Cell biology of von Willebrand factor.
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Animals↗
Biomedical subjects
D D Wagner
Publications and source records attributed to D D Wagner.
PADGEM protein: a receptor that mediates the interaction of activated platelets with neutrophils and monocytes.
PADGEM (platelet activation dependent granule-external membrane protein) is an integral membrane protein of the alpha granules of platelets and Weibel-Palade bodies of endothelial cells that is expressed on the plasma membrane upon cell activation and granule secretion. Activated platelets, but not resting platelets, bind to neutrophils, monocytes, HL60 cells, and U937 cells. This interaction is inhibited by anti-PADGEM antibodies, PADGEM, and EDTA; anti-GPIIb-IIIa, anti-thrombospondin, anti-GPIV, and thrombospondin produce no effect. Neutrophils and U937 cells, in contrast to Jurkatt cells, contain PADGEM recognition sites, as shown by binding of PADGEM contained in phospholipid vesicles. These results indicate that PADGEM mediates adhesion of activated platelets to monocytes and neutrophils. Therefore, PADGEM shares not only structural but also functional homology with ELAM-1 and MEL-14, members of a new family of vascular cell adhesion molecules.
Antibodies, Monoclonal↗
Plasma selenium-dependent glutathione peroxidase. Cell of origin and secretion.
Human plasma glutathione peroxidase (GSHPx) has been shown to be a glycosylated selenoprotein distinct enzymatically, structurally, and antigenically from known cellular glutathione peroxidases. The extracellular location of the enzyme and the fact that it is glycosylated suggested that it is a secreted protein. Utilizing mutually non-cross-reactive antibodies to human cellular and plasma GSHPx, we conducted a search to determine the tissue of origin for plasma GSHPx. The cells screened were endothelial cells because they are the main source of extracellular superoxide dismutase, HL-60 cells (myeloid cell line) because they are the main source of extracellular H2O2, and Hep G2 cells (hepatic cell line) because they are the source of many plasma proteins. Human umbilical vein endothelial cells were metabolically labeled with either [35S]methionine or [75Se]selenious acid, and HL-60 cells and Hep G2 cells were metabolically labeled with [75Se]selenious acid. Proteins were immunopurified from the labeled cells and their media with either anti-red blood cell (RBC) GSHPx IgG or with anti-plasma GSHPx IgG. Utilizing anti-RBC GSHPx IgG, only the cellular form of the enzyme was precipitated from all the cells tested but not from their media. When anti-plasma GSHPx IgG was applied to the cells and their media, a selenoprotein was precipitated only from the media of Hep G2 cells. When Hep G2 cells were incubated in the presence of the carboxylic ionophore monensin, an intracellular selenoprotein could be detected using anti-plasma GSHPx IgG. The precipitation of the cellular form from all three cell types was partially inhibited by preincubation of the anti-RBC GSHPx IgG with purified RBC GSHPx while the precipitation of the selenoprotein from the medium of Hep G2 cells by anti-plasma GSHPx IgG was prevented by preincubation of the antibody with purified plasma GSHPx. We suggest that plasma GSHPx is synthesized by and secreted from hepatic cells. This is, to the best of our knowledge, the only known selenoprotein with a defined function that has been shown to be synthesized for secretion by mammalian cells.
Cell Line↗
In vitro multimerization of von Willebrand factor is triggered by low pH. Importance of the propolypeptide and free sulfhydryls.
Large von Willebrand factor (vWf) multimers are assembled by the formation of disulfide bonds between dimers in trans Golgi and post-Golgi compartments. We were able to reproduce this process in a cell-free system using purified vWf dimers. The multimers formed in vitro extended to 5 x 10(6) Da and were similar in size distribution to those secreted constitutively by endothelial cells in culture. Multimerization occurred only at acidic pH with an optimum at pH 5.8 and needed the continued presence of an acidic pH for it to proceed. Pro-vWf dimers multimerized, whereas mature dimers failed to assemble into large multimers. Multimerization required the propolypeptide to be a contiguous part of pro-vWf subunits since free propolypeptide did not promote multimerization of mature dimers. In addition, multimers formed in the presence of both pro-vWf and mature vWf dimers incorporated only pro-vWf dimers. Two out of six available monoclonal antibodies to the prosequence inhibited multimerization. Multimerization was also inhibited by chemical blocking of free sulfhydryl(s). The free sulfhydryl(s) were localized to the mature region of the pro-vWf by examination of the mature subunit and the propolypeptide derived by proteolytic cleavage of pro-vWf subunits.
