Processing and Characterization of Recombinant von Willebrand Factor Expressed in Different Cell Types Using a Vaccinia Virus Vector

1992 ◽  
Vol 67 (01) ◽  
pp. 154-160 ◽  
Author(s):  
P Meulien ◽  
M Nishino ◽  
C Mazurier ◽  
K Dott ◽  
G Piétu ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Von Willebrand Factor ◽  
Human Fibroblasts ◽  
Active Form ◽  
Cell Types ◽  
Biologically Active ◽  
L Cells ◽  
Von Willebrand ◽  
Different Cell Types ◽  
Willebrand Factor

SummaryThe cloning of the cDNA encoding von Willebrand factor (vWF) has revealed that it is synthesized as a large precursor (pre-pro-vWF) molecule and it is now clear that the prosequence or vWAgll is responsible for the intracellular multimerization of vWF. We have cloned the complete vWF cDNA and expressed it using a recombinant vaccinia virus as vector. We have characterized the structure and function of the recombinant vWF (rvWF) secreted from five different cell types: baby hamster kidney (BHK), Chinese hamster ovary (CHO), human fibroblasts (143B), mouse fibroblasts (L) and primary embryonic chicken cells. Forty-eight hours after infection, the quantity of vWF antigen found in the cell supernatant varied from 3 to 12 U/dl depending on the cell type. By SDS-agarose gel electrophoresis, the percentage of high molecular weight forms of vWF varied from 39 to 49% relative to normal plasma for BHK, CHO, 143B and chicken cells but was less than 10% for L cells. In all cell types, the two anodic subbands of each multimer were missing. The two cathodic subbands were easily detected only in BHK and L cells. By SDS-PAGE of reduced samples, pro-vWF was present in similar quantity to the fully processed vWF subunit in L cells, present in moderate amounts in BHK and CHO and in very low amounts in 143B and chicken cells. rvWF from all cells bound to collagen and to platelets in the presence of ristocetin, the latter showing a high correlation between binding efficiency and degree of multimerization. rvWF from all cells was also shown to bind to purified FVIII and in this case binding appeared to be independent of the degree of multimerization. We conclude that whereas vWF is naturally synthesized only by endothelial cells and megakaryocytes, it can be expressed in a biologically active form from various other cell types.

scholarly journals Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2349-2355 ◽  
Author(s):  
A Rehemtulla ◽  
RJ Kaufman
Keyword(s):  
Aspartic Acid ◽  
Von Willebrand Factor ◽  
Transmembrane Domain ◽  
Active Form ◽  
Proteolytic Processing ◽  
Processing Enzymes ◽  
Dibasic Amino Acid ◽  
Processing Activity ◽  
Von Willebrand ◽  
Willebrand Factor

Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (- LysArg-) at residue 763. By coexpression of vWF and various propeptide processing enzymes in COS-1 cells, we here demonstrate that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme PACE (furin). Processing of vWF by the yeast homologue of PACE, Kex2, was inefficient and not specific for the authentic site. Two additional recently identified mammalian propeptide-processing enzymes PC2 and PC3 had no detectable vWF-processing activity. The inability of PC2 and PC3 to cleave vWF was apparently not due to the absence of a transmembrane domain, since deletion of the transmembrane domain from PACE resulted in a secreted form which retained its propeptide processing activity within the secretory apparatus. The inability of PC2 and PC3 to process wild-type vWF or any of the vWF mutants described suggests different members of subtilisin-related propeptide- processing enzyme family have evolved to selectively recognize and cleave specific sets of substrates. In addition to paired dibasic residues at the propeptide cleavage site, many proteins, including vWF, also contain an arginine at the P4 position. We have generated mutant vWFs with substitutions at the P2 lysine and/or the P4 arginine to investigate their significance in substrate specificity. A conservative substitution of the P4 arginine by lysine resulted in a decrease in vWF processing by PACE, as did a nonconservative substitution to alanine. Substitution of the P2 lysine to aspartic acid decreased processing and little or no processing was detected when both the P4 and P2 were mutated to lysine and aspartic acid, respectively. These data indicate that both the P4 arginine and the P2 lysine play an important role in substrate recognition by PACE.

