von Willebrand Factor Biosynthesis and Processing" TANYA N. MAYADAS AND DENISA D. WAGNER b,cpd beenter of Hemostasis and Thrombosis Research Division of Hematology-Oncology New England Medical Center and 'Departments of Medicine, and dAnatomy and Cellular Biology Tufts University School of Medicine Boston, Massachusetts 021 1 I

INTRODUCTION von Willebrand factor (vWf) is a large adhesive glycoprotein that participates in a variety of interactions aiding in the formation of a platelet plug following injury of the blood vessel. vWf exerts its effects locally at the site of injury by promoting the attachment of platelets to the exposed subendothelium.In2 Although this may be its primary function, vWf, along with other proteins such as fibronectin, fibrinogen, and collagen, also mediates platelet-platelet interaction3 and the spreading of the attached platelets4 on the subendothelial surface, thus enabling them to withstand the high shear forces encountered in the vasculature. Recent evidence also shows the involvement of vWf in the adhesion of endothelial cells to the basement membrane.5 In addition to its function in adhesion, vWf serves as a carrier for factor VIII,6 thereby providing it protection against p r o t e ~ l y s i s . ~ ~ ~ vWf in plasma is composed of a structurally heterogeneous pool of disulfide bonded rnultirners9 that range in size from 0.5 to 20 million daltons. The largest vWf multimers are the most biologically active species both in vivo and in vitro, suggesting that the multivalency of binding sites presented by the repeating subunits of multimeric vWf is optimal for the adhesion and bridging functions of vWf in hemostasis. lo Accordingly, individuals with either decreased vWf levels or a selective loss of high-molecularweight vWf multimers exhibit prolonged bleeding times due to defective platelet plug formation." This hereditary bleeding disorder, first identified in 1926 by Erich von Willebrand, is called von Willebrand disease. The vWf subunit is synthesized with a large prosequence not found in the mature m ~ l t i m e r s . lThe ~ . ~ precursor ~ subunit is made up of four different regions (A-D). The internally homologous regions cover 80-90% of the amino acid sequence and are 'This work was supported by National Institutes of Health Grants R01 HL41002, PO HL 42443, T32 HL 07437 (T.N.M.) and by an established investigatorship award from the American Heart Association. 153



arranged in the following order: D I-D2-D'-D3-Al-A2-A3-D4-B l-B2-B3-Cl-C2. The propolypeptide is composed of the D1 and D 2 domains, and the remaining domains constitute the mature subunit.15 Cysteine residues, abundant in vWf (8.7%) with the exception of cysteine-poor A domains,I6 are similarly positioned among the domains, indicating that structural features have been maintained within these direct repeats. Biosynthesis of this large adhesive glycoprotein is limited to endothelial cells1' and megakaryocytes." Exercise or infusion with the vasopressin analogue DDAVP causes a transient increase in plasma vWf levels,'9 suggesting the presence of a readily releasable pool of vWf in these cell types. Storage of vWf has been demonstrated in platelet alpha-granule20,2'and in rod-shaped endothelial cell specific organelles known as the Weibel-Palade b ~ d i e s .vWf ~ ~ .is~unusual ~ in that it is the only adhesive protein that is stored in cells other than platelets. Weibel-Palade bodies can be induced to release their contents after stimulation of endothelial cells with various secretagogues (regulated ~ e c r e t i o n ) However, .~~ the majority of vWf is secreted by a pathway that is directly linked to synthesis (constitutive s e c r e t i ~ n ) .vWf ~ ~ secreted via these two pathways differs both structurally and f ~ n c t i o n a l l y . vWf ~ ~ - ~secreted ~ constitutively is composed of all-molecular-weight multimers, whereas vWf released from Weibel-Palade bodies is composed of only the largest, most biologically potent m ~ l t i r n e r s vWf . ~ ~ present in plasma and vWf contributed by both platelets and endothelial cells appear to be necessary for optimal platelet a d h e s i ~ n . ' , ~The ~ - ~induction ~ of vWf release from the storage granules offers an important mechanism of enhancing platelet adhesion at the site of injury. vWf in megakaryocytes and endothelial cells is similarly processed3' and undergoes extensive posttranslational modifications such as carbohydrate addition, processing and sulfation, dimerization, multimerization, and prosequence cleavage. The multimeric structure of vWf is particularly interesting in terms of both vWf function and protein processing. First, the enhanced activity of vWf that results from its multimerization demonstrates an interesting mechanism of further increasing the biological efficacy of vWf subunits that alone contain all the binding sites needed for function. Second, vWf processing is of interest in cell biology, since multimerization of vWf in trans- and postGolgi compartments is peculiar to this protein, considering that all other oligomeric proteins known to polymerize intracellularly assemble and form disulfide bonds in the endoplasmic reticulum (ER). This review will focus on vWf processing events leading to assembly of the large vWf multimers.

