Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

scholarly journals Homocysteine inhibits von Willebrand factor processing and secretion by preventing transport from the endoplasmic reticulum

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 683-689 ◽  
Author(s):  
SR Lentz ◽  
JE Sadler
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Transport ◽  
Cell Protein ◽  
Carboxyl Terminal ◽  
Insulinoma Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Intracellular protein transport in endothelial cells is selectively inhibited by homocysteine, a thiol amino acid associated with both thrombosis and atherosclerosis. In a previous study, homocysteine decreased cell surface expression of the surface transmembrane glycoprotein thrombomodulin without decreasing secretion of another endothelial cell protein, plasminogen activator inhibitor-1. To define further the effects of homocysteine on protein transport, we examined the processing and secretion of the multimeric glycoprotein von Willebrand factor (vWF) in human umbilical vein endothelial cells. Incubation with 2 mmol/L homocysteine resulted in complete loss of vWF multimers and prevented asparagine-linked oligosaccharide maturation, propeptide cleavage, and secretion; these effects are consistent with impaired exit from the endoplasmic reticulum (ER). Dimerization was only partially inhibited, suggesting that homocysteine causes retention of provWF in the ER without preventing dimer formation. In pulse-chase incubations, intracellular provWF was degraded before exiting the ER in homocysteine-treated cells. Homocysteine also inhibited the processing and secretion of a carboxyl-terminal truncation mutant of human provWF expressed in rat insulinoma cells, indicating that retention in the endoplasmic reticulum can be mediated by regions of provWF apart from the carboxyl-terminal 20-Kd segment. These results suggest that retention of secretory proteins in the ER is regulated by redox mechanisms and imply that the intracellular transport of multiple endothelial cell proteins may be altered in patients with homocystinuria.

Requirement for Both D Domains of the Propolypeptide in von Willebrand Factor Muitimerization and Storage

1993 ◽  
Vol 70 (06) ◽  
pp. 1053-1057 ◽  
Author(s):  
Agnès M Journet ◽  
Simin Saffaripour ◽  
Denisa D Wagner
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Deletion Mutants ◽  
Relative Importance ◽  
D2 Domain ◽  
Constitutive Secretion ◽  
Von Willebrand ◽  
And Storage ◽  
Willebrand Factor

SummaryBiosynthesis of the adhesive glycoprotein von Willebrand factor (vWf) by endothelial cells results in constitutive secretion of small multimers and storage of the largest multimers in rodshaped granules called Weibel-Palade bodies. This pattern is reproduced by expression of pro-vWf in heterologous cells with a regulated pathway of secretion, that store the recombinant protein in similar elongated granules. In these cells, deletion of the vWf prosequence prevents vWf storage. The prosequence, composed of two homologous domains (D1 and D2), actively participates in vWf multimer formation as well. We expressed deletion mutants lacking either the D1 domain (D2vWf) or the D2 domain (D1vWf) in various cell lines to analyze the relative importance of each domain in vWf muitimerization and storage. Both proteins were secreted efficiently without being retained in the endoplasmic reticulum. Despite this, neither multimerized past the dimer stage and they were not stored. We conclude that several segments of the prosequence are jointly involved in vWf muitimerization and storage.

PERTURBATION OF CULTURED ENDOTHELIAL CELLS ALTERS CELLULAR VON WILLEBRAND FACTOR DISTRIBUTION

1987 ◽  
Author(s):  
J H Reinders ◽  
C L Verweii ◽  
J A V Mourlk ◽  
Ph G de Groot
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Phorbol Esters ◽  
Term Treatment ◽  
Cellular Distribution ◽  
Long Term Treatment ◽  
Von Willebrand ◽  
Willebrand Factor

