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.     

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.     

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.     

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.     

Composition of the von Willebrand factor storage organelle (Weibel- Palade body) isolated from cultured human umbilical vein endothelial cells

von Willebrand factor (VWF) is a large, adhesive glycoprotein that is biosynthesized and secreted by cultured endothelial cells (EC). Although these cells constitutively release VWF, they also contain a storage pool of this protein that can be rapidly mobilized. In this study, a dense organelle fraction was isolated from cultured umbilical vein endothelial cells by centrifugation on a self-generated Percoll gradient. Stimulation of EC by 4-phorbol 12-myristate 13-acetate (PMA) resulted in the disappearance of this organelle fraction and the synchronous loss of Weibel-Palade bodies as judged by immunoelectron microscopy. Electrophoretic and serologic analyses of biosynthetically labeled dense organelle fraction revealed that it is comprised almost exclusively of VWF and its cleaved pro sequence. These two polypeptides were similarly localized exclusively to Weibel-Palade bodies by ultrastructural immunocytochemistry. The identity of the dense organelle as the Weibel-Palade body was further established by direct morphological examination of the dense organelle fraction. The VWF derived from this organelle is distributed among unusually high molecular weight multimers composed of fully processed monomeric subunits and is rapidly and quantitatively secreted in unmodified form after PMA stimulation. These studies: establish that the Weibel-Palade body is the endothelial-specific storage organelle for regulated VWF secretion; demonstrate that in cultured EC, the VWF concentrated in secretory organelles is of unusually high molecular weight and that this material may be rapidly mobilized in unmodified form; imply that proteolytic processing of VWF involved in regulated secretion takes place after translocation to the secretory organelle; provide a basis for further studies of intracellular protein trafficking in EC.     

Carboxypeptidase E does not mediate von Willebrand factor targeting to storage granules

Sorting of von Willebrand factor precursor (pro-vWf) from the trans-Golgi network to secretory granules (Weibel-Palade bodies) is critical for its conversion to the biologically active highly multimeric form, as well as for regulated secretion by the endothelial cells. When expressed in hormone-secretory cells, vWf is also recognized as a stored protein and is directed to storage granules. Recently, carboxypeptidase E (CPE) was proposed as a granular sorting receptor for prohormones (Cool et al., Cell 88: 73, 1997). To explore whether CPE is also involved in pro-vWf sorting, we initially examined its expression in human umbilical vein endothelial cells. A specific message for CPE and the protein itself were detected making it a plausible candidate as a targeting receptor for vWf in endothelium. To investigate this possibility, we used mice lacking CPE. The highly multimeric forms, subunit composition and plasma levels of vWf in CPE-deficient mice were similar to those of their wild-type littermates. vWf was also found in alpha-granules of platelets and in Weibel-Palade bodies of endothelial cells obtained from the CPE-deficient mice. Furthermore, vWf was released from the cultured CPE-deficient endothelial cells after stimulation with a secretagogue. We conclude that CPE is not essential for sorting vWf to the regulated secretory pathway. Thus, a CPE-independent mechanism must exist for protein sorting to storage granules.     

Induction of specific storage organelles by von Willebrand factor propolypeptide

Endothelial cells store the multimeric adhesive glycoprotein von Willebrand factor (vWf), which promotes the formation of a platelet plug at the site of vessel injury. To investigate the packaging of vWf into the granules called Weibel-Palade bodies, we expressed pro-vWf cDNA and cDNA lacking the prosequence in a variety of cell lines. Storage granules formed only in cells that contain a regulated pathway of secretion. Furthermore, packaging required the prosequence. Pro-vWf, lacking the C-terminal region involved in interchain disulfide bonding, formed granules. We conclude that the signal for storage is universal in that an adhesive glycoprotein can be stored by a hormone-secreting cell; the storage of vWf is independent of its covalent multimeric structure; the unusual rod shape of Weibel-Palade bodies is due to vWf; and the vWf propolypeptide is necessary for the formation of vWf storage granules.     