Cells, Cultured↗
von Willebrand factor biosynthesis and partitioning between constitutive and regulated pathways of secretion after thrombin stimulation.
The major part of von Willebrand factor (vWf) synthesized in cultured endothelial cells is secreted constitutively without stimulation and consists of all multimeric forms of vWf. In contrast, stimulation with secretagogues such as thrombin results in the release of vWf from the storage pool, the Weibel-Palade bodies which contain only the largest, most biologically potent multimeric forms of vWf. We wished to determine whether the signal for release of vWf might also function as a signal for replenishment of the vWf by enhancing de novo biosynthesis and if replenishment of the vWf storage pool involved a diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. vWf mRNA and protein levels in unstimulated human umbilical vein endothelial cells were compared with cells that were briefly stimulated with 1 U/mL thrombin for 15 minutes and then incubated without thrombin for periods up to 72 hours. A comparison was also made between unstimulated cells and cells continuously exposed to thrombin for up to 48 hours. Thrombin stimulation, brief or continuous, had no significant effect on subsequent biosynthesis of vWf protein or vWf-specific mRNA. Since thrombin releases vWf only from the storage pool, we examined the possibility of diversion of newly synthesized vWf from the constitutive pathway to the regulated pathway. Cells were pulse-labeled, incubated for 15 minutes with and without thrombin, chased for various periods in unlabeled media, and briefly restimulated with thrombin. No significant redistribution of vWf between the two pathways was observed as a result of thrombin stimulation for the time periods tested.(ABSTRACT TRUNCATED AT 250 WORDS)
Blotting, Northern↗
Differing polarity of the constitutive and regulated secretory pathways for von Willebrand factor in endothelial cells.
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.
Ammonium Chloride↗
Mediation of fibrin-induced release of von Willebrand factor from cultured endothelial cells by the fibrin beta chain.
The exposure of endothelial cells (EC) to fibrin has been shown to stimulate the rapid release of von Willebrand factor (vWf) from storage sites in Weibel-Palade bodies. We have now investigated the fibrin structural features required for stimulation of release. The role of fibrinopeptide cleavage was examined by preparing fibrin with thrombin to remove both fibrinopeptide A (FPA) and fibrinopeptide B (FPB) and with reptilase or Agkistrodon contortrix procoagulant to selectively remove FPA or FPB, respectively. vWf release was found to require FPB cleavage, whereas removal of FPA and Factor XIIIa cross-linking of fibrin were without effect. The dependence of release on FPB cleavage suggested that a site involving the NH2 terminus of the beta chain could mediate vWf secretion. To test this hypothesis, B beta chain derivatives were prepared and examined for their capacity to induce release. Purified B beta chain had no effect on release at a concentration of 20 nM but stimulated release from 26 +/- 6% of cells at 200 nM, the maximum solubility. However, after thrombin cleavage of FPB, release occurred from 36 +/- 9% of cells at 20 nM and from 60 +/- 7% at 200 nM, both significantly greater than before cleavage. FPB and B beta 1-42 showed no activity, whereas beta 15-42, representing the NH2 terminus of the thrombin cleaved beta chain, stimulated significant release at concentrations of 0.1 and 1 mM. We conclude that FPB cleavage from fibrin is required for stimulation of vWf release from EC and that this is mediated by a site that includes the NH2 terminus of the beta chain.
Cells, Cultured↗
PADGEM (GMP140) is a component of Weibel-Palade bodies of human endothelial cells.
PADGEM protein (PADGEM), also known as GMP140, is a platelet alpha-granule membrane protein that is translocated to the external membrane after platelet activation. Although the biosynthesis of this protein was originally thought to be confined to megakaryocytes, the synthesis of PADGEM in endothelial cells was recently demonstrated (McEver et al: Blood 70:1974a, 1987). We now describe the subcellular localization of this protein in endothelial cells. Immunofluorescence staining of permeabilized human umbilical vein endothelial cells with KC4, a well characterized monoclonal antibody to PADGEM, showed positively stained elongated structures similar in distribution and shape to Weibel-Palade bodies. Their identity as Weibel-Palade bodies was confirmed by double label immunofluorescence using KC4 and a polyclonal antiserum to von Willebrand factor (vWf), a protein known to be specifically stored in these organelles. All Weibel-Palade bodies were found to contain PADGEM. In contrast to strong perinuclear staining produced with anti-vWf antibodies, no significant perinuclear staining was obtained with KC4, indicating that relatively little PADGEM is present in the endoplasmic reticulum and in the Golgi apparatus. In endothelial cells treated with secretagogues that stimulate vWf release the elongated structures positive for PADGEM disappeared, further identifying these structures as Weibel-Palade bodies. This observation extends the parallels between Weibel-Palade bodies and alpha-granules and suggests a possible functional association between vWf and PADGEM.
