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

Haberichter, Sandra L.; Jacobi, Paula M.; Montgomery, Robert R.

doi:10.1182/blood-2002-07-2281

Cited by 46 publications

(51 citation statements)

References 44 publications

(43 reference statements)

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“…VWF-regulated storage is VWFpp dependent: VWFpp independently traffics to the regulated secretory pathway and secondarily traffics VWF to storage through a noncovalent association. 15,20 The mutated propeptide, N528S-VWFpp, trafficked to storage granules indicating that the mutation does not interfere with the VWFpp storage signal ( Figure 5). N528S-VWFpp did not facilitate trafficking of coexpressed mature VWF to storage granules, suggesting that a disrupted VWFpp-VWF interaction contributes to the lack of regulated storage.…”

Section: Discussionmentioning

confidence: 97%

“…, and K9-VWD-aortic endothelial cells (AECs) were cultured and transfected as described. 20,21 HEK293 and 293T cells were transiently transfected using TransIT-293 Transfection Reagent (Mirus Bio) according to the manufacturer's instructions. K9-VWD-AECs were nucleofected as described.…”

Section: Antibodiesmentioning

confidence: 99%

“…on May 10, 2018. by guest www.bloodjournal.org From confocal laser scanning microscopy in the Imaging Core of the Medical College of Wisconsin using a Leica TCS SP2 confocal laser imaging system as previously described. 20 Transfected cells grown on 2-well chamber slides were fixed using 3.7% (vol/vol) buffered formalin, permeabilized in 1% Triton X-100 (in 20mM N-2-hydroxyethylpiperazine-NЈ-2-ethanesulfonic acid, 300mM sucrose, 50mM NaCl, 3mM MgCl 2 ⅐ 6 H 2 O, pH 7.0), and then blocked with 2% normal goat serum in Hanks balanced salt solution (HBSS). Cells were incubated at 4°C overnight in 2 to 5 g/mL primary antibodies (see "Antibodies") diluted in HBSS containing 1% BSA.…”

Section: Mutation N528s and Storage Of Vwf 4581mentioning

confidence: 99%

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

Haberichter

Budde²,

Obser

et al. 2010

Blood

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We 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 ␣-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. (Blood. 2010;115(22): 4580-4587)

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Section: Discussionmentioning

confidence: 97%

Section: Antibodiesmentioning

confidence: 99%

Section: Mutation N528s and Storage Of Vwf 4581mentioning

confidence: 99%

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

Haberichter

Budde²,

Obser

et al. 2010

Blood

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“…6 and Table II). As the Arg-1205 residue is not part of the recently described propeptide binding site (residues Thr-866 to Ile-907) (43), it is possible that the Arg-1205 to His mutation displays allosteric effects.…”