Endothelial cells, cultured from human umbilical veins, synthesize von Willebrand Factor (vWF), that is stored by the cells in Weibel-Palade bodies, secreted into the medium and incorporated into the extracellular matrix underneath the cells. We have studied the influence of perturbation by phorbol esters and thrombin on the cellular distribution of vWF. Short-term (< 1 hour) treatment of endothelial cells with phorbol ester PMA or thrombin resulted in the release of cellular stored vWF. Long-term treatment with perturbants evoked a distinct change in the endothelial cell distribution of vWF, evident 24 to 48 hours after exposure. While the contents of the vWF storage vesicles were gradually restored within 48 hours, enhanced amounts of vWF were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for vWF. The number as well as the size of vWF storage granules in the cells increased after exposure to perturbants. The perturbed cells responded to stimuli in releasing stored vWF, the amounts secreted were even greater than those in control cells. The extracellular matrix lost its vWF contents as the result of PMA or thrombin treatment, by blocking deposition of vWF in the matrix, not by enhancing degradation of matrix vWF. In perfusion experiments, the adhesion of washed platelets onto the isolated matrix of perturbed cells was considerable less than that in controls. Addition of vWF to the perfusate overcame this impairment. Thus, perturbation of endothelial cells changes the cellular distribution of vWF.Supported in part by ZWO grants 13-30-31 and 13-90-91 and Netherlands Heart Foundation grant 28.004.

scholarly journals Differential Expression of CD31 and Von Willebrand Factor on Endothelial Cells in Different Regions of the Human Brain: Potential Implications for Cerebral Malaria Pathogenesis

2020 ◽  
Vol 10 (1) ◽  
pp. 31 ◽  
Author(s):  
Smart Ikechukwu Mbagwu ◽  
Luis Filgueira
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Human Brain ◽  
Expression Pattern ◽  
Von Willebrand Factor ◽  
Brain Regions ◽  
And Function ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
The Brain

Cerebral microvascular endothelial cells (CMVECs) line the vascular system of the brain and are the chief cells in the formation and function of the blood brain barrier (BBB). These cells are heterogeneous along the cerebral vasculature and any dysfunctional state in these cells can result in a local loss of function of the BBB in any region of the brain. There is currently no report on the distribution and variation of the CMVECs in different brain regions in humans. This study investigated microcirculation in the adult human brain by the characterization of the expression pattern of brain endothelial cell markers in different brain regions. Five different brain regions consisting of the visual cortex, the hippocampus, the precentral gyrus, the postcentral gyrus, and the rhinal cortex obtained from three normal adult human brain specimens were studied and analyzed for the expression of the endothelial cell markers: cluster of differentiation 31 (CD31) and von-Willebrand-Factor (vWF) through immunohistochemistry. We observed differences in the expression pattern of CD31 and vWF between the gray matter and the white matter in the brain regions. Furthermore, there were also regional variations in the pattern of expression of the endothelial cell biomarkers. Thus, this suggests differences in the nature of vascularization in various regions of the human brain. These observations also suggest the existence of variation in structure and function of different brain regions, which could reflect in the pathophysiological outcomes in a diseased state.

scholarly journals The Interaction of the Platelet and von Willebrand Factor

1977 ◽  
Author(s):  
D.N. Fass ◽  
F. Booyse ◽  
J.C. Lewis ◽  
E. J. W. Bowie
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Cultures ◽  
Von Willebrand Factor ◽  
Sandwich Technique ◽  
Aortic Endothelial Cells ◽  
Confluent Culture ◽  
Endothelial Cell Cultures ◽  
Von Willebrand ◽  
Willebrand Factor

A culture of pig aortic endothelial cells was used for experiments to investigate the interaction between the platelet and von Willebrand factor. An antibody was raised in rabbits to purified porcine von Willebrand factor. A semi-confluent culture of pig endothelial cells was stained immunofluorescently by the sandwich technique using anti-Willebrand factor IgG. An extensive extracellular meshwork of microfilaments was revealed. In endothelial cell cultures from von Willebrand pigs, no immunoreactive microfilaments were found. Immunoelectronmicro-scopy with peroxidase linked antibody has been used to identify similar filaments in normal pig endothelial cells. Washed platelets were shown to adhere to semiconfluent or damaged normal endothelial cell cultures. If the cultures had been previously incubated with anti-Willebrand factor IgG, the washed platelets did not adhere. There was no adherence of platelets when they were added to semiconfluent or damaged von Willebrand endothelial cells.

