Annu. Rev. Cell Bioi. 1990.6." 217-46 Copyright © 1990 by Annual Reviews Inc. All rights reserved


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CELL BIOLOGY OF Annu. Rev. Cell. Biol. 1990.6:217-242. Downloaded from by NORTH CAROLINA STATE UNIVERSITY on 01/17/13. For personal use only.


Center for Hemostasis and Thrombosis Research, Division of Hematology-Oncology, New England Medical Center and Department of Medicine, Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, Massachusetts 02111 KEY WORDS:

adhesive glycoprotein, protein processing, intracellular protein transport, storage granules, regulated secretion

CONTENTS INTRODUCTION..............................................................................................................


vWF DOMAIN STRUCTURE ..................................................................................... ..........


.. ,""'" Endoplasmic Reticulum."",""""""',.... ,""""""""',.,""""""""" .. "."""""""""".,." Golgi and Post-Golgi Compartments,."""",,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,."""""""""""""""

222 224 225


............................................. ..................••........................ ..... ,

vWF STORAGE",,,,,,,,,,..,,,,,,,,,,,,,,,,,,, .. ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.,,,,,,,,,,,,,,,,,,,,,.,,,,,,,,,,,,,


vWF SECRETION"., ... """"""""""""""",,,,,,.,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,.,,,


"""" " .. """""""" ..... """"""".,, .. ,,.,,""" vWf/Factor VIII Complex . . . . , ...................... " .... , . . . . . . . ..................................... v Wf Interactions with the Extracellular Matrix "" """" . . . . . ,," .. ,, .... ,,""""" ...... ,,"'" v Wf and Platelet Adhesion""""""".." ...... " .. """"""". . . . " .. "",, ..... ,,"""""",,.,,""""

232 233 233 235

""""""""""""""""'''' '''''''''''' ''' .. ,"",", ...... ,""""',"',.,.,.,.




......... .







INTRODUCTION von Willebrand factor (vWf) is an unusual adhesive glycoprotein whose function in injury repair is limited to blood vessels. It is synthesized only by endothelial cells (Jaffe et al 1973) and by megakaryocytes, which are the precursors of platelets (Nachman et al 1977). In the body, there are three pools of vWf: (a) soluble plasma vWf; (b) basement membrane 217 0743-4634/90/111 5-0217$02.00

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2 18


(extracellular matrix) vWf; and (c) cellular vWf found in storage granules of endothelial cells and platelets. All three pools appear to contribute to adhesion of platelets and formation of platelet plug during blood vessel injury (Tschopp et a11974; Turritto et a11985; Bowie et a11986; Gralnick et al 1986). In the intact vessel, the basement membrane vWf may also promote endothelial cell adhesion (Dejana et al 1989)-a difficult task considering the shear stress of blood flow and the importance of main­ taining an intact nonthrombogenic surface of endothelial vessel lining. The plasma vWf has an additional role in hemostasis in that it carries Factor VIII (antihemophilic factor) thereby protecting it against proteolysis (Zimmerman et al 19 71; Weiss et al 19 77). During vascular injury, vWf interacts with two sets of receptors on platelets. First, the solid phase vWf attaches nonactivated platelets to the injured area by binding to the glycoprotein Ib-IX complex on the platelet surface (Caen et al 19 76; Sakariassen et al 1986). Second, upon platelet activation, the soluble plasma vWf or vWf released from the granules promotes platelet aggregation by crosslinking the glycoprotein lIb-IlIa integrin receptors on neighboring platelets (Turitto et al 1984; De Marco et al 1986). While only vWf can bind glycoprotein Ib-IX, vWf competes for the IIb-IlIa receptor with other adhesive proteins such as fibrinogen and fibronectin (Plow & Ginsberg 1989). An unusual characteristic of vWf is its molecular heterogeneity. vWf is found in plasma as a series of disulfide-bonded multimers (Ruggeri & Zimmerman 1981), ranging in size from 0. 5 to 20 million Daltons, built of a single subunit (Chopek et al 1986). The largest multimers are the best suited to mediate platelet-basement membrane and platelet-platelet inter­ actions probably because of the multiplicity of available binding sites (Zimmerman et al 1983). vWf was named after Dr. Erich von Willebrand, who in 192 6 described a bleeding disorder distinct from hemophilia that was later recognized to be caused by decreased synthesis of or defects in vWf (Ruggeri & Zimmerman 1987). In its mild form, von Willebrand disease is the most common hereditary bleeding disorder in humans ( 0.8% frequency) (Rodeghiero et al 1987). It is most often transmitted in an autosomal dominant pattern. The occurrence of severe von Willebrand disease with complete lack of the protein, such as originally described by von Willebrand, indicates that, unlike other adhesive glycoproteins, vWf is not likely to be involved in embryonic development. Although the subject of many clinical studies, vWf has also proven to be an interesting molecule for cell biologists. It was used to investigate processes such as protein processing, intracellular transport, targeting to storage granules, and regulated secretion. It will be these cellular processes



plus some of the protein's interactions after secretion that will be the main topics of this review.