GLYCOSYLATION The large precursor vWf subunit (pro-vWf) is modified in the ER by the addition of high-mannose carbohydrate side chains to twelve sites located on each mature subunitI6and to at least one of four potential sites located on the propolypeptide. These carbohydrate-modified vWf subunits are sensitive in vitro to the enzyme endoglycosidase H.32Following the transport of vWf to the Golgi apparatus, the mannose residues are trimmed, and galactose and sialic acid are subsequently added to form complextype carbohydrates that result in decreased mobility of the pro-vWf subunit in SDS polyacrylamide gels. Some of the N-linked carbohydrate side chains on vWf are also sulfated, a process that occurs in the Golgi apparatus.33The conversion of the highmannose carbohydrate to the complex type renders them insensitive to endoglycosidase H.32Examination of the susceptibility of vWf to endoglycosidase H in relation to the



various stages of vWf processing has provided a useful tool in determining the temporal sequence and location of vWf processing steps such as dimerization, multimerization, and prosequence cleavage. In addition to N-linked oligosaccharides, the mature vWf subunit also has 10 0-linked oligosaccharide sites on threonine and serine residue^.'^ The N-linked and 0-linked carbohydrates present on plasma vWf account for approximately 18.7% by weight of the vWf molecule and are clustered predominantly on the N and C terminal portions of the glycopr~tein.’~ Although a unified function for carbohydrates present on glycoproteins is not yet clear, we have found that treatment of human endothelial cells with the antibiotic tunicamycin, which prevents N-linked glycosylation, results in the accumulation of pro-vWf monomers in the ER, indicating that perhaps N-linked carbohydrate addition onto vWf is important in inducing or maintaining the conformation of the monomer needed for dimerization. 36 Although these studies on human umbilical vein endothelial cells (HUVEC) suggest that the formation of vWf dimers is dependent on prior carbohydrate addition, this requirement may be species specific since bovine endothelial cells cultured in tunicamycin process and secrete vWf n0rmal1y.l~The processing of highmannose to complex-type carbohydrates is not required for the late processing steps in vWf synthesis, since treatment of HUVEC with swainsonine, an inhibitor of the Golgi enzyme mannosidase 11,’’ does not effect the multimerization or prosequence cleavage of V W ~ . Although ’~ investigators have reported that removal of terminal sugars in vitro results in the appearance of lower-molecular-weight m u l t i m e r ~Federici , ~ ~ ~ ~ and colleaguesm have found that the sialic acid and galactose moieties on vWf are not necessary for maintaining the vWf multimeric structures but are required for protecting vWf against proteolytic degradation.

VON WILLEBRAND FACTOR DIMERIZATION AND MULTIMERIZATION vWf undergoes two distinct intracellular polymerization steps, which are compartmentalized into different organelles: dimerization occurs in the ER,’2,41where disulfide bond formation is known to occur in many proteins; whereas multimerization of dimers is a trans-Golgi and/or post-Golgi event36 (FIG. 1). The separation of these two vWf polymerization steps could theoretically confer a greater degree of complexity that would allow for finer regulation of these two processes. The differential compartmentalization also suggests that different enzymes or mechanisms may be involved in these processes. Mechanisms of disulfide linkage in the ER may involve spontaneous oxidation of free sulfhydryls, followed by disulfide interchange catalyzed by protein disulfide isomerase (PDI),42 an enzyme resident in the ER. The mechanism of formation of disulfide bond formation in the trans-Golgi and post-Golgi compartments is not known; it may occur through a sulfhydryl disulfide exchange system involving glutathione. vWf dimerization occurs through an unknown number of intersubunit disulfide bonds in the C-terminal portion of the m01ecule.~~~” These disulfide bonds reside in the last 150 residues of the vWf s ~ b u n i t . ~ ’Partial ,~~.~ localization ~ of interdimer disulfide bonds achieved by analysis of peptides produced by V8 Sraphylococcus aureus protease digestion and tryptic digestion of vWf indicates that at least one cysteine residue within residues 283-695 in the D3 domain may form an interchain disulfide c r o ~ s - l i n k , ~ ~ * ~ ~ and that (an)other interdimer disulfide bond(s) is/are located within the A1 domain (Sixma et QZ., unpublished observation).