Regulated von Willebrand factor (vWf) secretion is restored by pro-vWf expression in a transfectable endothelial cell line

Von Willebrand factor (vWf) is a glycoprotein involved in primary hemostasis and synthesized in endothelial cells (EC). vWf is stored in secretory granules specific for EC called Weibel-Palade bodies (WPb). Studies on the molecular mechanisms of vWf storage and acute release are hampered by the limitations of the available endothelial cell culture models. We created a suitable model by stable transfection of the vWf-negative ECV304 endothelial cell line with pro-vWf cDNA. Pro-vWf was normally cleaved to mature vWf and stored in WPb. Acute vWf release occurred in response to the calcium ionophore A23187. Thus, vWf expression is sufficient to restore functional secretory granules in ECV304 cells. We used this model to study the role of WPb in the storage of tissue-type plasminogen activator (t-PA), a key fibrinolytic enzyme that is acutely released by EC, but whose intracellular storage compartment is still a matter of debate. We observed that restoration of WPb in ECV304 cells results in the targeting of t-PA to these storage granules.     

Biosynthesis of von Willebrand protein by human endothelial cells: processing steps and their intracellular localization

Biosynthesis of von Willebrand protein by human umbilical vein endothelial cells involved distinct processing steps marked by the presence of several intermediate molecular species. Examination of endoglycosidase H sensitivity of these intracellular intermediates indicated that the processing steps occurred in at least two separate cellular compartments. In the pre-Golgi apparatus (most probably the endoplasmic reticulum), the high mannose carbohydrates were added onto the precursor monomer chains and the 260,000-mol-wt monomers dimerized by interchain disulfide bond formation. The other processing steps have been localized to the Golgi apparatus and later compartments (e.g., Weibel-Palade bodies). High mannose carbohydrate was converted to the complex type, leading to the appearance of a larger precursor subunit of 275,000 mol wt. The 275,000-mol-wt species was not formed if carbohydrate processing was inhibited by the ionophore monensin. From the large pool of dimers of precursor subunits, the high molecular weight multimers were built. These dimer molecules appeared to have free sulfhydryls which might have been involved in the interdimer disulfide bond formation. Simultaneously with multimerization, the precursor subunits were cleaved to the 220,000-mol-wt form. The cleavage of the pro-sequence was not likely to be an absolute requirement for von Willebrand protein multimerization or secretion, as the 275,000-mol-wt precursor subunit was present in secreted high molecular weight multimers of the protein.     

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.     

An Endothelial Storage Granule for Tissue-Type Plasminogen Activator

In previous studies we have shown that, after stimulation by a receptor ligand such as thrombin, tissue-type plasminogen activator (tPA) and von Willebrand factor (vWf) will be acutely released from human umbilical vein endothelial cells (HUVEC). However, the mechanisms involved in the secretion of these two proteins differ in some respects, suggesting that the two proteins may be stored in different secretory granules.

By density gradient centrifugation of rat lung homogenates, a particle was identified that contained nearly all tPA activity and antigen. This particle had an average density of 1.11–1.12 g/ml, both in Nycodenz density gradients and in sucrose density gradients. A similar density distribution of tPA was found for a rat endothelial cell line and for HUVEC. After thrombin stimulation of HUVEC to induce tPA secretion, the amount of tPA present in high-density fractions decreased, concomitant with the release of tPA into the culture medium and a shift in the density distribution of P-selectin.

vWf, known to be stored in Weibel-Palade bodies, showed an identical distribution to tPA in Nycodenz gradients. In contrast, the distribution in sucrose gradients of vWf from both rat and human lung was very different from that of tPA, suggesting that tPA and vWf were not present in the same particle.

Using double-immunofluorescence staining of HUVEC, tPA- and vWf-containing particles showed a different distribution by confocal microscopy. The distribution of tPA also differed from the distribution of tissue factor pathway inhibitor, endothelin-1, and caveolin. By immunoelectronmicroscopy, immunoreactive tPA could be demonstrated in small vesicles morphologically different from the larger Weibel-Palade bodies. It is concluded that tPA in endothelial cells is stored in a not-previously-described, small and dense (d = 1.11– 1.12 g/ml) vesicle, which is different from a Weibel-Palade body.

