scholarly journals Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.

1989 ◽  
Vol 108 (4) ◽  
pp. 1283-1289 ◽  
Author(s):  
L A Sporn ◽  
V J Marder ◽  
D D Wagner
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Molecular Form ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Polycarbonate Membrane ◽  
Secretory Pathways ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) is secreted from endothelial cells by one of two pathways-a constitutive pathway and a regulated pathway originating from the Weibel-Palade bodies. The molecular form of vWf from each of these pathways differs, with the most biologically potent molecules being released from Weibel-Palade bodies (Loesberg, C., M. D. Gonsalves, J. Zandbergen, C. Willems, W. G. Van Aken, H. V. Stel, J. A. Van Mourik, and P. G. DeGroot. 1983. Biochim. Biophys. Acta. 763:160-168; Sporn, L. A., V. J. Marder, and D. D. Wagner. 1987. Cell. 46:185-190). We investigated the polarity of the two secretory pathways using human umbilical vein endothelial cells cultured on polycarbonate membrane filters which allowed sampling of media from both the apical and basolateral compartments. After metabolic labeling of cells, vWf (constitutively secreted during a 10-min period or released during a 10-min treatment with a secretagogue) was purified from the apical and basolateral chambers and subjected to gel analysis. Approximately equal amounts of vWf were constitutively secreted into both chambers, and therefore this secretory pathway appeared to be nonpolarized. On the contrary, an average of 90% of vWf released from Weibel-Palade bodies after treatment with the calcium ionophore A23187 or PMA appeared in the basolateral chamber, indicating that the regulated pathway of secretion is highly polarized. Thrombin, a secretagogue which promotes disruption of the endothelial monolayer, led to release of vWf from cells with no apparent polarity. The presence of microtubule-depolymerizing agents nocodazol and colchicine inhibited the polarized release of vWf. Ammonium chloride treatment did not disrupt the polarity of the regulated secretory pathway, indicating that maintenance of low pH in intracellular compartments was not required for the polarized delivery of preformed Weibel-Palade bodies to the plasma membrane.

scholarly journals Endothelial Cell “Memory” of Inflammatory Stimulation: Human Venular Endothelial Cells Store Interleukin 8 in Weibel-Palade Bodies

1998 ◽  
Vol 188 (9) ◽  
pp. 1757-1762 ◽  
Author(s):  
Barbara Wolff ◽  
Alan R. Burns ◽  
James Middleton ◽  
Antal Rot
Keyword(s):  
Endothelial Cells ◽  
Secretory Pathway ◽  
De Novo ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Inflammatory Stimuli ◽  
Regulated Secretory Pathway ◽  
Von Willebrand ◽  
Willebrand Factor

The expression and secretion of interleukin (IL)-8, the prototype member of the C-X-C subfamily of chemokines, can be induced by diverse inflammatory stimuli in many cells, including endothelial cells (EC). Upon de novo synthesis, IL-8 localizes intracellularly in the Golgi apparatus, from where it is secreted. In addition to this constitutive secretory pathway, we describe a depot storage and separate regulated secretory pathway of IL-8 in EC. The prolonged stimulation of primary human EC with inflammatory mediators resulted in the accumulation of IL-8 in Weibel-Palade bodies, where it colocalized with von Willebrand factor. IL-8 was retained in these storage organelles for several days after the removal of the stimulus and could be released by EC secretagogues such as phorbol myristate acetate, the calcium ionophore A23187, and histamine. These findings suggest that storage of IL-8 in Weibel-Palade bodies may serve as the EC “memory” of a preceding inflammatory insult, which then enables the cells to secrete IL-8 immediately without de novo protein synthesis.

