Two Cys residues essential for von Willebrand factor multimer assembly in the Golgi

Von Willebrand factor (VWF) dimerizes through C-terminal CK domains, and VWF dimers assemble into multimers in the Golgi by forming intersubunit disulfide bonds between D3 domains. This unusual oxidoreductase reaction requires the VWF propeptide (domains D1D2), which acts as an endogenous pH-dependent chaperone. The cysteines involved in multimer assembly were characterized by using a VWF construct that encodes the N-terminal D1D2D'D3 domains. Modification with thiol-specific reagents demonstrated that secreted D'D3 monomer contained reduced Cys, whereas D'D3 dimer and propeptide did not. Reduced Cys in the D'D3 monomer were alkylated with N-ethylmaleimide and analyzed by mass spectrometry. All 52 Cys within the D'D3 region were observed, and only Cys(1099) and Cys(1142) were modified by N-ethylmaleimide. When introduced into the D1D2D'D3 construct, the mutation C1099A or C1142A markedly impaired the formation of D'D3 dimers, and the double mutation prevented dimerization. In full-length VWF, the mutations C1099A and C1099A/C1142A prevented multimer assembly; the mutation C1142A allowed the formation of almost exclusively dimers, with few tetramers and no multimers larger than hexamers. Therefore, Cys(1099) and Cys(1142) are essential for the oxidoreductase mechanism of VWF multimerization. Cys(1142) is reported to form a Cys(1142)-Cys(1142) intersubunit bond, suggesting that Cys(1099) also participates in a Cys(1099)-Cys(1099) disulfide bond between D3 domains. This arrangement of intersubunit disulfide bonds implies that the dimeric N-terminal D'D3 domains of VWF subunits align in a parallel orientation within VWF multimers.     

Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.     

von Willebrand factor (VWF) propeptide binding to VWF D'D3 domain attenuates platelet activation and adhesion

Noncovalent association between the von Willebrand factor (VWF) propeptide (VWFpp) and mature VWF aids N-terminal multimerization and protein compartmentalization in storage granules. This association is currently thought to dissipate after secretion into blood. In the present study, we examined this proposition by quantifying the affinity and kinetics of VWFpp binding to mature VWF using surface plasmon resonance and by developing novel anti-VWF D'D3 mAbs. Our results show that the only binding site for VWFpp in mature VWF is in its D'D3 domain. At pH 6.2 and 10mM Ca(2+), conditions mimicking intracellular compartments, VWFpp-VWF binding occurs with high affinity (K(D) = 0.2nM, k(off) = 8 × 10(-5) s(-1)). Significant, albeit weaker, binding (K(D) = 25nM, k(off) = 4 × 10(-3) s(-1)) occurs under physiologic conditions of pH 7.4 and 2.5mM Ca(2+). This interaction was also observed in human plasma (K(D) = 50nM). The addition of recombinant VWFpp in both flow-chamber-based platelet adhesion assays and viscometer-based shear-induced platelet aggregation and activation studies reduced platelet adhesion and activation partially. Anti-D'D3 mAb DD3.1, which blocks VWFpp binding to VWF-D'D3, also abrogated platelet adhesion, as shown by shear-induced platelet aggregation and activation studies. Our data demonstrate that VWFpp binding to mature VWF occurs in the circulation, which can regulate the hemostatic potential of VWF by reducing VWF binding to platelet GpIbα.     

Intersection of mechanisms of type 2A VWD through defects in VWF multimerization, secretion, ADAMTS-13 susceptibility, and regulated storage