     

Regulatory Components of the Alternative Complement Pathway in Endothelial Cell Cytoplasm,Factor H and Factor I,Are Not Packaged in Weibel-Palade Bodies

It was recently reported that factor H, a regulatory component of the alternative complement pathway, is stored with von Willebrand factor (VWF) in the Weibel-Palade bodies of endothelial cells. If this were to be the case, it would have therapeutic importance for patients with the atypical hemolytic-uremic syndrome that can be caused either by a heterozygous defect in the factor H gene or by the presence of an autoantibody against factor H. The in vivo Weibel-Palade body secretagogue, des-amino-D-arginine vasopressin (DDAVP), would be expected to increase transiently the circulating factor H levels, in addition to increasing the circulating levels of VWF. We describe experiments demonstrating that factor H is released from endothelial cell cytoplasm without a secondary storage site. These experiments showed that factor H is not stored with VWF in endothelial cell Weibel-Palade bodies, and is not secreted in response in vitro in response to the Weibel-Palade body secretagogue, histamine. Furthermore, the in vivo Weibel-Palade body secretagogue, DDAVP does not increase the circulating factor H levels concomitantly with DDAVP-induced increased VWF. Factor I, a regulatory component of the alternative complement pathway that is functionally related to factor H, is also located in endothelial cell cytoplasm, and is also not present in endothelial cell Weibel-Palade bodies. Our data demonstrate that the factor H and factor I regulatory proteins of the alternative complement pathway are not stored in Weibel-Palade bodies. DDAVP induces the secretion into human plasma of VWF —- but not factor H.     

Biogenesis of von Willebrand factor-containing organelles in heterologous transfected CV-1 cells.

Von Willebrand factor (vWF) is a multimeric protein involved in the adhesion of platelets to an injured vessel wall. vWF is synthesized by the endothelial cell and the megakaryocyte as a precursor protein (pro-vWF) that consists of four repeated domains, denoted D1-D2-D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2. Previously, we have defined the domains on the pro-vWF molecule involved in dimerization as well as the domains involved in multimer assembly of vWF dimers. In the endothelial cell, part of the vWF multimers is stored in specialized organelles, the Weibel-Palade bodies. By using immunoelectron microscopy, we demonstrate that upon expression of full-length vWF cDNA, vWF-containing organelles are encountered in monkey kidney CV-1 cells that are morphologically similar to the endothelial-specific Weibel-Palade bodies. Expression in CV-1 cells of a series of vWF cDNA deletion mutants, lacking one or more domains, revealed that only those vWF mutant proteins that are able to assemble into multimers are encountered in dense-cored vesicles. Our data show that this process is independent of a particular domain on vWF and indicate that a 'condensed', multimeric vWF is required for targeting to the Weibel-Palade body.     

The physiological function of von Willebrand's factor depends on its tubular storage in endothelial Weibel-Palade bodies

Weibel-Palade bodies are the 1-5 microm long rod-shaped storage organelles of endothelial cells. We have investigated the determinants and functional significance of this shape. We find that the folding of the hemostatic protein von Willebrand's factor (VWF) into tubules underpins the rod-like shape of Weibel-Palade bodies. Further, while the propeptide and the N-terminal domains of mature VWF are sufficient to form tubules, their maintenance relies on a pH-dependent interaction between the two. We show that the tubular conformation of VWF is essential for a rapid unfurling of 100 microm long, platelet-catching VWF filaments when exposed to neutral pH after exocytosis in cell culture and in living blood vessels. If tubules are disassembled prior to exocytosis, then short or tangled filaments are released and platelet recruitment is reduced. Thus, a 100-fold compaction of VWF into tubules determines the unique shape of Weibel-Palade bodies and is critical to this protein's hemostatic function.     

A 130-kDa protein on endothelial cells binds to amino acids 15-42 of the B beta chain of fibrinogen.