Endoplasmic Reticulum

Pro-vWf Dimer

vWf Multimer + Free Propolypeptide


Pro-vWf Multimer

Golgi Apparatus/ Weibel-Palade Bodies

are formed in two steps dimer formation and multimenzation of dimers The cleavage of the propolypeptide most hkely occurs after multimenzation The free propolypeptide circulates in plasma as a noncovalent dimer

FIGURE 1. Schematic representation of polymemtion and cleavage steps leading to formation of processed vWf multimers Disulfide-linked multimers











8X *n *








Recent evidence by Bonfanti and Wagner (unpublished observations) and by Voorberg et suggests that prior dimerization of monomers via C-terminal disulfide linkages is not a prerequisite for the formation of disulfide bonds at the N-termini of the subunits that lead to multimerization. C-terminal truncated vWf expressed in heterologous cells was unable to dimerize in the ER, since portions of the mature subunit containing cysteine residues that participate in intersubunit disulfide bonds were missing; but this vWf was, nevertheless, capable of forming disulfide bonds at the N-termini of the protein to produce “N-linked dimers.” These results further demonstrate that dimerization and multimerization are independent events. Observations from several studies, including results on the effects of tunicamycin on vWf b i o ~ y n t h e s i (discussed s~~ earlier), suggest that the vWf molecule may be recognized by E R regulatory proteins, such as BiP,48 which may retain the vWf subunits until dimerization occurs. These recognition elements may possibly reside in portions of the C-terminal regions of the pro-vWf subunit, since heterologous cells transfected with cDNA constructs encoding the p r ~ p o l y p e p t i d e the , ~ ~propolypeptide plus D’, D 3 domains,47and the pro-vWf subunit lacking approximately 20,000 Da from the Cterminal end (Bonfanti and Wagner, unpublished observation) express truncated vWf monomers that are secreted. The proposed association of monomeric vWf with BiP in the E R is not peculiar to vWf, since the association of BiP with protomers has been demonstrated for several other proteins.5s52 For example, the heavy chain of immunoglobulin remains associated with BiP until the light chain binds. The failure of the light chain to do so results in the retention of the heavy chain in the ER.5’ vWf dimerization is followed by the formation of interdimer disulfide bonds to form a series of oligomers and the cleavage of the prosequence. A pharmacological approach was used to localize vWf polymerization and prosequence cleavage processes within the cell. vWf processing was examined in endothelial cells treated with monensin, a carboxylic ionophore that specifically disrupts the Golgi apparatus.54Monensin treatment results in the secretion of pro-vWf dimers sensitive to endoglycosidase H digestion, confirming that they had not acquired complex carbohydrates, a Golgi-localized processing step. Therefore, the disruption of Golgi function results in the inhibition of the processing steps that have also been shown by pulse chase experiments to occur late in vWf maturation; vWf multimerization and prosequence cleavage.3s Since free sulfhydryls located on the mature portion of the pro-vWf subunitS5are not present after m u l t i m e r i ~ a t i o n ,it~ ~is, ~likely ~ that multimerization occurs through oxidation or disulfide interchange involving the free sulfhydryls that may ultimately form the intersubunit disulfide bond(s). The importance of the free sulfhydryls in multimer assembly was shown in in vitro multirnerization studies. Pro-vWf dimers that were exposed to sulfhydryl blocking agents such as N-ethylmaleimide and iodoacetic acid failed to r n u l t i m e r i ~ e . ~ ~ vWf multimerization is arrested after secretion. Therefore, there are aspects of the trans-Golgi apparatus and secretory granules, the sites of vWf polymerization, that are paramount to multimerization of vWf. Intraorganelle conditions such as pH, protein concentration, metal ion concentrations, and redox potentials are likely to play an important part in the formation of disulfide bonds between vWf subunits. I n addition, the large propolypeptide has been well established as crucial to the multimerization process, but the mechanism by which it facilitates this process is not known. Treatment of HUVEC with ammonium chloride, a weak base that increases the pH in acidic organelles, results in the secretion of mostly mature vWf dimers and some pro-vWf dimers. Therefore, while vWf multimerization is inhibited, prosequence cleavage is not significantly affected.36 Since the trans-Golgi cisternae and secretory vesicles in several cells are the sites of active proton pumping and maintain an acidic pH,58359it can be concluded that vWf multimers assemble in these organelles and that