Type 2A VWD is characterized by the absence of large VWF multimers and decreased platelet-binding function. Historically, type 2A variants are subdivided into group 1, which have impaired assembly and secretion of VWF multimers, or group 2, which have normal secretion of VWF multimers and increased ADAMTS13 proteolysis. Type 2A VWD patients recruited through the T. S. Zimmerman Program for the Molecular and Clinical Biology of VWD study were characterized phenotypically and potential mutations identified in the VWF D2, D3, A1, and A2 domains. We examined type 2A variants and their interaction with WT-VWF through expression studies. We assessed secretion/intracellular retention, multimerization, regulated storage, and ADAMTS13 proteolysis. Whereas some variants fit into the traditional group 1 or 2 categories, others did not fall clearly into either category. We determined that loss of Weibel-Palade body formation is associated with markedly reduced secretion. Mutations involving cysteines were likely to cause abnormalities in multimer structure but not necessarily secretion. When coexpressed with wild-type VWF, type 2A variants negatively affected one or more mechanisms important for normal VWF processing. Type 2A VWD appears to result from a complex intersection of mechanisms that include: (1) intracellular retention or degradation of VWF, (2) defective multimerization, (3) loss of regulated storage, and (4) increased proteolysis by ADAMTS13.     

Changing insights in the diagnosis and classification of autosomal recessive and dominant von Willebrand diseases 1980-2015

The European Clinical Laboratory and Molecular (ECLM) criteria define 10 distinct Willebrand diseases (VWD): recessive type 3, severe 1, 2C and 2N; dominant VWD type 1 secretion/clearance defect, 2A, 2B, 2E, 2M and 2D; and mild type 1 VWD (usually carriers of recessive VWD). Recessive severe 1 and 2C VWD are characterized by secretion and multimerization defects caused by mutations in the D1-D2 domain. Recessive 2N VWD is a mild hemophilia due to D’-FVIII-von Willebrand factor (VWF) binding site mutations. Dominant 2E VWD caused by heterozygous missense mutations in the D3 domain is featured by a secretion-clearance-multimerization VWF defect. Dominant VWD type 2M due to loss of function mutations in the A1 domain is characterized by decreased ristocetin-induced platelet aggregation and VWF:RCo, normal VWF multimers and VWF:CB, a poor response of VWF:RCo and good response of VWF:CB to desmopressin (DDAVP). Dominant VWD type 2A induced by heterozygous mutations in the A2 domain results in hypersensitivity of VWF for proteolysis by ADAMTS13 into VWF degradation products, resulting in loss of large VWF multimers with triplet structure of each individual VWF band. Dominant VWD type 2B due to a gain of function mutation in the A1 domain is featured by spontaneous interaction between platelet glycoprotein Ib (GPIb) and mutated VWF A1 followed by increased proteolysis with loss of large VWF multimers and presence of each VWF band. A new category of dominant VWD type 1 secretion or clearance defect due to mutations in the D3 domain or D4-C1-C5 domains consists of two groups: Those with normal or smeary pattern of VWF multimers.     

The von Willebrand factor-reducing activity of thrombospondin-1 is located in the calcium-binding/C-terminal sequence and requires a free thiol at position 974

Plasma von Willebrand factor (VWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury; however, only the very large VWF multimers are effective in promoting platelet adhesion in flowing blood. The multimeric size of VWF can be controlled by the glycoprotein, thrombospondin-1 (TSP-1), which facilitates reduction of the disulfide bonds that hold VWF multimers together. The TSP family of extracellular glycoproteins consists of 5 members in vertebrates, TSP-1 through TSP-4 and TSP-5/COMP. TSP-1 and TSP-2 are structurally similar trimeric proteins composed of disulfide-linked 150-kDa monomers. Recombinant pieces of TSP-1 and TSP-2 incorporating combinations of domains that span the entire subunit were produced in insect cells and examined for VWF reductase activity. VWF reductase activity was present in the Ca(++)-binding repeats and C-terminal sequence of TSP-1, but not of TSP-2. Alkylation of Cys974 in the C-terminal TSP-1 construct, which is a serine in TSP-2, ablated VWF reductase activity. These results imply that the reductase function of TSP-1 centers around Cys974 in the C-terminal sequence.     