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vWF DOMAIN STRUCTURE vWf multimers seen in the electron microscope after rotary shadowing appear as unbranched, loosely coiled, or extended thin strands (Figure 1, top) (Fowler et a11985; Slayter et aI 1985). The extended molecules are 13 J-lm long, a size approaching the diameter of a platelet and greatly surpassing the length of a fibronectin or fibrinogen molecule. A distinctive periodicity can be recognized in these vWf molecules, as depicted sche­ matically in Figure 1. The building block protomer (between arrowheads) is identical in appearance to the smallest circulating vWf species (Fretto et al 1986), which is a dimer. The electron micrographs of vWf multimers show that the subunits are organized in a head-to-head and tail-to-tail fashion and that the molecules are composed of globular regions connected by thin flexible rods (Fowler & Fretto 1989). Circular dichroism studies of the multimers have demonstrated regions rich in IX helix and f3 pleated sheet structure, as well as a high percentage of random coil configuration (Loscalzo & Handin 1984).


�'� i







mature subunit-'OS',-,S=2-=S=-3



Factor lZIII







• Collagen

Figure 1 Domain structure of pro-vWf. Top: Electron micrograph of a rotary shadowed von Willebrand factor multi mer and a model representing its likely structure. The dimeric building block of the multimer is indicated between arrowheads. Reproduced with permission from Fowler et al 1985. Bottom: The organization of repeated units in the pro-vWf sequence. The locations of binding sites for other molecules are indicated by black rectangles and those of interchain disulfide bonds by -S-S-. Adapted from Sadler et al 1985 and Baruch et al 1 989.

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The primary structure of vWf has been determined both by direct amino acid sequence analysis (Titani et al 1986 ) and from the corresponding cD NA. vWf has been cloned from endothelial cell cD NA libraries by four different groups (Ginsburg et al 198 5; Lynch et a1198 5; Sadler et a1198 5; Verweij et al 198 5). Northern blotting analysis of endothelial cell R NA showed that vWf mR NA was about 9 kb (Sadler 1989). The vWf messenger was detected neither in kidney, nor in many cultured cell types that do not secrete vWf (Ginsburg et al 198 5) . The open reading frame i n vWf cD NA predicts a 2813-amino acid polypeptide as the primary translation product (Figure 1, bottom), and the transcriptional start site was mapped 24 5 nucleotides upstream from the initiator methionine (Collins et al 1987 ). Studies on biosynthesis of vWf by endothelial cells have predicted that the vWf subunit is first synthesized as a much larger precursor (Wagner & Marder 1983; Lynch et al 198 3 ). The existence of a prosequence was confirmed by the vWf cD NA clone analysis, which shows that the encoded primary translation product is 76 3 residues larger than the mature vWf subunit, which is known to contain only 20 50 amino acids (Titani et al 198 6). Since the predicted carboxyl­ terminal sequence of the presursor is identical to that of plasma vWf (Titani et al 1986), all proteolytic processing must occur at the amino terminal end of the precursor (Figure 1). The amino terminal extension contains a typical 22-amino-acid-long signal peptide followed by a sequence that is identical (Fay et al 1986 ) to a Mr 100, 000 plasma glyco­ protein of unknown function called vW Antigen II (Montgomery & Zimmerman 19 78). This identity explains why vW Antigen II was also previously found to be deficient in plasma of patients with severe von Willebrand disease (Montgomery & Zimmerman 1978). Therefore, it appears that after proteolytic cleavage the vWf propolypeptide circulates in plasma independently of the vWf multimers. Similar to other pro­ sequences, the propeptide is cleaved after paired basic amino acids Lys­ Arg; whether or not any further trimming of the propeptide carboxyl­ terminal end occurs is not known. Four types of repeated domains (A-D ) exhibiting internal homologies are found in the pro-vWf subunit (Figure 1) and account for approximately 90% of the protein sequence (for review see Sadler 1989 ). Most parts of the vWf sequence are rich in cysteine residues, with 8. 3% cysteine the most common amino acid in pro-vWf. The central A domains are relatively poor in cysteine residues: they contain only 6 Cys among 614 residues. All cysteines appear to be parts of disulfide bridges, since no free sulfhydryls are found either on plasma vWf multimers (Legaz et al 19 73; Kirby & Mills 19 7 5), or on the free propolypeptide (Mayadas & Wagner 1989 ). The interchain disulfide bonds that link the vWf subunits are localized to two



regions: residue 283-695 from the N-terminal end of the mature subunit, and within the last 142 residues of the mature subunit (Fretto et al 1986; Marti et al 1987). The number of interchain disulfide bonds at the two sites is not known. The mature subunit has a mass of