Section: Table II Affinity Constants For the Interactions Between Vwfmentioning

confidence: 99%

An Experimental Model to Study the in Vivo Survival of von Willebrand Factor

Lenting

Westein

Terraube

et al. 2004

Journal of Biological Chemistry

132

189

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To explore the molecular basis of von Willebrand factor (VWF) clearance, an experimental model employing VWF-deficient mice was developed. Biodistribution was examined by the injection of radiolabeled VWF, which was primarily directed to the liver with minor amounts in other organs. Disappearance of VWF from plasma was characterized by a rapid initial phase (t1 ⁄2 ␣ ‫؍‬ 13 min) and a slow secondary phase (t1 ⁄2 ␤ ‫؍‬ 3 h), with a mean residence time (MRT) of 2.8 h. A similar clearance was observed for VWF consisting of only high or low molecular weight multimers, indicating that, in our experimental model, clearance is independent of multimeric distribution. This allowed us to compare the survival of fulllength VWF to truncated variants. Deletion of both the amino-terminal D -D3 and carboxyl-terminal D4-CK domains resulted in a fragment with a similar clearance to wild-type VWF. Deletion of only the D -D3 region was associated with an almost 2-fold lower recovery and increased clearance (MRT ‫؍‬ 1.6 h), whereas deletion of only the D4-CK region resulted in a significantly reduced clearance (MRT ‫؍‬ 4.5 h, p < 0.02). These results point to a role of the D -D3 region in preventing clearance of VWF. Furthermore, replacement of D3 domain residue Arg-1205 by His resulted in a markedly increased clearance (MRT ‫؍‬ 0.3 h; p ‫؍‬ 0.004). Therefore, this mutation seems to abrogate the protective effect of the D -D3 region. In vitro analysis of this mutant also revealed a 2-fold reduced affinity for VWF propeptide at low pH, showing that mutation of Arg-1205 results not only in an increased clearance rate but is also associated with an impaired pH-dependent interaction with VWF propeptide.von Willebrand factor (VWF) 1 is a multimeric plasma protein that participates in the hemostatic process. The absence of functional VWF is associated with an abnormal bleeding tendency known as von Willebrand Disease (VWD) (1, 2). VWF contributes to hemostasis in a dual manner. First, it promotes the adhesion of platelets at sites of vascular injury by acting as a molecular bridge between the sub-endothelial collagen matrix and the platelet-surface receptor complex glycoprotein (Gp) Ib␣/IX/V (3). In addition, VWF serves as a carrier protein for coagulation factor VIII (FVIII) in the circulation. This chaperone function results in stabilization of the FVIII heterodimeric structure (4) and protection of FVIII from premature clearance by the low density lipoprotein receptor-related protein (5, 6).VWF is produced and stored in endothelial cells and megakaryocytes. It is synthesized as pre-pro-VWF, a single chain polypeptide with the domain structure

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“…68 It is noteworthy that Du et al have shown that a lentiviral vector using the ITGA2B gene promoter driving expression of a molecule encoding a fragment of the VWF propeptide and D2 domain (which helps traffic the molecule for storage within a-granules) fused to BDD FVIII successfully confined a majority of FVIII specifically within the platelet a-granule compartment of platelets to establish hemostasis for canine hemophilia A. 31,69,70 These results indicate that the addition of an a-granule targeting peptide may add an additional level of safety for treating patients with preexisting inhibitory antibodies because sequestration of FVIII within platelet a-granules may help to concentrate FVIII within granules and prevent leakage of FVIII into the plasma from the cytoplasmic compartment. This result is consistent with studies performed in mice affected with von Willebrand disease that stored FVIII less efficiently within a-granules due the absence of von Willebrand factor (carrier protein for BLOOD, 10 MARCH 2016 x VOLUME 127, NUMBER 10 PLATELET-TARGETED THERAPY 1263…”

Section: Ectopic Delivery Of Coagulation Factors From Plateletsmentioning

confidence: 99%

Megakaryocyte- and megakaryocyte precursor–related gene therapies

Wilcox

2016

Blood

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Hematopoietic stem cells (HSCs) can be safely collected from the body, genetically modified, and re-infused into a patient with the goal to express the transgene product for an individual’s lifetime. Hematologic defects that can be corrected with an allogeneic bone marrow transplant can theoretically also be treated with gene replacement therapy. Because some genetic disorders affect distinct cell lineages, researchers are utilizing HSC gene transfer techniques using lineage-specific endogenous gene promoters to confine transgene expression to individual cell types (eg, ITGA2B for inherited platelet defects). HSCs appear to be an ideal target for platelet gene therapy because they can differentiate into megakaryocytes which are capable of forming several thousand anucleate platelets that circulate within blood vessels to establish hemostasis by repairing vascular injury. Platelets play an essential role in other biological processes (immune response, angiogenesis) as well as diseased states (atherosclerosis, cancer, thrombosis). Thus, recent advances in genetic manipulation of megakaryocytes could lead to new and improved therapies for treating a variety of disorders. In summary, genetic manipulation of megakaryocytes has progressed to the point where clinically relevant strategies are being developed for human trials for genetic disorders affecting platelets. Nevertheless, challenges still need to be overcome to perfect this field; therefore, strategies to increase the safety and benefit of megakaryocyte gene therapy will be discussed.

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

Cited by 46 publications

References 44 publications

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

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

An Experimental Model to Study the in Vivo Survival of von Willebrand Factor

Megakaryocyte- and megakaryocyte precursor–related gene therapies

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