this process proceeds via a low pH-requiring mechanism. In addition, the differential susceptibility to ammonium chloride of multimerization and prosequence cleavage indicates that the two processes occur i n d e ~ e n d e n t l yStudies . ~ ~ of in vitro vWf multimerization confirmed a requirement for acidic pH (FIG.2): The continuous presence of pH 5.8 is optimal for in vitro multimerizationS5and correlates well with the pH often found in trans-Golgi and post-Golgi compartments. Since no other enzymes or cellular components are present during in v i m multimerization, it is likely that acidic pH has a direct effect on the vWf and that all the information necessary for multimerization is contained within the pro-vWf molecule. The change in pH encountered by vWf dimers transported from the ER, which is at neutral pH, through to the trans-Golgi

FIGURE 2. The Effect of pH on multimerization of pro-vWf dimers in v i m and a comparison of pro-vWf dimers multimerized in vitro with multimers secreted constitutivelyby HUVEC. ProvWf dimers were obtained from culture media of metabolically labeled endothelial cells treated with 1 pM monensin. Panel A: samples of pro-vWf dimers were placed in buffer of varying pH. Control starting samples were kept refrigerated (St.). The samples incubated at 37 "C for 4 h were electrophoresed nonreduced on a 2% agarose gel, the autoradiograph of which is shown. Only dimers incubated at pH below 6.2 formed multimers. Panel B: samples of pro-vWf dimers before (- ip vitro) and after (+ in vitro) incubation at pH 5.8 were compared to a sample of vWf multimers constitutively secreted by metabolically labeled HUVEC. (From Mayadas and Wagner.ssReprinted by permission from Journal of Biological Chemistry.)

compartment, which maintains an acidic pH, may be the triggering event in vWf multimerization. The longitudinally oriented tubular structures found in Weibel-Palade bodies probably represent highly condensed, organized polymers of v W ~Perhaps . ~ ~ the local concentrations of vWf at sites of polymerization are sufficiently high to favor noncovalent associations, thereby facilitating disulfide bond formation. The role of noncovalent forces in vWf protomer association has been implied by Loscalzo er a1.,@'who demonstrated that at concentrations of vWf exceeding that of plasma, vWf multimeric structure is preserved in aqueous solution even after partial reduction with beta-mercaptoethanol. However, dilution of the beta-mercaptoethanol-treated sample results in a decrease in the amount of multimeric vWf with the appearance of vWf protomers. This



