrombin levels were normal and not significantly different between the 2 groups (INR 1.14 vs1.12, PTT 29.9 vs31.5 secs, Antithrombin 103 vs12 IU). However the median D-Dimer level was significantly higher in the HIV related TTP group (3.66 mg/l, range 0.95–6.95 vs.0.28mg/l, range 0.22–0.3. p=0.0024)Discussion This study confirms our clinical impression that extremely high D-Dimer levels, in the absence of any other abnormalities in coagulation parameters, appear to be a consistent feature of HIV related TTP. In our experience this finding is often helpful in suggesting the diagnosis. The D-Dimer levels appear to be significantly higher than those in patients with idiopathic TTP although the small numbers of HIV negative patients in this study limits the reliability of the comparison and few reported case series give sufficiently detailed data to confirm this finding. It is possible that the very high D-Dimer levels noted in HIV positive patients reflect differences in the pathogenesis of TTP in HIV infection. Endothelial damage with loss of its antithrombotic properties may result in both localized coagulation activation (as evidenced by raised D-Dimer levels) and release of stored Von Willebrand Factor. The consequent relative deficiency of Von Willebrand Cleaving Protease could then result in accumulation of high molecular weight multimers and platelet activation with clinical outcomes similar to that seen in “classical” TTP where an absolute deficiency in the protease (either congenital or acquired due to the presence of antibodies) is implicated.

Induction of megakaryocytes to synthesize and store a releasable pool of human factor VIII

von Willebrand factor (VWF) is a complex plasma glycoprotein that modulates platelet adhesion at the site of a vascular injury, and it also serves as a carrier protein for factor (F)VIII. As megakaryocytes are the only hematopoietic lineage to naturally synthesize and store VWF within alpha-granules, this study was performed to determine if expression of a FVIII transgene in megakaryocytes could lead to trafficking and storage of FVIII with VWF in platelet alpha-granules. Isolex selected CD34+ cells from human G-CSF mobilized peripheral blood cells (PBC) and murine bone marrow were transduced with a retrovirus encoding the B-domain deleted form of human FVIII (BDD-FVIII). Cells were then induced with cytokines to form a population of multiple lineages including megakaryocytes. Chromogenic analysis of culture supernatant from FVIII-transduced human cells demonstrated synthesis of functional FVIII. Treatment of cells with agonists of platelet activation (ADP, epinephrine, and thrombin receptor-activating peptide) resulted in the release of VWF antigen and active FVIII into the supernatant from transduced cells. Immunofluorescence analysis of cultured human and murine megakaryocytes revealed a punctate pattern of staining for FVIII that was consistent with staining for VWF. Electron microscopy of transduced megakaryocytes using immunogold-conjugated antibodies colocalized FVIII and VWF within the alpha-granules. FVIII retained its association with VWF in human platelets isolated from the peripheral blood of NOD/SCID mice at 2-6 weeks post-transplant of transduced human PBC. These results suggest feasibility for the development of a locally inducible secretory pool of FVIII in platelets of patients with hemophilia A.

Weibel-Palade bodies recruit Rab27 by a content-driven, maturation-dependent mechanism that is independent of cell type.

The identification of organelles is crucial for efficient cellular function, yet the basic underlying mechanisms by which this might occur have not been established. One group of proteins likely to be central to organelle identity is the Rab family of small GTPases. We have thus investigated Rab recruitment to membranes using endothelial cells as a model system. We report that Weibel-Palade bodies, the Von Willebrand Factor storage compartment of human umbilical vein endothelial cells, contain Rab27a. We have also found that Weibel-Palade body-like structures induced in HEK-293 cells by the expression of von Willebrand factor can recruit endogenous Rab27a. In the absence of von Willebrand Factor, Rab27a is not lysosome associated, indicating that it can distinguish between the Weibel-Palade-body-like organelle and a classical lysosome. Finally, a time course of Weibel-Palade-body formation was established using a green-fluorescent version of von Willebrand factor. Newly formed Weibel-Palade bodies lack Rab27a, which is acquired some hours after initial appearance of the cigar-shaped organelle. We conclude that a lumenal cargo protein drives the recruitment of Rab27a to the organelle membrane by a novel mechanism that is indirect, maturation-dependent and cell-type independent.

Critical independent regions in the VWF propeptide and mature VWF that enable normal VWF storage

Von Willebrand factor (VWF) is synthesized in endothelial cells, where it is stored in Weibel-Palade bodies. Administration of 1-desamino-8-D-arginine-vasopressin (DDAVP) to patients with type 1 von Willebrand disease and to healthy individuals causes a rapid increase in plasma VWF levels. This increase is the result of stimulated release of VWF from Weibel-Palade bodies in certain beds of endothelial cells. The VWF propeptide (VWFpp) targets VWF to storage granules through a noncovalent association. The nature of the VWFpp/VWF interaction was investigated by using cross-species differences in VWF storage. While canine VWFpp traffics to storage granules and facilitates the multimerization of human VWF, it does not direct human VWF to storage granules. Since storage takes place after furin cleavage, this defect appears to be due to the defective interaction of canine VWFpp and human VWF. To determine the regions within VWFpp and VWF important for this VWFpp/VWF association and costorage, a series of human-canine chimeric VWFpp and propeptide-deleted VWF (Deltapro) constructs were produced and expressed in AtT-20 cells. The intracellular localization of coexpressed proteins was examined by confocal microscopy. Two amino acids, 416 in VWFpp and 869 in the mature VWF molecule, were identified as being critical for the association and granular storage of VWF.

An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant

AbstractWe describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre–pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid–cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post–1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.