Helical self-assembly of a mucin segment suggests an evolutionary origin for von Willebrand factor tubules

The glycoprotein von Willebrand factor (VWF) contributes to hemostasis by stanching injuries in blood vessel walls. A distinctive feature of VWF is its assembly into long, helical tubules in endothelial cells prior to secretion. When VWF is released into the bloodstream, these tubules unfurl to release linear polymers that bind subendothelial collagen at wound sites, recruit platelets, and initiate the clotting cascade. VWF evolved from gel-forming mucins, the polymeric glycoproteins that coat and protect exposed epithelia. Despite the divergent function of VWF in blood vessel repair, sequence conservation and shared domain organization imply that VWF retained key aspects of the mucin bioassembly mechanism. Here, we show using cryo-electron microscopy that the ability to form tubules, a property hitherto thought to have arisen as a VWF adaptation to the vasculature, is a feature of the amino-terminal region of mucin. This segment of the human intestinal gel-forming mucin (MUC2) was found to self-assemble into tubules with a striking resemblance to those of VWF itself. To facilitate a comparison, we determined the residue-resolution structure of tubules formed by the homologous segment of VWF. The structures of the MUC2 and VWF tubules revealed the flexible joints and the intermolecular interactions required for tubule formation. Steric constraints in full-length MUC2 suggest that linear filaments, a previously observed supramolecular assembly form, are more likely than tubules to be the physiological mucin storage intermediate. Nevertheless, MUC2 tubules indicate a possible evolutionary origin for VWF tubules and elucidate design principles present in mucins and VWF.