Alteration in GPIIb/IIIa Binding of VWD-Associated von Willebrand Factor Variants with C-Terminal Missense Mutations

2019 ◽  
Vol 119 (07) ◽  
pp. 1102-1111
Author(s):  
Gesa König ◽  
Tobias Obser ◽  
Olivier Marggraf ◽  
Sonja Schneppenheim ◽  
Ulrich Budde ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Active Form ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Missense Mutations ◽  
Primary Hemostasis ◽  
Platelet Receptor ◽  
Adhesive Proteins ◽  
Von Willebrand ◽  
Willebrand Factor

AbstractThe platelet receptor glycoprotein (GP) IIb/IIIa, formed by integrins αIIb and β3, plays an important role in platelet adhesion and aggregation. Its major binding site is the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. Upon platelet activation, inside-out signaling activates the complex permitting binding to RGD motif containing proteins, such as von Willebrand factor (VWF). VWF is a large multidomain plasma GP essential to primary hemostasis, which can directly interact with platelets because it exhibits binding sites for GPIbα and GPIIb/IIIa in its A1 and C4 domain, respectively. A vast variety of VWF variants have been identified in which domain-specific mutations affect distinct functions of VWF but reduced GPIIb/IIIa binding has barely been studied so far. Here, we strived to investigate the influence of C domain mutations, which have been identified in patients diagnosed with von Willebrand disease (VWD), on VWF–GPIIb/IIIa interaction. To determine binding to membrane-incorporated GPIIb/IIIa in the absence of GPIbα, we developed and validated a cell-based binding assay which uses HEK293 cells stably expressing a constitutively active form of the GPIIb/IIIa receptor complex on their plasma membrane. By employing this assay, we measured GPIIb/IIIa binding of 14 VWF C domain mutants identified in VWD patients. Mutants p.Cys2257Arg, p.Gly2441Cys, p.Cys2477Tyr, and p.Pro2722Ala exhibited significantly reduced binding. Summarizing, we have developed a useful research tool to specifically investigate GPIIb/IIIa interaction with its protein binding partners and identified four VWF variants that exhibit impaired GPIIb/IIIa binding. At least in the homozygous state, this defect could contribute to the VWD phenotype.

Acute Activation of Endothelium Induces Circulation of Active Von Willebrand Factor in HELLP Sydrome.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2658-2658
Author(s):  
Janine J. Hulstein ◽  
Pieter J. van Runnard Heimel ◽  
Arie Franx ◽  
Peter J. Lenting ◽  
Hein W. Bruinse ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Pregnant Women ◽  
Von Willebrand Factor ◽  
Hellp Syndrome ◽  
Active Form ◽  
Immunosorbant Assay ◽  
Binding Conformation ◽  
Elevated Liver Enzymes ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand Factor (VWF) is unable to interact spontaneously with platelets under physiological conditions. To induce this interaction, a conversion of the VWF A1-domain into a Glycoprotein Ibα (GpIbα)-binding conformation is required. We recently described a nanobody, designated AU/VWFa-11 that preferentially recognizes the GpIbα-binding conformation (Hulstein et al. 2005 Blood, in press). Using an AU/VWFa-11 based immunosorbant assay, we found that the active form of VWF circulates in VWD type 2B and Thrombotic Thrombocytopenic Purpura. Although these diseases have different phenotypical appearances, both are characterized by thrombocytopenia. Another disease associated with a low platelet count is HELLP (hemolysis, elevated liver enzymes and low platelets) syndrome, a severe form of preeclampsia that compromises pregnancy. In this study, we have compared the levels of activated VWF in the plasma of healthy pregnant women (n=9), patients suffering from preeclampsia (n=6) and patients with HELLP syndrome (n=14) at similar gestational age. In HELLP syndrome, the levels of activated VWF were increased 1.8-fold compared to healthy volunteers (p=0.0001). Moreover, these levels were also increased 1.5-fold compared to the patients suffering from preeclampsia. In order to get insight into the origin of the increased activated VWF levels, a number of other parameters were determined. VWF antigen levels were found to be increased in normal pregnancy (165% ± 32.5), as expected. In preeclampsia and HELLP syndrome these levels were even further increased (178% ± 89.8 and 338% ± 89.3, respectively). In healthy pregnant women and in preeclampsia, VWF propeptide levels were concomitantly increased. In contrast, in HELLP syndrome propeptide levels were increased up to 3-fold compared to propeptide levels in healthy pregnant women (p<0.0001) and 1.8-fold compared to patients with preeclampsia (p<0.04). When we calculated the propeptide /VWF antigen ratio, this ratio was found to be within the normal range in healthy pregnant women (0.11 ± 0.04) and preeclampsia (0.12 ± 0.03). However, this ratio was significantly increased in HELLP syndrome (0.17 ± 0.05, p<0.05). Because these increased ratios may represent secretion of VWF from the Weibel Palade bodies, these data suggests that acute activation of the endothelium occurs in HELLP syndrome and is the source of circulating active VWF. Therefore, we determined the amounts of active VWF secreted by human umbilical vein endothelial cells (HUVEC) in vitro. Medium containing constitutionally secreted VWF and medium from PMA stimulated endothelial cells was collected and the amount of active VWF was measured in the AU/VWFa-11-based immunosorbant assay. A 1.6-fold increase in activated VWF levels was found in medium containing constitutionally secreted VWF compared to normal pool plasma (p<0.05). The amount of activated VWF was increased even up to 2.2-fold in medium of the stimulated endothelial cells (p<0.001) indicating that activation of endothelium in HELLP could indeed result in increased levels of activated VWF. In conclusion, acute activation of the endothelium in HELLP syndrome results in increased amounts of activated VWF. This might well explain the thrombocytopenia associated with HELLP syndrome.