data suggests that disulfide linkages are necessary to maintain the multimeric structure and activity of vWf at plasma concentrations but that at increased vWf concentrations, noncovalent forces of association may play a role in maintaining the polymeric size of vWf. Noncovalent interactions may occur between the D1 and D 2 domains of the prosequence and between the D' domains of the mature vWf (FIG.1). This is based on observations that the deletion of either the p r ~ p o l y p e p t i d e , ~(FIG. ~ , ~ ' 3) or of the D' domain4' results in the expression of only vWf dimers, indicating the importance of these regions in pro-vWf multimerization. Wise and colleagues noted that the propolypeptide interferes with binding of factor VIII to the D' region of the pro-vWf subunit,62 which may in addition suggest a noncovalent interaction between the propolypeptide and the D' domain that may consequently prevent factor VIII binding. We have recently shown that vWf multimerization is a metal-dependent process. Ethylenediaminetetraacetic acid (EDTA) added to dimers exposed to conditions normally optimal for in vitro multimerization results in the inhibition of polymerization. However, the addition of excess CaCl, to the EDTA-containing buffers restored multimerization (our unpublished observations). The requirement of Ca+, ions in multimerization correlates well with high Ca+2concentrations observed in secretory vesicle^.^' The Ca+, ions required in the multimerization process may function as ion bridges to hold together two molecules or two different regions of the same molecule in a proper steric relationship to one another so as to allow covalent or noncovalent interactions between the molecules. Metal-induced polymerization has been demonstrated for various proteins. For example, secretogranin I1 was induced to aggregate in vitro in the presence of CaCI, and acidic P H . ~ The role of the large propolypeptide in multimerization has been well established by both recombinant D N A approaches and in vitro multimerization. Verweij et a1.61 and Wise et u I . demonstrated ~~ that the expression of recombinant pro-vWf cDNA in COS cells leads to the synthesis and secretion of processed large-molecular-weight multimers, whereas the expression of cDNA in which the sequences coding for the propolypeptide are deleted results in the secretion of only dimers (FIG.3). These studies show further that although the propolypeptide is important in multimerization, it does not play a role in the formation of vWf dimers. In addition, Wise et al.49demonstrated that in COS cells the vWf propolypeptide can facilitate multimer assembly when it is expressed independently of mature vWf subunits. However, in vitro the free propolypeptide does not aid in the multimerization of mature dimers, suggesting that in this system the propolypeptide needs to be colinear with the mature dimers to promote their multirnerizati~n.~~ Although the mechanisms by which the propolypeptide promotes multimerization is not known, we can deduce from the in vitro multimerization of pro-vWf dimers that the propolypeptide is directly involved in the multimerization process, rather than in the targeting of the protein to a cellular compartment where the relevant enzymes may be located.55 The propolypeptide may play a structural and/or a catalytic role. These two proposed roles are not mutually exclusive, and their coordination may be necessary for efficient multimerization. The possible role of the propolypeptide in a structural capacity may involve a pH- or metal ion-induced conformational change in the propolypeptide portion of the pro-vWf molecule, thereby allowing the interaction of propolypeptides of different molecules (FIG. 1). The association of the propolypeptide may help to form the interchain disulfide bonds between the D3 and A domains of the mature subunit. Support for this hypothesis is shown by the presence in plasma of free propolypeptide that is, indeed, self-associated as a noncovalently linked dimer.65 A proposed catalytic role for the propolypeptideS5 would be to promote multimerization by disulfide interchange, an activity similar to the PD142found in the ER.66This was proposed because the assembly of disulfide-bonded multimers in vWf occurs only at



acidic pH, which is not optimal for spontaneous disulfide bond formation. Since 1987, several proteins previously identified as having functions quite distinct from disulfide isomerization have been shown to share sequence homology and functional identity with PDI.42For example, the beta subunit of prolyl-4-hydroxylase, which in a complex with alpha subunits aids in the hydroxylation of prolyl residues, has been shown to be identical with PDI and to exhibit isomerase a ~ t i v i t y . ~ ~The - " beta subunit of two hormones, follitropin and lutropin, containing homology with the PDI active site, were shown to have isomerase activity in vitro: a possible disulfide interchange between the hormones and the receptor may possibly initiate a conformational change in the receptor that is required for signal transd~ction.~' Comparison of the PDI sequences with the propolypeptide indicated homology between the active site of PDI (Cys-Gly-HisC Y S ) ,repeated ~~ twice in PDI, and a sequence present in both the D1 and D2 domains of the vWf propolypeptide (Cys-Gly-Leu-Cy~).~~ This vicinal disulfide Cys-Gly-X-Cys may have the capacity to catalyze disulfide interchange leading to vWf multimer assembly.

FIGURE 3. Expression of pro-vWf and mature vWf in COS cells. Approximately 1 x lo6 COS cells were transfected with an expression vector containing cDNA encoding for either pro-vWf or mature vWf (kindly provided by Evan Sadler, Washington University, St. Louis, MO). Twenty-four hours after transfection, cells were labeled with ['3]cysteine for 48 h. The media were collected and immune-precipitated with polyclonal vWf antiserum. The samples were analyzed on a 2% agarose gel, an autoradiograph of which is shown. Cells transfected with pro-vWf cDNA secreted the whole array of multimers, whereas COS cells expressing mature vWf synthesized only dimers. HMW = highmolecular- weight.

