Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Biggelaar, Maartje van den; Bouwens, Eveline A.; Kootstra, Neeltje A.; Hebbel, Robert P.; Voorberg, Jan; Mertens, Koen

doi:10.3324/haematol.13427

Cited by 34 publications

(39 citation statements)

References 48 publications

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“…This suggests that round WPBs are equally well released when compared to elongated WPB. This is in line with previous observations that WPBs containing FVIII, leading to the formation of spherical WPBs, can still release their cargo (Van den Biggelaar et al, 2009;Bouwens et al, 2011). Spherical WPBs in canine endothelial cells bearing the p.Tyr87Ser mutation, are rapidly releasing their cargo upon stimulation with appropriate secretagogues (Haberichter et al, 2005).…”

Section: Discussionsupporting

confidence: 79%

Storage and secretion of naturally occurring von Willebrand factor A domain variants

et al. 2014

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SummaryVon Willebrand disease (VWD) is a bleeding disorder characterized by reduced plasma von Willebrand factor (VWF) levels or functionally abnormal VWF. Low VWF plasma levels in VWD patients are the result of mutations in the VWF gene that lead to decreased synthesis, impaired secretion, increased clearance or a combination thereof. However, expression studies of variants located in the A domains of VWF are limited. We therefore characterized the biosynthesis of VWF mutations, located in the VWF A1-A3 domains, that were found in families diagnosed with VWD. Human Embryonic Kidney 293 (HEK293) cells were transiently transfected with plasmids encoding full-length wild-type VWF or mutant VWF. Six mutations in the A1-A3 domains were expressed. We found that all mutants, except one, showed impaired formation of elongated pseudo-Weibel-Palade bodies (WPB). In addition, two mutations also showed reduced numbers of pseudo-WPB, even in the heterozygous state, and increased endoplasmic reticulum retention, which is in accordance with the impaired regulated secretion seen in patients. Regulated secretion upon stimulation of transfected cells reproduced the in vivo situation, indicating that HEK293 cells expressing VWF variants found in patients with VWD can be used to properly assess defects in regulated secretion.

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Section: Discussionsupporting

confidence: 79%

Storage and secretion of naturally occurring von Willebrand factor A domain variants

et al. 2014

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“…These cells are a good alternative for human umbilical vein endothelial cells; they are easily isolated from peripheral blood (19,20) and are endothelial progenitor cells with all the typical endothelial characteristics, including formation of Weibel-Palade bodies (20,25). Stimulation of BOECs with histamine resulted in the release of VWF multimers, of which some remained anchored to the surface of the endothelial cells.…”

Section: Vwf Strings Anchored To the Surface Of Boecs Are Specificallymentioning

confidence: 99%

Local Elongation of Endothelial Cell-anchored von Willebrand Factor Strings Precedes ADAMTS13 Protein-mediated Proteolysis

Ceunynck

Rocha

Feys

et al. 2011

Journal of Biological Chemistry

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Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. By following changes in VWF string length, we demonstrated that VWF strings are cleaved multiple times, successively shortening string length in the function of time and generating fragments ranging in size from 5 to over 100 m. These are larger than generally observed in normal plasma, indicating that further proteolysis takes place in circulation. Interestingly, in 89% of all cleavage events, VWF strings elongate precisely at the cleavage site before ADAMTS13 proteolysis. These local elongations are a general characteristic of VWF strings, independent of the presence of ADAMTS13. Furthermore, large elongations, ranging in size from 1.4 to 40 m, occur at different sites in space and time. In conclusion, ADAMTS13-mediated proteolysis of VWF strings under flow is preceded by large elongations of the string at the cleavage site. These elongations may lead to the simultaneous exposure of many exosites, thereby facilitating ADAMTS13-mediated cleavage.von Willebrand factor (VWF) 6 is a large multimeric glycoprotein that plays an important role in hemostasis (1). Apart from serving as a protecting carrier for factor VIII, the main function of VWF is mediating initial platelet adhesion to the damaged vessel wall by forming a bridge between subendothelial collagen and platelets in circulation (2-4). VWF is synthesized by endothelial cells (EC) and megakaryocytes as a multimeric protein ranging in size from 500 to more than 20,000 kDa (2, 5). VWF is secreted in blood either constitutively or upon stimulation from Weibel-Palade bodies in EC or from ␣-granules in platelets. Stimulation of EC results in the release of ultralarge (UL), hyper-reactive VWF multimers (Ͼ20,000 kDa), some of which remain attached to the endothelial surface. If unprocessed, these UL-VWF multimers can spontaneously bind and agglutinate platelets generating widespread microthrombi in circulation, leading to thrombotic thrombocytopenic purpura (6 -8). In normal hemostasis, UL-VWF multimers are rapidly cleaved by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif, member 13) into smaller but still hemostatically active multimers (9). The interaction between ADAMTS13 and VWF occurs through a unique mechanism that involves conformational changes in VWF. The scissile bond in the VWF A2 domain (Tyr-1605-Met-1606 bond) is cryptic in the folded VWF molecule and only becomes accessible for ADAMTS13 proteolysis when VWF undergoes a conformational change (10, 11).In the absence of ADAMTS13, UL-VWF multimers decorated with platelets (VWF strings) persist on the endothelial surface both in vitro and in vivo for several minutes (12, 13). Interestingly, only a subset of VWF strings is decorated with platelets. These VWF strings can form bu...