Calcimycin↗
Temperature-sensitive steps in the secretory pathway for von Willebrand factor in endothelial cells.
The effect of reduced temperature on the post-translational processing and stimulated release of von Willebrand factor (vWf) from human umbilical vein endothelial cells was studied. Following pulse-labeling, cells were incubated for 4 h at 18 degrees C or 20 degrees C. Post-translational processing was reversibly arrested at the dimer stage, dimers were composed of Endo H-sensitive precursor subunits, and no vWf was detected in the culture medium. This block was reversible, since warming cells to 37 degrees C relieved it and resulted in the appearance of fully processed vWf in the cells and the culture medium. The same results were obtained when cells were incubated with carbonyl cyanide m-chlorophenol hydrazone or dinitrophenol which inhibit mitochondrial oxidative phosphorylation, known to block exit of secretory proteins from the endoplasmic reticulum (ER). This indicated that ER exit is not required for the complete dimerization of vWf. Reduced temperature (18 degrees C and 20 degrees C) also reversibly and nearly completely inhibited the secretagogue-induced release of vWf from Weibel-Palade bodies without affecting the microtubular cytoskeleton. We add reduced temperature to the list of useful tools for the study of the vWf secretory pathway in endothelial cells.
Calcimycin↗
Molecular and cellular biology of von Willebrand factor.
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Cell Adhesion↗
Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.
The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.
Antibodies↗
Fibrin induces release of von Willebrand factor from endothelial cells.
Addition of fibrinogen to human umbilical vein endothelial cells in culture resulted in release of von Willebrand factor (vWf) from Weibel-Palade bodies that was temporally related to formation of fibrin in the medium. Whereas no release occurred before gelation, the formation of fibrin was associated with disappearance of Weibel-Palade bodies and development of extracellular patches of immunofluorescence typical of vWf release. Release also occurred within 10 min of exposure to preformed fibrin but did not occur after exposure to washed red cells, clot liquor, or structurally different fibrin prepared with reptilase. Metabolically labeled vWf was immunopurified from the medium after release by fibrin and shown to consist of highly processed protein lacking pro-vWf subunits. The contribution of residual thrombin to release stimulated by fibrin was minimized by preparing fibrin clots with nonstimulatory concentrations of thrombin and by inhibiting residual thrombin with hirudin or heating. We conclude that fibrin formed at sites of vessel injury may function as a physiologic secretagogue for endothelial cells causing rapid release of stored vWf.
Blood Coagulation↗
von Willebrand factor released from Weibel-Palade bodies binds more avidly to extracellular matrix than that secreted constitutively.
Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies of cultured endothelial cells following treatment with a secretagogue (Sporn et al, Cell 46:185, 1986). These multimers were shown by immunofluorescent staining to bind more extensively to the extracellular matrix of human foreskin fibroblasts than constitutively secreted vWf, which is composed predominantly of dimeric molecules. Increased binding of A23187-released vWf was not due to another component present in the releasate, since releasate from which vWf was adsorbed, when added together with constitutively secreted vWf, did not promote binding. When iodinated plasma vWf was overlaid onto the fibroblasts, the large forms bound preferentially to the matrix. These results indicated that the enhanced binding of the vWf released from the Weibel-Palade bodies was likely due to its large multimeric size. It appears that multivalency is an important component of vWf interaction with the extracellular matrix, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the Weibel-Palade bodies, therefore, is not only especially suited for platelet binding, but also for interaction with the extracellular matrix.
Calcimycin↗
Intact microtubules are necessary for complete processing, storage and regulated secretion of von Willebrand factor by endothelial cells.
The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for 1 h with colchicine (10(-6) M) or nocodozole (10(-6) M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One-hour incubation in the absence of the drug was sufficient for microtubules to reform and restore the full capacity of the cells to release vWf. Long-term incubation (24 h) of endothelial cells with microtubule-destabilizing agents had a profound effect on vWf distribution. In control cells, vWf was localized to organelles in the perinuclear region (i.e., endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm, and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. Since Weibel-Palade bodies specifically contain the large multimers, we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.