VON WILLEBRAND FACTOR (vWF) PRO-POLYPEPTIDE IS REQUIRED FOR vWF MULTIMER FORMATION

1987 ◽  
Author(s):  
C L Verweij ◽  
M Hart ◽  
H Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Expression System ◽  
Proteolytic Processing ◽  
Information Encoding ◽  
Interchain Interactions ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor (vWF) is a multimeric plasma glycoprotein synthesized in vascular endothelial cells as a pre-pro-polypeptide with a highly repetitive domain structure, symbolized by the formula:(H)-D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-(0H).A heterologous expression system, consisting of a monkey kidney cell line (C0S-1), transfected with full-length vWF cDNA, is shown to mimic the constitutively, secretory pathway of vWF in endothelial cells. The assembly of pro-vWF into multimers and the proteolytic processing of these structures is found to oro-ceed along the following, consecutive steps. Pro-vWF subunits associate to form dimers, a process that does not involve the pro-polypeptide of pro-vWF. This observation is derived from transfection of C0S-1 cells with vWF cDNA, lacking the genetic information encoding the pro-polypeptide, composed of the domains D1 and D2. Pro-vWF dimers are linked intracellularly to form a regular series of multimeric structures that are secreted and cannot be distinguished from those released constitutively by endothelial cells. The presence of the pro-polypeptide, embedded in pro-vWF, is obligatory for multimerization since the deletion mutant lacking the D1 and D2 domains fails to assemble beyond the dimer stage. It is argued that the D domains are involved in interchain interactions.

scholarly journals von Willebrand factor multimerization and the polarity of secretory pathways in endothelial cells

Blood ◽  
2016 ◽  
Vol 128 (2) ◽  
pp. 277-285 ◽  
Author(s):  
Mafalda Lopes da Silva ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Secretory Pathways ◽  
Key Points ◽  
Von Willebrand ◽  
Willebrand Factor

Key Points The 3 endothelial secretory pathways—constitutive, basal, and regulated—release VWF in different multimeric states. Apical- and basolaterally-released VWF follow different secretory pathways, thus releasing differentially multimerized protein.

Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3322-3328 ◽  
Author(s):  
Patrick André ◽  
Cécile V. Denis ◽  
Jerry Ware ◽  
Simin Saffaripour ◽  
Richard O. Hynes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Glycoprotein ◽  
Ionophore A23187 ◽  
Stop And Go ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Low Shear

Abstract With the use of intravital microscopy, a new type of platelet–endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds−1) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm2·s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded withA23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in β3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet–endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibα (GPIbα) because the adhesion of murine platelets expressing exclusively the human GPIbα could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbα. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbα. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.

scholarly journals Multimeric composition of endothelial cell-derived von Willebrand factor

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2074-2076 ◽  
Author(s):  
HM Tsai ◽  
RL Nagel ◽  
VB Hatcher ◽  
II Sussman
Keyword(s):  
Endothelial Cell ◽  
Von Willebrand Factor ◽  
Culture Media ◽  
Umbilical Vein ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Buffer System ◽  
Ionophore A23187 ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography. Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells. The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma. When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet. The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes. It remained the only multimer in media conditioned for up to three days. Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition. The small multimer was never detected in the EC- conditioned media. These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation.

scholarly journals The roles of protein kinase C and intracellular Ca2+ in the secretion of von Willebrand factor from human vascular endothelial cells

1992 ◽  
Vol 286 (2) ◽  
pp. 631-635 ◽  
Author(s):  
M A Carew ◽  
E M Paleolog ◽  
J D Pearson
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase ◽  
Von Willebrand Factor ◽  
Secretory Pathway ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Selective Inhibition ◽  
Von Willebrand ◽  
Willebrand Factor

Secretion of von Willebrand factor (vWf) glycoprotein from storage granules in human umbilical-vein endothelial cells was studied in vitro. Either elevation of intracellular Ca2+ concentration ([Ca2+]i) with a Ca2+ ionophore or activation of protein kinase (PK) C by phorbol 12-myristate 13-acetate caused vWf secretion, and together the agents acted synergistically. However, when vWf release was stimulated by receptor-mediated agonists, selective inhibition of PKC had no effect on histamine-induced secretion and significantly elevated thrombin-induced secretion. Furthermore, ATP, which efficiently elevates [Ca2+]i in these cells, was a very poor effector of vWf release. We conclude that elevation of [Ca2+]i by physiological agonists is necessary for vWf release, but other signalling mechanisms, as yet uncharacterized, but not due to PKC activation, are required for full induction of the secretory pathway.

scholarly journals Proteolytic cleavage of human von Willebrand factor induced by enzyme(s) released from polymorphonuclear cells

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1281-1285
Author(s):  
EA Thompson ◽  
MA Howard
Keyword(s):  
Von Willebrand Factor ◽  
Cell Activation ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Polymorphonuclear Cells ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Von Willebrand ◽  
Willebrand Factor