Many extracellular proteins in higher organisms comprise multiple copies of characteristic structural domains. As these proteins evolved to their current forms, replication of primordial domains increased the physical spans of the proteins and allowed for functional diversification, such as the acquisition of multiple, distinct protein–protein interaction sites (1). Further increasing functional variation, whole gene duplications and exon shuffling gave rise to protein families such as collagens, laminins, and the large set of serine proteases involved in blood clot formation and degradation (2–4). By comparing structures of related modern-day proteins, it may be possible to deduce their ancestral origins and shed light on the fundamental principles by which new activities emerged, or by which new functionalities can be designed.One extracellular protein family that arose through domain replication and gene duplication is the gel-forming mucins, huge glycoproteins that coat and protect the epithelia in the lungs, intestines, and other organs. These mucins have three von Willebrand Factor type D (VWD) assemblies at the amino terminus and often another VWD assembly near the carboxyl terminus, together with von Willebrand Factor type C (VWC) domains and a carboxyl-terminal cystine knot (CTCK) (Fig. 1A). The central regions of gel-forming mucins are rich in proline, threonine, and serine (PTS) amino acid residues, providing numerous O-glycosylation sites. Some mucins also contain short, cysteine-rich CysD domains within the PTS region. Phylogenetic studies concluded that the architecture of the mucin amino terminus arose early in Ctenophora (comb jellies). In contrast, CysD domains evolved later, first appearing in Bilateria and found together with the PTS region in Deuterostomia (5, 6).Open in a separate windowFig. 1.Domain organization of MUC2 and VWF. (A) MUC2 and VWF primary structures. Human MUC2 has a PTS stretch of more than 2,300 amino acids with 60% threonine and 20% proline content, whereas VWF has VWA domains in this region. Protein lengths in amino acids are shown to the Right. The CTCK domain mediates disulfide-linked dimerization of the carboxy termini (17). (B) The map of the MUC2 amino-terminal ∼1,400 amino acids (N-term) is expanded to show all domains (50). Cradle refers to a cysteine-rich region that allows the D1 and D2 assemblies to embrace a D3 assembly from another subunit in the filament (22). Bridge refers to a longer cysteine-rich region that extends from the D2 assembly in one bead to the D3 assembly in the neighboring bead. Cysteines forming intersubunit disulfides during mucin assembly are indicated by yellow balls. (C) Three beads in the beaded filament of the MUC2 amino-terminal region (22) are shown colored according to panel B.A remarkable variation on the theme of mucins arose with the evolution of the complex vasculature in the vertebrate lineage (3, 7, 8). von Willebrand factor (VWF), a glycoprotein responsible for hemostasis, has a similar domain organization and is homologous to mucins in its amino- and carboxyl-terminal regions (5, 9, 10). Like mucins, VWF has three VWD assemblies at the amino terminus and one VWD assembly, in addition to VWC and CTCK domains, at the carboxyl terminus (Fig. 1A). However, VWF contains three von Willebrand Factor type A (VWA) domains in its central portion instead of the mucin PTS region. The VWA1, VWA2, and VWA3 domains have specific roles in blood clotting or VWF turnover (11–14). The shared domain architecture of the mucin and VWF termini contrasts with the different central regions, indicating that VWF evolved to utilize the capabilities of the ancestral mucin termini (9) while its central part was adapted for its distinct biological niche.The amino and carboxy termini of mucins and VWF mediate their assembly into higher order structures (15, 16). For both mucins and VWF, the CTCK domains of two polypeptides associate in the endoplasmic reticulum (ER) to generate intermolecularly disulfide-bonded dimers (17, 18). These dimers progress to the Golgi apparatus, which is a more acidic environment than the ER. Under acidic conditions, the amino termini of mucins and VWF form noncovalent intermolecular interactions that juxtapose VWD3 assemblies from different CTCK-linked dimers. The VWD3 assemblies then become disulfide bonded to one another to produce long, disulfide-linked polymers (19–22), although these polymers are thought to remain highly compact until secretion.Before release from the cell, VWF and mucin polymers are stored in dedicated secretory granules. Mucins are packed in spherical granules about 10 µm in diameter (22) and VWF in cigar-shaped compartments known as Weibel–Palade bodies (WPBs) ∼5 µm long and 0.3 µm wide in endothelial cells (23). Transmission electron microscopy (TEM) of WPBs showed that their distinct shape is due to quasi-crystalline bundles of aligned VWF tubules (21, 24–26). The amino-terminal region of VWF forms the tubular scaffold, while the VWA, VWD4, VWC, and CTCK domains are thought to project outward from the tubule (27, 28). VWF polymers unfurl from the tubules (29) and are secreted into the bloodstream in a latent form until they are activated by high shear at sites of vascular damage (30, 31). In contrast, mucins are secreted from their storage granules onto the epithelial surface, where an increase in pH and a decrease of calcium concentration cause a massive expansion of the glycoproteins and formation of hydrogels (32–36). To our knowledge, there have been no reports of a tubular storage form for mucins prior to secretion.We recently reported the cryo-electron microscopy (cryo-EM) structure of the amino-terminal region of the intestinal mucin MUC2 (Fig. 1B) and showed that it forms a beads-on-a-string filament (Fig. 1C). This filament scaffold is proposed to properly position the cysteines in the VWD3 assembly for formation of intermolecular disulfides as required for polymerization (22). The filament with the surrounding PTS regions and carboxy termini in full-length MUC2 likely constitutes the stored form of the glycoprotein at low pH prior to secretion from cells (22). The persistence length of the beaded string appeared to be short relative to the dimensions of the mucin storage granules, consistent with the apparent lack of long-range order in these granules. The different granular storage forms of mucins and VWF may reflect the unique selection pressures on their secretion and function. Evidence to date implied that VWF evolved de novo the capability of assembling a tubular scaffold.In this work, we show that a mucin segment has an intrinsic ability to form tubules, a property that may have been exploited during the evolution of VWF. We present residue-resolution structures of VWF and MUC2 amino-terminal segment tubules, enabling comparison between them and an analysis of the features that stabilize each one. Moreover, the elements that may suppress tubule formation in the context of full-length mucins are discussed.     