Factors which stimulate the release of von Willebrand factor (vWf) from endothelial cell Weibel-Palade bodies and which induce the expression of the leukocyte-binding adhesion molecule P-selectin (PADGEM, GMP-140, CD62) on the endothelial cell surface remain incompletely characterized. Fibrin but not fibrinogen is a potent stimulus for the release of stored von Willebrand factor from endothelial cells. Removal of fibrinopeptides A and B from fibrinogen occurs during the formation of fibrin, and the removal of fibrinopeptide B is a requirement for fibrin to induce vWf secretion. The cleavage of fibrinopeptide A by reptilase enzyme forms a fibrin gel yet it is incapable of stimulating Weibel-Palade body degranulation. As a consequence of removing fibrinopeptide B, B beta 15-42 becomes the new NH2 terminus of the beta chain of fibrin. We have shown that the peptide B beta 15-42 in solution inhibits the release of vWf stimulated by fibrin. In addition, B beta 15-42 coupled to ovalbumin supports the binding and spreading of endothelial cells, while a scrambled form of this peptide coupled to the same carrier does not. We investigated whether these determinants near the amino terminus of the beta chain of fibrin bind to a specific protein on the surface of endothelial cells. A 130-kDa protein was isolated from surface-labeled human umbilical vein endothelial cells by specific binding to B beta 15-42 immobilized on Sepharose. This glycoprotein was eluted with the B beta 15-42 peptide in solution but not with the scrambled form of this peptide. The fibrin-derived peptides B beta 19-26 and B beta 37-56-cysteine were also incapable of eluting the 130-kDa protein bound to immobilized B beta 15-42 as were the arginine-glycine-aspartic acid-serine RGDS tetrapeptide and EDTA. The 130-kDa protein is recognized neither by antibodies to the known integrins found on endothelial cells nor by antibodies to CD31 (endoCAM, PECAM-1), a member of the immunoglobulin family of receptors found on endothelial cells. The beta chain of fibrin thus contains a sequence near its amino terminus which specifically binds to what is likely a novel endothelial cell surface protein. This glycoprotein may promote endothelial cell adhesion to fibrin during the wound healing process and is a candidate for a receptor involved in fibrin-mediated release of Weibel-Palade bodies from endothelial cells.     

Subunit composition of oligomeric human von Willebrand factor

The oligomerization of human endothelial cell-synthesized von Willebrand factor (vWf) has been studied by gel chromatography in columns of Sephacryl S-500 and by discontinuous agarose gel electrophoresis. A quantitative recovery of high Mr vWf oligomers has been obtained after binding to a monoclonal anti-vWf-Sepharose adduct. This reagent has been used to analyze gel filtration chromatographic elution profiles of [35S]methionine-labeled culture medium and cell lysate. It was determined that high Mr oligomers are present in endothelial cell lysates as well as in the medium overlying these cells and are composed of Mr 225,000 subunits. When vWf oligomers were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of a reducing agent, the Mr 240,000 subunit (provWf) was not observed to oligomerize beyond the dimer stage to a significant degree. Therefore, vWf oligomerization appears to be facilitated by conversion of provWf subunits to mature vWf subunits, most likely by proteolytic removal of sequences unique to the intracellular precursor.     

Endothelium specific Weibel-Palade bodies in a continuous human cell line,EA.hy926

Summary Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.     

Rod-shaped tubulated bodies in the endothelia of mesenteric arteries of hypertensive rats.

Weibel-Palade bodies were found in the endothelial cells of the mesenteric arteries of hypertensive rats. They were spherical or rod-shaped and bounded by a single membrane. They measured approximately 0.1 to 0.2 mu in diameter and up to 0.4 mu in length. The majority of them showed microtubular subunits, measuring about 200 A in width. These bodies are thought to originate from Golgi apparatus.     

How to roll an endothelial cigar: the biogenesis of Weibel-Palade bodies

Weibel-Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar-shaped membrane-bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P-selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non-covalent interactions between pro- and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrand's disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.     

The propeptide of von Willebrand factor independently mediates the assembly of von Willebrand multimers

The biosynthesis of von Willebrand Factor (vWF) by vascular endothelial cells involves a complex series of processing steps that includes proteolytic cleavage of a 741-residue propeptide and the assembly of disulfide-linked multimers. Using a model system in which experimentally altered vWF cDNAs are expressed in COS-1 cells, we have shown that the vWF propeptide contains determinants that govern the assembly of vWF multimers. Furthermore, the role of the propeptide (in the assembly process) does not require it to be a contiguous part of the pro-vWF primary structure, since independently expressed propeptide was shown to promote the assembly of mature vWF subunits into multimers. Pulse-chase experiments indicated that the independently expressed propeptide formed a transient association with the mature vWF subunit inside the cell. Thus, it appears that the vWF propeptide segment can act in "trans" to direct the assembly of disulfide-linked vWF multimers.