scholarly journals ABO blood group is a determinant of von Willebrand factor protein levels in human pulmonary endothelial cells

2019 ◽  
Vol 73 (6) ◽  
pp. 347-349 ◽  
Author(s):  
Glenn P Murray ◽  
Steven R Post ◽  
Ginell R Post
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Blood Group ◽  
Von Willebrand Factor ◽  
Blood Type ◽  
Abo Blood Group ◽  
Blood Group Antigens ◽  
Protein Levels ◽  
Von Willebrand ◽  
Willebrand Factor

ABO blood group antigens are expressed on von Willebrand factor (VWF) and glycosylation patterns influence circulating VWF levels. The aim of this study was to examine the effect of ABO blood type on tissue-associated VWF protein levels. We selected 35 formalin-fixed paraffin-embedded pulmonary tissue blocks obtained at autopsy from decedents who died from pulmonary embolism with known ABO blood groups (O, A, B and AB phenotypes), prepared tissue microarrays (TMAs) and stained TMAs with antibodies to VWF and platelet/endothelial cell adhesion marker-1 (PECAM-1) as a marker of endothelial cells. A pixel count scoring algorithm was used to quantify VWF and PECAM-1 staining intensity in pulmonary arterioles in digitised images. Compared with type O, non-O individuals have a significantly higher amount of endothelial cell-associated VWF protein expression. VWF protein levels associated with pulmonary vascular endothelial cells is influenced by ABO antigenic determinants.

NAADP links histamine H1 receptors to secretion of von Willebrand factor in human endothelial cells

Blood ◽  
2011 ◽  
Vol 117 (18) ◽  
pp. 4968-4977 ◽  
Author(s):  
Bianca Esposito ◽  
Guido Gambara ◽  
Alexander M. Lewis ◽  
Fioretta Palombi ◽  
Alessio D'Alessio ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Von Willebrand Factor ◽  
Calcium Release ◽  
Umbilical Vein ◽  
Pore Channel ◽  
Human Endothelial Cells ◽  
Signaling Mechanism ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract A variety of endothelial agonist–induced responses are mediated by rises in intracellular Ca2+, suggesting that different Ca2+ signatures could fine-tune specific inflammatory and thrombotic activities. In search of new intracellular mechanisms modulating endothelial effector functions, we identified nicotinic acid adenine dinucleotide phosphate (NAADP) as a crucial second messenger in histamine-induced Ca2+ release via H1 receptors (H1R). NAADP is a potent intracellular messenger mobilizing Ca2+ from lysosome-like acidic compartments, functionally coupled to the endoplasmic reticulum. Using the human EA.hy926 endothelial cell line and primary human umbilical vein endothelial cells, we show that selective H1R activation increases intracellular NAADP levels and that H1R-induced calcium release involves both acidic organelles and the endoplasmic reticulum. To assess that NAADP links H1R to Ca2+-signaling we used both microinjection of self-inactivating concentrations of NAADP and the specific NAADP receptor antagonist, Ned-19, both of which completely abolished H1R-induced but not thrombin-induced Ca2+ mobilization. Interestingly, H1R-mediated von Willebrand factor (VWF) secretion was completely inhibited by treatment with Ned-19 and by siRNA knockdown of 2-pore channel NAADP receptors, whereas thrombin-induced VWF secretion failed to be affected. These findings demonstrate a novel and specific Ca2+-signaling mechanism activated through H1R in human endothelial cells, which reveals an obligatory role of NAADP in the control of VWF secretion.