Molecular Genetics of Type 2 von Willebrand Disease

Type 2 von Willebrand disease (VWD) is characterized by a wide heterogeneity of functional and structural defects. These abnormalities' cause either defective von Willebrand factor (VWF)-dependent platelet function in subtypes 2A, 2B, and 2M or defective VWF-factor VIII (FVIII) binding in subtype 2N. The diagnoses of types 2A, 2B, and 2M VWD may be guided by the observation of disproportionately low levels of ristocetin cofactor activity or collagen-binding capacity relative to VWF antigen. The abnormal platelet-dependent function is often associated with the absence of high molecular weight (HMW) multimers (type 2A, type 2B), but the HMW multimers may also be present (type 2M, some type 2B), and supranormal multimers may exist ("Vicenza" variant). The observation of a low FVIII-to-VWF:Ag ratio is a hallmark of type 2N VWD. in which the FVIII levels depend on the severity of the FVIII-binding defect. Today, the identification of mutations in particular domains of the pre-pro-VWF is helpful in classifying these variants and providing further insight into the structure-function relationship and the biosynthesis of VWF. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC VWD. Mutations located in the D' domain or in the N terminus of the D3 domain define type 2N VWD. Mutations in the D3 domain characterize Vicenza and IIE patients. Mutations in the A1 domain may modify the binding of VWF multimers to platelets, either increasing (type 2B) or decreasing (type 2M, 2A/2M) the affinity of VWF for platelets. In type 2A VWD, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding, impairing VWF secretion or increasing its susceptibility to proteolysis. Finally, a mutation localized in the carboxy-terminus CK domain, which is crucial for the dimerization of the VWF subunit, has been identified in a rare subtype 2A, formerly named IID.     

An IAP retrotransposon in the mouse ADAMTS13 gene creates ADAMTS13 variant proteins that are less effective in cleaving von Willebrand factor multimers

Severe deficiency of ADAMTS13, a von Willebrand factor (VWF)-cleaving metalloprotease, causes thrombotic thrombocytopenic purpura. When analyzed with VWF multimers, but not with an abbreviated VWF peptide (VWF73) as the substrate, the plasma ADAMTS13 activity levels of mouse strains segregated into a high and a low group that differed by approximately 10 fold. Low ADAMTS13 activity was detected in mice containing 2 alleles of intracisternal A-type particle (IAP) retrotransposon sequence in the ADAMTS13 gene. Molecular cloning of mouse ADAMTS13 identified 2 truncated variants (IAP-a and IAP-b) in the low-activity mice. Both of the IAP variants lacked the 2 carboxyl terminus thrombospondin type 1 repeat (TSR) and CUB domains of full-length ADAMTS13. The IAP-b variant also had splicing abnormalities affecting the spacer domain sequence and had miniscule enzymatic activity. Compared with full-length ADAMTS13, the IAP-a variant was approximately one ninth as active in cleaving VWF multimers but was only slightly less active in cleaving VWF73 peptide. Recombinant human ADAMTS13 was also less effective in cleaving VWF multimers than VWF73 when the C-terminal TSR sequence was deleted. In summary, the carboxyl terminus TSR sequence is important for cleaving VWF multimers. Assay results should be interpreted with caution when peptide substrates are used for analysis of variant ADAMTS13 proteins.     

Classification and characterization of hereditary types 2A, 2B, 2C, 2D, 2E, 2M, 2N, and 2U (unclassifiable) von Willebrand disease.

All variants of type 2 von Willebrand disease (VWD) patients, except 2N, show a defective von Willebrand factor (VWF) protein (on cross immunoelectrophoresis or multimeric analysis), decreased ratios for VWF:RCo/Ag and VWF:CB/Ag and prolonged bleeding time. The bleeding time is normal and FVIII:C levels are clearly lower than VWF:Ag in type 2N VWD. High resolution multimeric analysis of VWF in plasma demonstrates that proteolysis of VWF is increased in type 2A and 2B VWD with increased triplet structure of each visuable band (not present in types 2M and 2U), and that proteolysis of VWF is minimal in type 2C, 2D, and 2E variants that show aberrant multimeric structure of individual oligomers. VWD 2B differs from 2A by normal VWF in platelets, and increased ristocetine-induced platelet aggregation (RIPA). RIPA, which very likely reflects the VWF content of platelets, is normal in mild, decreased in moderate, and absent in severe type 2A VWD. RIPA is decreased or absent in 2M, 2U, 2C, and 2D, variable in 2E, and normal in 2N. VWD 2M is usually mild and characterized by decreased VWF:RCo and RIPA, a normal or near normal VWF multimeric pattern in a low resolution agarose gel. VWD 2A-like or unclassifiable (2U) is distinct from 2A and 2B and typically featured by low VWF:RCo and RIPA with the relative lack of high large VWF multimers. VWD type 2C is recessive and shows a characteristic multimeric pattern with a lack of high molecular weight multimers, the presence of one single-banded multimers instead of triplets caused by homozygosity or double hereozygosity for a mutation in the multimerization part of VWF gene. Autosomal dominant type 2D is rare and characterized by the lack of high molecular weight multimers and the presence of a characteristic intervening subband between individual oligimers due to mutation in the dimerization part of the VWF gene. In VWD type 2E, the large VWF multimers are missing and the pattern of the individual multimers shows only one clearly identifiable band, and there is no intervening band and no marked increase in the smallest oligomer. 2E appears to be less well defined, is usually autosomal dominant, and accounts for about one third of patients with 2A in a large cohort of VWD patients.     