270 kd determined

from its amino acid sequence and an estimated carbohydrate content of 18.7%. N- and O-linked carbohydrates are most abundant at both ends of the mature subunit (Titani et al 1989). Two Arg-Gly-Asp (RGD) sequences, common among adhesive glycoproteins (Ruoslahti & Piersch­

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bacher 1986), are present in the pro-vWf molecule (Figure I). The RGD sequence present in the Cl domain is part of the binding site for integrin receptors (Plow et a1 1984; Haverstick et a1 1985; Dejana et al 1989). The significance of the other RGD sequence located on the propeptide is not known. The A domains of vWf appear to be homologous to domains found in several other proteins such as complement factors B (20-24% homology) and C2, chicken cartilage matrix protein, at collagen type VI, and the a­ chains of the leukocyte adhesion molecules Mac- I, p 150, 95, and LFA-I (reviewed in Mancuso et al 1989). A strong similarity between short seg­ ments in domains Cl and C2 to a sequence in thrombospondin was found, but the segments are too short (10 amino acids ) to conclude that the proteins are homologous (Lawler & Hynes 1986). Interestingly, the same vWf regions (residues 1179-1237 and 1330-1397) are related to two seg­ ments in the

Dictyostelium discoideum prestalk D 11 protein (Barklis et al

1985) and are included in a larger region of about 70 amino acids, which has the same 9 cysteine positions as found in the corresponding thrombo­ spondin domain and is also present in al-procollagen types I and III (Hunt & Barker 1987). The D domains of vWf show homologies to both invertebrate and vertebrate vitellogenins (Baker 1988). The biological sig­ nificance of the homologies and similarities of vWf to other proteins described above is not known. Several functional domains have been identified on the pro-vWf subunit (Figure 1,

bottom) (for review see Baruch et al 1989). Two collagen-binding

sites have been located on the mature vWf subunit (Kalafatis et al 1987; Pareti et al 1987). The corresponding vWf fragments show structural homology and bind fibrillar collagen only in their native state-not when reduced and alkylated. There may be a third binding site for collagen, located on the propolypeptide (Takagi et al 1989), although in contrast to mature vWf, the propolypeptide is not detected in collagen-containing extracellular matrices (Wagner et al 1987a ). Two heparin-binding sites were identified on the mature vWf subunit (Fretto et al 1986; Fujimura et aI 1987). Surprisingly, deletion of the Al domain, which contains only one of the two binding sites, completely abolished the binding to heparin



thus indicating that this binding site may be essential

(J. J. Sixma et aI,

unpublished information). The factor VIII binding domain is located in the N-terminal portion of the vWf subunit between amino acids 1-2 72 (Foster et al 1987). The epitope for a monoclonal antibody that inhibits factor VIII binding to vWf was mapped to residues 78- 96 (Bahou et al 1989). The vWf molecule interacts with the sulfated N-terminal portion of the factor VIn light chain (Foster et a1 1988; Lollar et aI 1988). The binding

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site for glycoprotein Ib -IX on vWf is likely to be formed by two short sequences of about 15 residues separated by 2 05 residues and maintained in close proximity by disulfide bonds (Mohri et al 1988) (Figure 1). This site appears to interact with the N-terminal portions of the O!-chain of glycoprotein Ib (Vincente et aI 1988). The interaction with glycoprotein IbIX is completely abolished by deletion of the A l domain

(1. J. Sixma et

aI, unpublished information). The binding region of vWf to glycoprotein lIb-IlIa on activated platelets is located on the C-terminal portion of the mature subunit. Since this recognition site can be specifically inhibited by RGD sequence-containing peptides, it is likely that this site includes the RGD sequence (Plow et a11 985; Haverstick et aI1 985). The RGD sequence is also likely to be the recognition site on vWf for integrins of endothelial

cells (Dejana et al 1989). The vWf gene has been localized to chromosome 12 (Ginsburg et al 1985; Verweij et aI1985), and a partial pseudogene has been identified on chromosome 2 2 (Shelton-Inloes et al 1987). The gene is 178 kb in length and contains 52 exons. The signal peptide and propolypeptide are encoded by 17 exons, and the mature subunit and 3' noncoding region are encoded by the remaining 35 exons. Segments that encode homologous domains have similar exonic structures, consistent with their evolution by gene segment duplication (Mancuso et al 1 989).