PROTEOLYTIC CLEAVAGE vWf undergoes at least two proteolytic processing steps to yield the mature subunit (FIG.1). The N-terminus signal peptide, comprising 22 r e s i d ~ e s , is ' ~typical of signal peptides present on proteins directed into the ER and is cleaved cotranslationally by the signal peptidase resident in the ER.The second cleavage step is the cleavage of the vWf propolypeptide, which is a very late processing event and appears to occur both in the trans-Golgi and secretory granules.36Within the regulated pathway of secretion, the propolypeptide cleavage likely occurs after packaging of vWf in the Weibel Palade bodies, since mature subunits were found stored in stoichiometric amounts with the free p r o p ~ l y p e p t i d e The . ~ ~ vWf propolypeptide cleavage occurs between Arg and Ser residues in the sequence L~s(762)-Arg(763)-Ser(764),~' similar to the paired basic residues present at the cleavage site of many pro hormone^.^^ Whether the propolypeptide is further proteolytically processed at its C terminal is not known. vWf propolypep-



tide cleavage is not required for vWf multimerization, and, as is the case with insulin proce~sing,’~ there is no data to suggest that cleavage is a prerequisite for intracellular protein transport: COS cells transfected with cDNA encoding pro-vWf mutated at its dibasic cleavage site synthesized vWf that multimerized normally and was ~ e c r e t e d ; ~ ~ . ~ ’ endothelial cells constitutively secrete multimers that are partially composed of uncleaved p r e c u r s ~ r ;a~ partial ~ . ~ ~ persistence of uncleaved pro-vWf subunits has also been described in plasma multimers of a variant form of VWD;’~and finally, the multimers can be formed after in vitro multimerization of pro-vWf dimers in a system where no prosequence cleavage occurs.55

REGULATION OF VON WILLEBRAND FACTOR BIOSYNTHESIS The mechanism of vWf tissue-specific expression is not known. In addition to the tissue-specific regulation of expression, there is evidence that vWf biosynthesis may be hormonally regulated. For example, dexamethasone has a negative effect on vWf biosynthesis in cultured endothelial cells,77whereas estrogen treatment increases vWf synthesis.78 This data is corroborated by clinical evidence: pregnancy results in an increase in vWf plasma levels, whereas patients with hypothyroidism exhibit the phenotype of acquired von Willebrand d i ~ e a s e . ’ ~ . ~ ~ Regulation of vWf biosynthesis following stimulation of endothelial cells with thrombin was studied by the authors and by Reinders and colleagues. We found that Weibel-Palade body depletion as a result of thrombin stimulation does not serve as a signal for compensatory increase in vWf biosynthesis. Thrombin stimulation also does not affect the partitioning of vWf between the constitutive pathway and the regulated pathway, which comprises approximately 5% of vWf produced by HUVEC. Instead, replenishment of Weibel-Palade bodies may take place at a steady state rate similar to that of unstimulated cells.81 Reinders et d 8 *studied the effect of PMA (phorbol myristate acetate) and thrombin on the distribution of vWf in HUVEC. They showed that PMA treatment or one-hour pretreatment with thrombin results in an enhanced accumulation of vWf in the medium, with no increase in vWf-specific mRNA. Although the authors conclude that the enhanced vWf in the media was due to increased de novo synthesis by more efficient translation of existing vWf-specific mRNA, it is also possible that the enhanced accumulation of vWf in the media reflects secretion of preformed vWf pools originating from Weibel-Palade bodies. One might speculate that perhaps the immediate replenishment of Weibel-Palade bodies after their release is not desirable, since the endothelial cells would then be in a position to be restimulated by physiological stimuli still present in the vicinity of the developing clot or at the site of inflammation. This would result in an uncontrolled release of biologically potent vWf multimers at the site of injury and could result in excessive platelet aggregation. vWf is unique in that it is synthesized as an intact protomer, and yet the biologic activity of the secreted protein is regulated by its multimeric state, which in turn is controlled by intracellular pH, ion concentration, and other likely regulators yet to be discovered. Therefore, theoretically, normal hemostasis as related to the biosynthesis of vWf may be seen as a regulated process in which biological activity is determined not only by the genetically encoded protein sequence but by posttranslational processing steps that are dependent on the intracellular milieu.



ACKNOWLEDGMENT We thank Drs. scripts.

M. Pannekoek and J. J. Sixma for providing unpublished manu-

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von Willebrand factor biosynthesis and processing.

von Willebrand Factor Biosynthesis and Processing" TANYA N. MAYADAS AND DENISA D. WAGNER b,cpd beenter of Hemostasis and Thrombosis Research Division...
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