Structural organization of Weibel-Palade bodies revealed by cryo-EM of vitrified endothelial cells

In endothelial cells, the multifunctional blood glycoprotein von Willebrand Factor (VWF) is stored for rapid exocytic release in specialized secretory granules called Weibel-Palade bodies (WPBs). Electron cryomicroscopy at the thin periphery of whole, vitrified human umbilical vein endothelial cells (HUVECs) is used to directly image WPBs and their interaction with a 3D network of closely apposed membranous organelles, membrane tubules, and filaments. Fourier analysis of images and tomographic reconstruction show that VWF is packaged as a helix in WPBs. The helical signature of VWF tubules is used to identify VWF-containing organelles and characterize their paracrystalline order in low dose images. We build a 3D model of a WPB in which individual VWF helices can bend, but in which the paracrystalline packing of VWF tubules, closely wrapped by the WPB membrane, is associated with the rod-like morphology of the granules.     

Functional architecture of Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of these organelles. WPB exocytosis plays a role in the pathophysiology and treatment of von Willebrand disease and may have impact on common hematologic and cardiovascular disorders. This review summarizes the major advances made on the biogenesis and exocytosis of WPBs and places these recent discoveries in the context of von Willebrand disease.     

Interplay between ADAMTS13 and von Willebrand factor in inherited and acquired thrombotic microangiopathies

The presence of unusually large multimers of von Willebrand factor (VWF) is thought to be a major pathogenic factor for thrombotic thrombocytopenic purpura (TTP). ADAMTS13 is a protease that regulates the multimeric size and function of VWF by cleaving VWF. Hence, congenital or acquired deficiency of ADAMTS13 causes life-threatening illness of TTP. Mutations in the ADAMTS13 gene cause inherited TTP, and the development of autoantibodies that inhibit ADAMTS13 activity frequently are associated with acquired TTP. ADAMTS13 consists of 1,427 amino acid residues and is composed of multiple structural and functional domains, containing a signal peptide, a propeptide, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 (Tsp1) motif, a cysteine-rich domain, a spacer domain, seven additional Tsp1 repeats, and two CUB domains. In particular, the cysteine-rich/spacer domains are essential for VWF cleavage and are the principal epitopes recognized by autoantibodies in patients with acquired TTP. Therefore, it is likely that these domains are involved in the recognition and binding of ADAMTS13 to VWF. ADAMTS13 circulates in the blood in an active state, and efficiently cleaves unfold form of VWF induced under shear stress caused by blood flow, preventing the accumulation of pathogenic unusually large VWF multimers (ULVWF). Thus, ADAMTS13 helps maintain vascular homeostasis by preventing the excess thrombus formation.     