scholarly journals The role of the 5′-flanking region in the cell-specific transcription of the human von Willebrand factor gene

1993 ◽  
Vol 293 (3) ◽  
pp. 641-648 ◽  
Author(s):  
V Ferreira ◽  
Z Assouline ◽  
J L Schwachtgen ◽  
B R Bahnak ◽  
D Meyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Von Willebrand Factor ◽  
Transcriptional Activity ◽  
Cell Types ◽  
Flanking Region ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Cat Gene ◽  
Cpae Cells

Transcriptional regulation of the human von Willebrand factor (vWF) gene was investigated in calf pulmonary artery endothelial (CPAE), HeLa, COS 7 and Hep G2 cells. Various lengths of flanking sequences extending up to 2123 bp 5′ of the transcription initiation site and containing 19 bp of the first exon, were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and these constructs were assayed in transient transfection assays. Sequences up to 89 bp upstream of the cap site showed transcriptional activity in all cell types. Sequences between -147 and -419 bp markedly reduced CAT activity in CPAE cells and abolished it in other cell lines. A domain from -592 to -810 bp generated low levels of expression only in CPAE cells. This transcriptional activity was repressed with constructs containing 1041 to 1240 bp upstream of the cap site. Endothelial cell-specific transcription was restored by a construct that contained 1286 bp upstream of the cap site. The additional 46 bp upstream of the negative regulatory domain were within the 5′ end of an inverse human Alu-family DNA repeat. RNAase-protection assays confirmed the correct transcriptional initiation. The sequence between -89 and -420 contained at least one negative regulatory element able to repress the CAT gene expression controlled by the heterologous thymidine kinase promoter in all cell types. A construct that included the sequence from -89 to -1286 bp increased the transcriptional activity directed by the thymidine kinase promoter only in CPAE cells. These results indicate that negative and positive elements in the 5′-flanking region interact to regulate vWF gene expression.