Reduction of von Willebrand Factor by Endothelial Cells

2000 ◽  
Vol 84 (09) ◽  
pp. 506-513 ◽  
Author(s):  
Lijuan Xie ◽  
Colin Chesterman ◽  
Philip Hogg
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Protein Disulfide Isomerase ◽  
Umbilical Vein ◽  
Human Umbilical Vein ◽  
Reducing Activity ◽  
Von Willebrand ◽  
Protein Disulfide ◽  
Willebrand Factor

SummaryThe haemostatic activity of plasma von Willebrand factor (vWF) is a function of multimer size. Only the large vWF multimers are effective in promoting platelet adhesion to a site of vascular injury. We observed that the conditioned medium of cultured human umbilical vein, human microvascular and bovine aortic endothelial cells contained an activity which reduced the average multimer size of plasma or purified vWF. The average multimer size of vWF produced endogenously by human umbilical vein endothelial cells was similarly reduced following secretion. The reducing activity was ablated by pre-treatment with heat or the thiol blocking agents, iodoacetamide, N-ethylmaleimide or E-64, but not by a range of specific serine-, cysteine-, aspartic-, or metalloproteinase inhibitors. Reduction in vWF multimer size was associated with formation of new thiols in vWF and there was no evidence for additional proteolytic processing of vWF. The reducing activity was associated with a protein with an anionic pI that binds heparin and contains reactive thiol(s). These results suggested that the interchain disulfide bonds that link the vWF homodimers near the N-termini were being reduced by a vWF reductase secreted by endothelial cells. In support of this hypothesis, incubation of vWF with the protein reductants, protein disulfide isomerase and thioredoxin, resulted in formation of new thiols in vWF and reduction in the average multimer size of vWF. These findings may have consequences for control of vWF haemostatic activity. Abbreviations: BAEC, bovine aortic endothelial cell; BSA, bovine serum albumin; cm, conditioned medium; GSH, reduced glutathione; HUVEC, human umbilical vein endothelial cell; HDMVEC, human dermal microvascular endothelial cell; IAM, iodoacetamide; MPB, 3-(N-maleimidylpropionyl)biocytin; NEM, N-ethylmaleimide; PDI, protein disulfide isomerase; PVDF, polyvinylidene difluoride; vWF, von Willebrand factor.

scholarly journals Relative Importance of the Glycoprotein Ib-Binding Domain and the RGD Sequence of von Willebrand Factor for Its Interaction With Endothelial Cells

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2335-2344 ◽  
Author(s):  
Christelle Perrault ◽  
Hanneke Lankhof ◽  
Dominique Pidard ◽  
Danièle Kerbiriou-Nabias ◽  
Jan J. Sixma ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Binding Domain ◽  
Rgd Sequence ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Endothelial Cell Adhesion

Endothelial cell adhesion to von Willebrand Factor is mainly mediated through an interaction between the αvβ3 integrin and the RGD sequence of von Willebrand factor (vWF ). To define the potential involvement of glycoprotein Ibα (GPIbα) as an endothelial vWF receptor, we compared cell adhesion to three recombinant vWF, the wild-type (WT-rvWF ) and two mutants, RGGS-rvWF (D1746G), defective for binding to platelet αIIbβ3, and ΔA1-rvWF with a deletion between amino-acids 478 and 716, which does not bind to platelet GPIbα. Adhesion of human umbilical vein endothelial cells to purified vWF recombinants was measured by automatized cell counting using an image analyzer. Whereas cell adhesion to ΔA1-rvWF was unchanged compared with WT-rvWF, reaching a plateau of 40% total cells at a concentration of 2.5 μg/mL rvWF, adhesion to RGGS-rvWF was only 10% of total cells. Cell stimulation by tumor necrosis factor-α (TNFα), reported to upregulate the expression of the putative endothelial GPIbα, did not modify adhesion to these rvWF. Monoclonal antibodies to vWF or GPIbα, blocking vWF interaction with platelet GPIbα, were unable to inhibit endothelial cell adhesion to rvWF. In contrast, antibody 9 to vWF, blocking the αvβ3-dependent endothelial cell adhesion to plasma vWF, inhibited adhesion to WT-rvWF as efficiently as to ΔA1-rvWF (50% inhibition at a concentration of 11 and 15 μg/mL, respectively). In agreement with the fact that endothelial cell adhesion to vWF appeared independent of the GPIbα-binding domain, we were unable to detect endothelial surface expression of GPIbα by flow cytometry or in cell lysates by immunoprecipitation followed by immunoblotting. Moreover, expression of GPIbα mRNA was undetectable in endothelial cells, even after stimulation by TNFα. These studies indicate that GPIbα is not expressed in human cultured endothelial cells and is not involved in adhesion to vWF-containing surfaces. Thus, in static conditions, cultured endothelial cells adhere to vWF through an αvβ3-dependent, GPIbα-independent mechanism.

