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2166 Part XII: Hemostasis and Thrombosis Chapter 126: von Willebrand Disease 2167

middle portion of the VWF structural gene, spanning exons 23 to 34 subunits for multimer assembly. However, the propeptide facilitates
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and the intervening noncoding sequences. VWFP1 is approximately multimer assembly even when coexpressed as a separate molecule from
97 percent identical in sequence to the authentic VWF gene, indicating the mature VWF monomer. 56,57
that it is of fairly recent evolutionary origin. Gene conversion involv- Propeptide cleavage occurs late in VWF synthesis or just prior to
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ing the VWFP1 pseudogene, possibly through recombination with the secretion. Cleavage occurs adjacent to two basic amino acids, Lys-Arg
large homologous exon 28 sequence, has been proposed as a mecha- at positions –2 and –1. An Arg at position –4 is also required for rec-
nism for introducing mutations into the VWF gene. 28–31 ognition by the intracellular protease responsible for propeptide cleav-
VWF is synthesized exclusively in megakaryocytes and endothelial age. Multimerization and propeptide cleavage are not linked to each
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cells and, as a result, has frequently been used as a specific histochemical other. The multimers secreted by cultured endothelial cells contain both
marker to identify cells of endothelial cell origin. Although generally pro-VWF and mature subunits, 59,60 and recombinant VWF with a point
assumed to mark all endothelial cells, VWF is expressed at widely vary- mutation inhibiting propeptide cleavage is still assembled into normal
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ing levels among endothelial cells, depending on the size and location of multimer structures. Although propeptide cleavage appears to occur
the associated blood vessel. 32,33 A careful survey in the mouse identified primarily intracellularly, cleavage may also occur after secretion.
wide differences in the level of VWF mRNA, with 5 to 50 times higher VWF is stored in tubular structures within the α-granules of
concentrations in the lung and brain, particularly in small vessels, than platelets and within the Weibel-Palade bodies in endothelial cells 62,63
in comparable vessels in the liver and kidney. In general, the higher lev- (reviewed in Ref. 64). These large VWF structures form by tubular pack-
els of VWF mRNA and antigen were found in the endothelial cells of ing of the VWF N-terminal domains within the secretory granules.
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large vessels rather than in microvasculature, and in venous rather than Weibel-Palade bodies are derived from the Golgi apparatus and are
arterial endothelial cells. 33 found in most endothelial cells, although the number varies consider-
Specific DNA sequences within or near the proximal promoter ably between endothelial cell beds. It has been shown that VWF and
of the VWF gene appear to be required for endothelial-specific gene FVIII colocalize in storage granules. Although VWF is not required
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expression, 34–39 although it is likely that additional important regulatory to traffic FVIII to platelets, VWF appears to play a role in trafficking
elements exist outside of this region, some of which may lie at a great FVIII to Weibel-Palade bodies in endothelial cells. 67,68 Weibel-Palade
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distance. VWF is expressed in most, but not all, endothelial cells, and bodies mature as they move to the periphery of the cell in an ordered
this vascular-bed specific gene expression program is likely a result of process dependent on Rab proteins and Rab effector proteins, which
the concerted action of multiple regulatory elements. Endothelial VWF act as chaperones and organizers of the various stages of Weibel-Palade
gene expression also appears to be upregulated by exposure to shear body maturity and subsequent exocytosis (reviewed in Ref. 69). The
stress. The length of a polymorphic GT repeat in the proximal VWF transmembrane glycoprotein P-selectin is also found in the membranes
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promoter correlates with the magnitude of this response, and several of both the α-granule and the Weibel-Palade body. The VWF D’D3
other more distal DNA sequences are predicted to be involved in a shear domain has been shown in vitro to associate with P-selectin and to be
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stress response. However, this GT repeat does not appear to influence necessary for the recruitment of P-selectin to Weibel-Palade bodies.
circulating VWF levels. 43 There appears to be heterogeneity within Weibel-Palade body popula-
tions both in relative content of VWF and P-selectin and in response
VON WILLEBRAND FACTOR BIOSYNTHESIS to regulated secretion by different stimuli. In addition to VWF and
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The processing steps involved in the biosynthesis of VWF are similar P-selectin, the Weibel-Palade body also contains tissue-type plasmino-
in megakaryocytes and endothelial cells (reviewed in Refs. 46 and 47). gen activator (t-PA), a thrombolytic secreted protein that also may
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VWF is first synthesized as a large precursor monomer polypeptide, be released distinctly from VWF, and several other proteins that are
depicted schematically in Fig. 126–1. VWF is unusually rich in cysteine, known to participate in inflammation or angiogenesis (for a complete
which accounts for 8.3 percent of its amino acid content. All cysteines list of Weibel-Palade contents see Ref. 74).
in the mature VWF molecule are thought to be involved in disulfide VWF is secreted from endothelial cells continuously via consti-
bonds, although these bonds may be exposed in circulating mature tutive and constitutive-like (or basal) pathways and upon stimulated
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VWF by shear stress. Pro-VWF monomers are assembled into dimers release of storage granules via a classic regulated pathway. 46,75 Regulated
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through disulfide bonds at both C termini, and only dimers are exported secretion of VWF from its storage site in the Weibel-Palade body is
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from the endoplasmic reticulum (ER). 48,50,51 triggered by a number of secretagogues, including thrombin, fibrin,
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Glycosylation begins in the ER, with 12 potential N-linked glycosy- histamine, the C5b-9 complement complex, and several inflamma-
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lation sites present on the mature subunit and three on the propeptide. tory cytokines. Recent in vitro data suggests that there may also be
Extensive additional posttranslational modification of VWF occurs in suppression of regulated VWF secretion by statins. 81,82 The secretagogue
the Golgi apparatus, including the addition of multiple O-linked car- desmopressin acetate (DDAVP), a vasopressin analogue, is used clini-
bohydrate structures, sulfation, and multimerization through the for- cally for its capacity to cause a marked release of VWF and FVIII in vivo
mation of disulfide bonds at the N termini of adjacent dimers. It is by acting through type 2 vasopressin receptors to induce secretion from
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unusual for a protein to undergo extensive disulfide bond formation the Weibel-Palade bodies in endothelial cells. Constitutive-like secre-
at this late stage, and this process appears to be catalyzed by disulfide tion of VWF occurs evenly at the luminal and abluminal surface, while
isomerase activity present within the VWFpp. Mutations at either of regulated secretion from the Weibel-Palade body is highly polarized
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two specific cysteines within the propeptide that are thought to be crit- in the luminal direction (Fig. 126–2). 75,84 While constitutively secreted
ical for disulfide isomerase activity, or a shift in the spacing between multimers are of relatively small size, the multimers stored within the
them, results in loss of multimer formation. An intermediate species Weibel-Palade body are the largest, most biologically potent form. 85,86
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with disulfide bonds between the propeptide and VWF D’D3 domain The VWF stored in platelet α-granules is also enriched for large mul-
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appears briefly in either the late ER or early Golgi, which may position timers. The N-terminal D domains appear to be required for VWF
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these domains for subsequent multimerization. The multimerization storage, with deletion of any of the individual domains resulting in
process appears to require the slightly acidic environment of the distal constitutive secretion. 88,89 It also appears that cleavage of the VWFpp is
Golgi. The VWFpp self-associates and may also serve to align VWF required for efficient formation of storage granules. 90
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Kaushansky_chapter 126_p2163-2182.indd 2166 9/21/15 3:14 PM