In vivo fragmentation of the von Willebrand factor antigen (vWF:Ag) molecule has been demonstrated on radiocrossed immunoelectrophoresis (CIE) in the plasma from patients with disseminated intravascular coagulation, in factor VIII concentrates, and in normal serum. Experiments reported here show that polymorphonuclear (PMN) cells contain a non-calcium-dependent protease(s) that when released and incubated with vWF:Ag results in an additional vWF:Ag peak on radio- CIE. Production of fragments of vWF:Ag by incubation with PMN cells occurred in a time-dependent manner. The protease(s) responsible was inhibited by diisopropyl fluorophosphate, soybean trypsin inhibitor, and aprotinin, but not by benzamidine, azide, epicron, or hirudin. Citrate, EDTA, and leupeptin also had no effect on the PMN cell enzyme's activity, indicating that the enzyme(s) is not calcium dependent. The PMN cell enzyme responsible for vWF:Ag fragmentation is located intracellularly and released by freezethaw lysis or cell activation by calcium or the calcium ionophore A23187.

scholarly journals von Willebrand factor is present on the surface of platelets stimulated in plasma by ADP

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1362-1366
Author(s):  
B Adelman ◽  
P Carlson ◽  
P Powers
Keyword(s):  
Binding Site ◽  
Von Willebrand Factor ◽  
Platelet Rich Plasma ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Surface Binding ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Willebrand Factor

von Willebrand factor (vWf) can bind to glycoprotein (GP) IIb/IIIa on activated platelets. The significance of this interaction is unclear, however, because it has not been possible to detect vWf binding to GPIIb/IIIa on platelets stimulated in plasma. We have developed an indirect, flow cytometry assay that uses fluorescein-labeled antibodies to detect vWf and fibrinogen on platelets. Using this assay, we found vWf on the surface of platelets stimulated in plasma by ADP. The number of platelets that bound vWf increased in proportion to ADP concentration and incubation time. Washed platelets in a protein-free buffer activated by 1 mumol/L calcium ionophore A23187 or 10 mumol/L ADP also bound vWf, suggesting that we were detecting surface binding of alpha-granule-derived vWf. Monoclonal antibodies against the vWf binding site on GPIb (6D1) and the vWf and fibrinogen binding sites on GPIIb/IIIa (LJP5 and LJ-CP8, respectively) were used to characterize the mechanism of vWf binding to stimulated platelets. Ristocetin- induced binding of vWf was inhibited by 6D1, and ADP-induced binding of fibrinogen was inhibited by LJ-CP8. None of these antibodies inhibited ADP-induced vWf binding. Aspirin and prostaglandin E1 also inhibited ADP-induced binding of vWf in platelet-rich plasma. During platelet activation in plasma, vWf derived from alpha-granules becomes bound to the platelet surface possibly being transferred already associated with a binding site.

Von Willebrand Factor in Cultured Human Vascular Endothelial Cells from Adult and Umbilical Cord Arteries and Veins

1986 ◽  
Vol 56 (02) ◽  
pp. 189-192 ◽  
Author(s):  
Pauline B van Wachem ◽  
Jan Hendrik Reinders ◽  
Marijke F van Buul-Wortelboer ◽  
Philip G de Groot ◽  
Willem G van Aken ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Vena Cava ◽  
Ionophore A23187 ◽  
Cultured Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Arteries And Veins

SummaryEndothelial cells were cultured from various human arteries and veins, obtained from adult individuals and from umbilical cords. We compared the storage and secretion of von Willebrand factor by endothelial cells from umbilical veins with that of endothelial cells cultured from a number of adult vessels, including aorta, arteria iliaca, vena saphena magna and vena cava. There were no differences in the way the cultured endothelial cells handled the von Willebrand factor they synthesized. Endothelial cells from the various vessels responded to stimuli in secreting stored von Willebrand factor. The cells also responded to thrombin and ionophore A23187 in producing enhanced amounts of prostacyclin. Thus, cultured umbilical vein endothelial cells have properties that are very similar to those of cultured endothelial cells of various other origins. It is concluded that foetal venous cells provide a representative model for studies of endothelial cell von Willebrand factor biosynthesis and prostacyclin production.

Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3322-3328 ◽  
Author(s):  
Patrick André ◽  
Cécile V. Denis ◽  
Jerry Ware ◽  
Simin Saffaripour ◽  
Richard O. Hynes ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Platelet Adhesion ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Platelet Glycoprotein ◽  
Ionophore A23187 ◽  
Stop And Go ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
Low Shear

With the use of intravital microscopy, a new type of platelet–endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds−1) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm2·s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded withA23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in β3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet–endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibα (GPIbα) because the adhesion of murine platelets expressing exclusively the human GPIbα could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbα. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbα. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.

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