Interaction of ADAMTS13 with the Endothelial Cell Surface.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3710-3710 ◽  
Author(s):  
Anthony Vomund ◽  
Elaine M. Majerus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Plasma Concentrations ◽  
Fluorescein Isothiocyanate ◽  
Endothelial Cell Surface ◽  
Sds Page ◽  
Carboxyl Terminal ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract ADAMTS13 proteolysis of von Willebrand Factor (VWF) generates smaller multimers that are less likely to promote blood clotting. Deficiency of ADAMTS13 leads to thrombotic thrombocytopenic purpura, a frequently fatal disease, characterized by microangiopathic hemolytic anemia and thrombocytopenia. ADAMTS13 has a characteristic domain structure that includes metalloprotease and disintegrin domains, a thrombospondin type 1 repeat (TSR), cysteine-rich and spacer domains, 7 additional TSRs, and 2 carboxyl-terminal CUB domains. The ADAMTS13 substrate, VWF, is synthesized in endothelial cells and forms large multimers within the cell. These large multimers are secreted and adhere to the endothelial cell surface where they can bind platelets flowing in blood leading to thrombosis. ADAMTS13 has been shown to cleave VWF on the surface of endothelial cells, but it is unclear if ADAMTS13 also interacts with the endothelial cell surface. We have used iodinated ADAMTS13, fluorescence-activated cell sorting (FACS), and biochemical analysis using flow conditions to demonstrate that ADAMTS13 does interact with the endothelial cell surface. Iodinated ADAMTS13 bound the endothelial cell surface at 4oC. This binding was specific since the binding was inhibited in the presence of 40-fold excess unlabeled ADAMTS13. Binding of ADAMTS13 to the cell surface was time-dependent with maximal binding occurring within two hours. The binding was also reversible; the half-time for dissociation was four hours. Binding was inhibited by heparin but not by dextran sulfate. The Kd of binding to endothelial cells was 75 nM (range 40–100 nM). FACS analysis also demonstrated binding of ADAMTS13 to endothelial cells. A fluorescein isothiocyanate labeled anti-epitope antibody bound to endothelial cells in the presence but not the absence of ADAMTS13. A polyclonal antibody to VWF inhibited binding of ADAMTS13 to VWF, but this antibody did not affect binding of ADAMTS13 to endothelial cells, suggesting that ADAMTS13 interacts with endothelial cells independently of VWF. Studies with C-terminal truncation constructs of ADAMTS13 indicated that the carboxyl-terminal TSRs are important for binding since constructs terminating with the metalloprotease domain, the first TSR, or the sixth TSR failed to compete with full-length ADAMTS13 for binding to endothelial cells, but constructs terminating with either the seventh or eighth TSR did compete for binding. Lastly, recombinant ADAMTS13 was found to be associated with endothelial cells in flow experiments. Endothelial cells were perfused with medium containing plasma concentrations of ADAMTS13 (1 μg/ml) at 10 dynes/cm2. After perfusion, the endothelial cells were washed and bound ADAMTS13 was identified from whole cell lysates through SDS-PAGE and immunoblotting with an anti-V5 epitope antibody. ADAMTS13 was found associated with endothelial cells after perfusion. Binding of ADAMTS13 to the endothelial cells prior to perfusion led to enhanced proteolysis of VWF as compared to addition of ADAMTS13 during perfusion only. This suggests that the interaction of ADAMTS13 with endothelial cells is important since it enhances the cleavage of VWF as compared to that of ADAMTS13 in solution.

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