Determinants of von Willebrand Factor Function

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. SCI-17-SCI-17
Author(s):  
Cécile V. Denis ◽  
Olivier D. Christophe ◽  
Peter J. Lenting
Keyword(s):  
Von Willebrand Factor ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Active Form ◽  
Regulatory Pathways ◽  
Spontaneous Interaction ◽  
Platelet Binding ◽  
Platelet Thrombus ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Abstract SCI-17 Platelet thrombus formation is a multistep process involving a number of molecular players, including von Willebrand factor (vWF). vWF is an adhesive multimeric protein that acts as a molecular bridge between the subendothelium and the glycoprotein Ib/IX/V receptor complex on platelets. Furthermore, vWF promotes the expansion of the platelet plug by cross-linking platelets via binding to integrin αIIbβ3. It is important to keep in mind that before participating in the formation of platelet-rich thrombi, vWF and platelets coexist in the circulation without interacting with each other. For optimal function, it is essential that vWF-platelet interactions occur in a timely way, that is, not too early and not too late. In the former case, spontaneous interaction may lead to intravascular thrombosis, while in the latter, hemorrhagic complications may arise. In order to reach this fine balance of regulation, a number of mechanisms are in place that contribute to control vWF function. In the last few years, considerable progress has been made in either revealing or better understanding such determinants. Physiologically, most of these mechanisms are dedicated to the prevention of excessive vWF-platelet interactions. These include shear-stress-mediated vWF conformational changes that lead to exposure or nonexposure of the platelet-binding site and cleavage sites on the vWF molecule. Intramolecular shielding of the vWF-platelet binding domain by adjacent domains also contributes to vWF reactivity. A major determinant of vWF function is related to its multimeric size, which can be controlled by proteolysis by ADAMTS13 and by other proteases, such as granzyme B or neutrophil elastase. The thiol reductase activity of ADAMTS13 toward vWF also contributes to multimer regulation. Finally, interaction of vWF with plasma proteins such as β2-glycoprotein I, or with endothelial proteins such as osteoprotegerin and galectins, can also participate in keeping vWF from binding excessively to platelets. Pathologically, dysregulations of the above-mentioned mechanisms may lead to either an overly active form of vWF or, in contrast, to an inactive protein. Additional determinants can also become prominent, such as the presence of mutations in the vWF sequence, leading to the genetic bleeding disorder known as von Willebrand disease. Determinants affecting vWF-platelet function have been studied extensively, as vWF participation in platelet thrombus formation is its best known and most important role. However, rather fascinating mechanisms have been identified that can modulate other functions of vWF. An example thereof is the recent identification of vWF cleavage by ADAM28 expressed by carcinoma cells in order to escape the proapoptotic action of vWF on such cells. Another example is the regulation of the Factor VIII binding capacity of vWF that can be controlled by cleavage by granzyme M. Identification of these various regulatory pathways now opens new avenues to act upon in order to better control the fine balance between the prohemostatic and the prothrombotic roles of vWF. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Preferred sequence requirements for cleavage of pro-von Willebrand factor by propeptide-processing enzymes

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2349-2355 ◽  
Author(s):  
A Rehemtulla ◽  
RJ Kaufman
Keyword(s):  
Aspartic Acid ◽  
Von Willebrand Factor ◽  
Transmembrane Domain ◽  
Active Form ◽  
Proteolytic Processing ◽  
Processing Enzymes ◽  
Dibasic Amino Acid ◽  
Processing Activity ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Maturation of pro-von Willebrand factor (vWF) to its active form requires proteolytic processing after a pair of dibasic amino acids (- LysArg-) at residue 763. By coexpression of vWF and various propeptide processing enzymes in COS-1 cells, we here demonstrate that vWF is preferentially processed by the paired dibasic amino acid-cleaving enzyme PACE (furin). Processing of vWF by the yeast homologue of PACE, Kex2, was inefficient and not specific for the authentic site. Two additional recently identified mammalian propeptide-processing enzymes PC2 and PC3 had no detectable vWF-processing activity. The inability of PC2 and PC3 to cleave vWF was apparently not due to the absence of a transmembrane domain, since deletion of the transmembrane domain from PACE resulted in a secreted form which retained its propeptide processing activity within the secretory apparatus. The inability of PC2 and PC3 to process wild-type vWF or any of the vWF mutants described suggests different members of subtilisin-related propeptide- processing enzyme family have evolved to selectively recognize and cleave specific sets of substrates. In addition to paired dibasic residues at the propeptide cleavage site, many proteins, including vWF, also contain an arginine at the P4 position. We have generated mutant vWFs with substitutions at the P2 lysine and/or the P4 arginine to investigate their significance in substrate specificity. A conservative substitution of the P4 arginine by lysine resulted in a decrease in vWF processing by PACE, as did a nonconservative substitution to alanine. Substitution of the P2 lysine to aspartic acid decreased processing and little or no processing was detected when both the P4 and P2 were mutated to lysine and aspartic acid, respectively. These data indicate that both the P4 arginine and the P2 lysine play an important role in substrate recognition by PACE.

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