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“…In particular, hematopoietic stem cells (HSCs) merit consideration as target cells for hemophilia gene therapy since they can self-renew and differentiate into all the distinct lympho-hematopoietic lineages. An additional benefit of targeting HSCs is the possibility to induce immune hypo-responsiveness or, ideally, immunological tolerance to the transgene product, as was demonstrated for FVIII [1,77] Megakaryocytes and their platelet progeny are attractive targets for hemophilia A gene therapy, because platelets play a crucial role in primary hemostasis and it is beneficial to express FVIII in cells that synthesize and store its natural carrier protein VWF [1,82,83,[86][87][88][89]. Consequently, ectopic FVIII expression in megakaryocytes resulted in co-localized storage of FVIII with VWF in the α-granules of platelets, constituting a releasable pool of FVIII/VWF complexes [1,83].…”

Section: Ex Vivo LV Gene Therapy: Hscmentioning

confidence: 99%

“…An additional advantage of BOECs as hemophilia gene therapy targets that they express and store the von Willebrand factor (VWF) in their cytosolic Weibel-Palade bodies. VWF is a natural carrier protein for factor FVIII, therefore co-storage and subsequent release of the VWF/FVIII complex has the benefit of secreting large amounts of FVIII at sites of vascular injury as well as directly increasing FVIII half-life by protecting FVIII from premature clearance and proteolytic degradation [89,98]. Intravenous administration of genetically modified BOECs resulted in sustained therapeutic or even supra-physiologic (1174 ng/mL) levels of FVIII over the 5 months in vivo in NOD/SCID mice [99].…”

Section: Ex Vivo LV Gene Therapy: Msc and Boecmentioning

confidence: 99%

Recent Advances and Future Perspectives in Lentiviral Gene Therapy for Hemophilia A and B

Mátrai¹,

Chuah²,

VandenDriessche³

2012

J Genet Syndr Gene Ther

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Hemophilia A and B are rare incurable hereditary diseases due to deficiencies in clotting factor VIII (FVIII) and factor IX (FIX), respectively. These genetic defects result in potentially life-threatening, uncontrolled bleeding episodes. Current treatment by protein substitution therapy does not constitute a cure making gene therapy an attractive alternative. Lentiviral vectors (LVs) have many distinctive features that make them especially well suited for FVIII or FIX gene delivery. This includes the lack of vector-specific pre-existing immunity, their ability to permanently transduce both dividing and non-dividing cells and their capacity to readily accommodate FIX and FVIII expression cassettes, consistent with their packaging capacity of 10 kb. LVs have been used to achieve sustained therapeutic clotting factor expression levels and hemostatic correction in preclinical hemophilic mouse models. The liver has been the target organ of choice for direct in vivo LV transduction of FVIII or FIX genes, resulting in sustained therapeutic effects. Nevertheless, the potential development of neutralizing antibodies to the clotting factors following ex vivo or in vivo gene therapy with LVs can preclude long-term phenotypic correction. These risks can be minimized by preventing ectopic expression in antigen-presenting cells. LVs are well suited to deliver the clotting factor genes into hematopoietic stem/progenitor cells, allowing for stable FVIII or FIX expression upon hematopoietic reconstitution. In addition, therapeutic FVIII and FIX expression levels have been achieved in vivo after transplantation of lentivirally transduced endothelial and mesenchymal stem/progenitor cells. Current challenges relate primarily to the translation of these findings to larger preclinical animal models and ultimately to patients suffering from hemophilia.

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Storage and regulated secretion of factor VIII in blood outgrowth endothelial cells

Cited by 34 publications

References 48 publications

Storage and secretion of naturally occurring von Willebrand factor A domain variants

Storage and secretion of naturally occurring von Willebrand factor A domain variants

Local Elongation of Endothelial Cell-anchored von Willebrand Factor Strings Precedes ADAMTS13 Protein-mediated Proteolysis

Recent Advances and Future Perspectives in Lentiviral Gene Therapy for Hemophilia A and B

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