vWF BIOSYNTHESIS vWf is certainly one of the largest and most structurally complex proteins described. The primary translate undergoes many posttranslational modi­ fications in distinct cellular compartments (Figure 2) before arriving at its final multimeric form. The same processing steps are followed in both megakaryocytes and EC, independently of their vascular bed of origin (Sporn et a1 1985; van Wachem et al 1986). Since vWf synthesis is restricted to these two cell types, the presence of intracellular vWf can be utilized to study the emergence of the EC lineage in early embryos (Yablonka­ Reuveni 1989). Laboratory and clinical evidence show that the rate of vWf biosynthesis may be hormonally regulated. For example, cultured


Endoplasmic Rectic ulum Initia I N-link ed Glycoaylation



Post Goigi Extracellular

N-linked Carbohydrate Processing and Sulfation

0."" Multimerization

Dim erlzatlon

::: :::::::::::: ::::::::::::

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Q-lInked Glycosylation


Prosequence Cleavage




Figure 2 Schematic representation of the processing steps involved in vWf biosynthesis and their proposed subcellular localization. Adapted from Handin & Wagner 1 989.

endothelial cells synthesize less vWf when incubated with dexamethasone (Piovella et aI1983), whereas treatment with estrogens (Harrison & McKee 1984) increases vWf synthesis. The amount of vWf in plasma increases dramatically during pregnancy, whereas hypothyroidism is associated with acquired von Willebrand disease (Dalton et al 1987; MacCallum et al 1987). The first successful culturing of endothelial cells derived from human umbilical vein (Jaffe et al 1973; Gimborne et al 1974) initiated the early studies on vWf biosynthesis. The current availability of vWf c-DNA allows its expression in other cell types and also the production of specific mutations in vWf. This is helpful in defining the vWf domains important for multimerization and targeting of the protein to different cellular compartments. There are at least two unusual features of vWf biosynthesis. First, the interchain disulfide bonds crosslinking the vWf polymers are not all formed in the same compartment. vWf dimers are formed at the traditional oligo­ merization site-the endoplasmic reticulum (ER), whereas the multimers are formed much later in vWf processing in the trans- and post-Golgi compartments. vWf is the only protein known to form interchain disulfide bonds in these late compartments. The second unusual feature is the large size of the vWf propolypeptide (100 kd) and the fact that it takes an active part in promoting vWf multimerization and possibly in directing the protein's transport to the storage granules.



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Endoplasmic Reticulum

Pulse-chase experiments have proven helpful in determining the temporal sequence of events in vWf biosynthesis by endothelial cells. Cotrans­ lationally, 12 high mannose N-linked oligosaccharide chains are added to the mature vWf (Titani et al 1989) and at least one is added to the propolypeptide. The glycosylated pro-vWf exists transiently as a monomer, but two hr after onset of labeling, all the subunits are converted to dimers. Dimerization occurs through the formation of an unknown number of disulfide bonds between the C-terminal ends of the pro-vWf subunits (Wagner et a1 1987b; Figure 1). Several observations indicate that this process takes place in the ER. First, the large intracellular pool of dimers has incompletely processed the oligosaccharide chains susceptible to digestion by the enzyme endoglycosidase H (Wagner & Marder 1984), which localizes this pool prior to the medial Golgi (Hubbard & Ivatt 1981); second, low temperature ( l8°C) and mitochondrial oxidative phos­ phorylation inhibitors, which are known to inhibit transit from the ER to the Golgi apparatus, do not prevent dimerization while they inhibit multimerization and'secretion of the protein (Roarke et al 1989); and finally, the carboxylic ionophore monesin, which affects the functioning of the Golgi apparatus (Tartakoff 1983), completely inhibits the late pro­ cessing steps in vWf biosynthesis (complex carbohydrate formation, multi­ merization and prosequence cleavage), but has no effect on dimerization (Wagner et aI 1 985). As is the case of other proteins (Lodish et al 1983), the exit of pro­ vWf molecules from the ER appears to be the'rate-limiting step in vWf biosynthesis: As soon as the metabolically labeled vWf with complex type oligosaccharide chains is detected in the cells, it is also found constitutively secreted. This takes about 120 min from the onset of labeling in human endothelial cells (Wagner & Marder 1984). In comparison, it takes less than 30 min: for fibronectin molecules to be processed and secreted (Choi &'Hynes 1979). The exit of pro-vWf from the ER appears to be tightly controlled. Monomeric vWf with processed glycans is not detected intracellularly nor is it found to be secreted from endothelial cells, which indicates that dimerization precedes exit from the ER. Monomeric vWf is also not secreted by other cell types in which vWf is expressed experimentally. Interestingly; when portions of the pro-vWf cDNA that do not encode the vWfC-terminalregion (where interchain disulfide bonds normally form) are expressed 'In tHese cells, they are found to be secreted. These include the free propolypeptide (Wise tt 'ai.l988);, -the propolypeptide 'plus D' D3 domains (Voorberg et al 1990); and thfiiPfiO-vWf subunit Iacking'ap-,

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proximately 20,000 Dalt

Cell biology of von Willebrand factor.

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