Benzimidazoles↗
Topology and order of formation of interchain disulfide bonds in von Willebrand factor.
Interchain disulfide bonds between the subunits in von Willebrand factor (vWf) dimers and in vWf multimers have been studied using some unique features of the cultured human umbilical vein endothelial cell system. Ammonium chloride inhibition of multimerization of vWf allowed selective examination of vWf dimeric molecules, and monoclonal antibody against the vWf propolypeptide was used to separate pro-vWf dimers from mature dimers. After cleavage of dimers and multimers with Staphylococcus aureus V-8 protease, the location of interchain disulfide bonds in amino (N)-terminal or carboxyl (C)-terminal fragments was determined by gel electrophoresis under reduced and nonreduced conditions. The first interchain disulfide bonds formed during dimerization are in the C-terminal region of the subunits, whereas interdimer disulfide bonds are located in the N-terminal portion. These data confirm recent electron microscopic projections of disulfide bond locations and provide support to the hypothetical role of the propolypeptide in the multimerization process.
Ammonium Chloride↗
Inducible secretion of large, biologically potent von Willebrand factor multimers.
von Willebrand factor (vWf) secreted constitutively by human endothelial cells was compared to that released from Weibel-Palade bodies after stimulation. The majority of constitutively secreted molecules were dimeric and contained both pro-vWf and mature subunits. In contrast, the vWf released by the calcium ionophore A23187 or thrombin consisted of only very large multimers of mature subunits. The large multimers are known to be more active in in vitro platelet binding assays, and their absence in vivo results in a bleeding disorder. Endothelial cells therefore concentrate a special subclass of very large and biologically potent vWf multimers in Weibel-Palade bodies, presumably available for release in response to vascular injury.
Calcimycin↗
Propolypeptide of von Willebrand factor circulates in blood and is identical to von Willebrand antigen II.
The generally mild bleeding disorder of von Willebrand disease is associated with abnormalities of two distinct plasma proteins, the large multimeric von Willebrand factor (vWF), which mediates platelet adhesion, and von Willebrand antigen II (vW AgII), which is of unknown function. The two proteins were found to have a common biosynthetic origin in endothelial cells and megakaryocytes, which explains their simultaneous absence in the severe form of this hereditary disease. Shared amino acid sequences from a 100-kilodalton plasma glycoprotein and from vW AgII are identical to amino acid sequences predicted from a complementary DNA clone encoding the 5' end of vWF. In addition, these proteins have identical molecular weights and immunologic cross reactivities. Monoclonal antibodies prepared against both proteins recognize epitopes on the pro-vWF subunit and on a 100-kilodalton protein that are not present on the mature vWF subunit in endothelial cell lysates. In contrast, polyclonal antibodies against vWF recognize both pro-vWF and vWF subunits. Thus, the 100-kilodalton plasma glycoprotein and vW AgII are identical proteins and represent an extremely large propolypeptide that is first cleaved from pro-vWF during intracellular processing and then released into plasma.
Amino Acid Sequence↗
Initial glycosylation and acidic pH in the Golgi apparatus are required for multimerization of von Willebrand factor.
Two conditions were identified that interfered with the complex polymerization process in biosynthesis of von Willebrand factor (vWf). Treatment of human umbilical vein endothelial cells with tunicamycin inhibited N-linked glycosylation of nascent vWf and the resulting pro-vWf monomers failed to dimerize. The single subunits accumulated in the endoplasmic reticulum and were neither processed further nor secreted. In the presence of a weak base (ammonium chloride or chloroquine), interdimer disulfide bond formation was inhibited in a dose-dependent manner. This process appeared therefore to be pH sensitive and likely to be initiated in the acidic trans-Golgi apparatus (Anderson, R. G. W., and R. K. Pathak, 1985, Cell, 40: 635-643). The weak base had no obvious effect on the other processing steps, i.e. dimerization, complex carbohydrate formation and sulfation, and produced only slight inhibition of prosequence cleavage. On the other hand, the weak base interfered with the targeting of newly synthesized vWf into Weibel-Palade bodies, with all of the vWf being secreted constitutively and none stored in the Weibel-Palade bodies. In summary, initial glycosylation of the nascent vWf protein and low pH in the trans-Golgi apparatus were important conditions for the successful polymerization of human vWf. Genetic defects disrupting any one of these conditions could result in the phenotype of von Willebrand disease.
Acetylglucosaminidase↗