Impact of mutations in the von Willebrand factor A2 domain on ADAMTS13-dependent proteolysis

Classical von Willebrand disease (VWD) type 2A, the most common qualitative defect of VWD, is caused by loss of high-molecular-weight multimers (HMWMs) of von Willebrand factor (VWF). Underlying mutations cluster in the A2 domain of VWF around its cleavage site for ADAMTS13. We investigated the impact of mutations commonly found in patients with VWD type 2A on ADAMTS13-dependent proteolysis of VWF. We used recombinant human ADAMTS13 (rhuADAMTS13) to digest recombinant full-length VWF and a VWF fragment spanning the VWF A1 through A3 domains, harboring 13 different VWD type 2A mutations (C1272S, G1505E, G1505R, S1506L, M1528V, R1569del, R1597W, V1607D, G1609R, I1628T, G1629E, G1631D, and E1638K). With the exception of G1505E and I1628T, all mutations in the VWF A2 domain increased specific proteolysis of VWF independent of the expression level. Proteolytic susceptibility of mutant VWF in vitro closely correlated with the in vivo phenotype in patients. The results imply that increased VWF susceptibility for ADAMTS13 is a constitutive property of classical VWD type 2A, thus explaining the pronounced proteolytic fragments and loss of HMWM seen in multimer analysis in patients.     

Functional self-association of von Willebrand factor during platelet adhesion under flow

We have used recombinant wild-type human von Willebrand factor (VWF) and deletion mutants lacking the A1 and A3 domains, as well as specific function-blocking monoclonal antibodies, to demonstrate a functionally relevant self-association at the interface of soluble and surface-bound VWF. Platelets perfused at the wall shear rate of 1,500 s(-1) over immobilized VWF lacking A1 domain function failed to become tethered to the surface when they were in a plasma-free suspension with erythrocytes, but adhered promptly if soluble VWF with functional A1 domain was added to the cells. The same results were observed when VWF was immobilized onto collagen through its A3 domain and soluble VWF with deleted A3 domain was added to the cells. Thus, VWF bound to glass or collagen sustains a process of homotypic self-association with soluble VWF multimers that, as a result, can mediate platelet adhesion. The latter finding demonstrates that direct immobilization on a substrate is not a strict requirement for VWF binding to platelet glycoprotein Ibalpha. The dynamic and reversible interaction of surface-bound and soluble VWF appears to be specifically homotypic, because immobilized BSA, human fibrinogen, and fibronectin cannot substitute for VWF in the process. Our findings highlight a newly recognized role of circulating VWF in the initiation of platelet adhesion. The self-assembly of VWF multimers on an injured vascular surface may provide a relevant contribution to the arrest of flowing platelets opposing hemodynamic forces, thus facilitating subsequent thrombus growth.     

Diagnosis and follow-up of thrombotic thrombocytopenic purpura by means of von Willebrand factor collagen binding assay.

Thrombotic thrombocytopenic purpura (TTP) is characterized by intravascular thrombosis leading to consumption of large or unusually large von Willebrand factor (VWF) multimers. The usefulness of VWF collagen binding (VWF:CB) assay was assessed in detecting the decrease/absence of large VWF multimers or the presence of abnormally large forms in patients with TTP. Nine patients with TTP were studied during the acute phase of the disorder and the absence of large VWF multimers was demonstrated by means of the VWF:CB assay. These findings were confirmed by VWF multimer pattern analysis; VWF:CB deficiency appeared to correlate with abnormalities in large VWF multimers. The diagnostic potency of VWF:CB was especially evident when the values were expressed as VWF:CB/VWF:Ag ratio. VWF:CB was also used during the follow-up of the disorder to document improvement or restoration of large VWF multimers. VWF:CB was able to detect the absence or decrease of large VWF multimers better than VWF ristocetin cofactor (VWF:RCo); in fact, VWF:CB was defective when large VWF multimers persisted to be decreased, in contrast with what observed with VWF:RCo. In conclusion, VWF:CB is a simple test that appears to be useful, together with clinical symptoms and reduced platelet count, for the diagnosis and follow-up of TTP.     

Sequence and structure relationships within von Willebrand factor

In the present study, we re-annotated von Willebrand factor (VWF), assigned its entire sequence to specific modules, and related these modules to structure using electron microscopy (EM). The D domains are assemblies of smaller modules visible as lobes in EM. Modules in the D-domain assemblies include von Willebrand D, 8-cysteine, trypsin inhibitor-like, E or fibronectin type 1-like domains, and a unique D4N module in D4. The D1-D2 prodomain shows 2 large connected assemblies, each containing smaller lobes. The previous B and C regions of VWF are re-annotated as 6 tandem von Willebrand C (VWC) and VWC-like domains. These 6 VWC domains correspond to 6 elongated domains that associate in pairs at acidic pH in the stem region of VWF dimeric bouquets. This correspondence is demonstrated by binding of integrin α(IIb)β(3) to the fourth module seen in EM, VWC4, which bears the VWF Arg-Gly-Asp motif. The C-terminal cystine knot domain dimerizes end-to-end in a manner predicted by homology to TGF-β and orients approximately perpendicular to the VWC domains in dimeric bouquets. Homologies of domains in VWF to domains in other proteins allow many disulfide bonds to be tentatively assigned, which may have functional implications.     

Reduced survival of type 2B von Willebrand factor, irrespective of large multimer representation or thrombocytopenia

Background

Type 2B von Willebrand factor (VWF) is characterized by gain of function mutations in the A1 domain inducing a greater affinity for platelet GPIb, possibly associated with the disappearance of large VWF multimers and thrombocytopenia.

Design and Methods

VWF survival was explored using 1-desamino-8-D-arginine vasopressin (DDAVP) in 18 patients with type 2B von Willebrand disease (VWD) and compared with their platelet count and large VWF multimer representation.

Results

A similarly significant shorter VWF survival, expressed as T_1/2elimination (T_1/2el), was observed in patients lacking large VWF multimers (type 2B) and in those with a normal multimer pattern (atypical type 2B) (4.47±0.41 h and 4.87±0.9 h, respectively, vs. normal 15.53±2.17 h) due mainly to a greater VWF clearance. The half-life of large VWF multimers, explored by VWF collagen binding (VWF:CB) activity, was likewise reduced. The similarly reduced VWF half-life was also confirmed by the increase in the VWF propeptide ratio (a useful tool for exploring VWF survival) which was found to be the same in type 2B and atypical type 2B patients. The post-DDAVP drop in platelet count occurred in all patients lacking large multimers but not in those with a normal multimer pattern. A correlation was always found between pre- and/or post-DDAVP thrombocytopenia and the lack of large VWF multimers in type 2B VWD while these were unrelated to the reduced VWF half-life.

Conclusions

In addition to demonstrating that a shorter VWF survival contributes to the type 2B and atypical type 2B VWD phenotype, our findings suggest that VWF clearance and proteolysis are independent phenomena.     

Plasma exchange with solvent/detergent-treated plasma of resistant thrombotic thrombocytopenic purpura

A patient with unremitting thrombotic thrombocytopenic purpura (TTP), with circulating von Willebrand factor (VWF) multimers of unusually high molecular weight, and refractory to standard plasma exchange therapy, was treated with solvent detergent (S/D) plasma. The patient achieved a sustained clinical and haematological remission, with normal VWF multimeric profile. Spontaneous remission of this patient's condition could not be excluded but would appear unlikely. S/D plasma was efficacious and potentially safe for repeated large-volume plasma exchange with respect to viral safety and reduction of anaphylactoid reactions. We have assayed coagulation factors and physiological inhibitors of haemostasis in S/D plasma, which were comparable to normal plasma except in the distribution of VWF multimers.
The use of S/D plasma, previously reported in paediatric chronic relapsing TTP, should be assessed in further cases of adult TTP in the context of a clinical trial.     

Integrin alpha(v)beta(3) on human endothelial cells binds von Willebrand factor strings under fluid shear stress

Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin alpha(v)beta(3) in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin alpha(v)beta(3) reduced the number of VWF strings formed. In addition, integrin alpha(v) decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the alpha(v) subunit reduced the expression of cell-surface integrin alpha(v)beta(3) and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca(2+) and Mg(2+) but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin alpha(v)beta(3) on human endothelial cells.