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.     

Multiple domains of ADAMTS13 are targeted by autoantibodies against ADAMTS13 in patients with acquired idiopathic thrombotic thrombocytopenic purpura

Background

Type G immunoglobulins against ADAMTS13 are the primary cause of acquired (idiopathic) thrombotic thrombocytopenic purpura. However, the domains of ADAMTS13 which the type G anti-ADAMT13 immunoglobulins target have not been investigated in a large cohort of patients with thrombotic thrombocytopenic purpura.

Design and Methods

Sixty-seven patients with acquired idiopathic thrombotic thrombocytopenic purpura were prospectively collected from three major U.S. centers. An enzyme-linked immunosorbent assay determined plasma concentrations of anti-ADAMTS13 type G immunoglobulins, whereas immunoprecipitation plus western blotting determined the binding domains of these type G immunoglobulins.

Results

Plasma anti-ADAMTS13 type G immunoglobulins from 67 patients all bound full-length ADAMTS13 and a variant truncated after the eighth TSP1 repeat (delCUB). Approximately 97% (65/67) of patients harbored type G immunoglobulins targeted against a variant truncated after the spacer domain (MDTCS). However, only 12% of patients’ samples reacted with a variant lacking the Cys-rich and spacer domains (MDT). In addition, approximately 37%, 31%, and 46% of patients’ type G immunoglobulins interacted with the ADAMTS13 fragment containing TSP1 2-8 repeats (T2-8), CUB domains, and TSP1 5-8 repeats plus CUB domains (T5-8CUB), respectively. The presence of type G immunoglobulins targeted against the T2-8 and/or CUB domains was inversely correlated with the patients’ platelet counts on admission.

Conclusions

This multicenter study further demonstrated that the multiple domains of ADAMTS13, particularly the Cys-rich and spacer domains, are frequently targeted by anti-ADAMTS13 type G immunoglobulins in patients with acquired (idiopathic) thrombotic thrombocytopenic purpura. Our data shed more light on the pathogenesis of acquired thrombotic thrombocytopenic purpura and provide further rationales for adjunctive immunotherapy.     

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.     

Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura

Severe deficiency of the von Willebrand factor (VWF)-cleaving protease ADAMTS13 can lead to thrombotic thrombocytopenic purpura (TTP), a disease associated with the widespread formation of platelet-rich thrombi in many organs. Autoantibodies that inactivate ADAMTS13 are the most frequent cause of acquired TTP. Little is known about epitope specificity and reactivity of anti-ADAMTS13 antibodies. In this study, a series of ADAMTS13 domains were expressed in Escherichia coli, and the reactivity of purified recombinant fragments with anti-ADAMTS13 auto-antibodies from 25 patients with severe ADAMTS13 deficiency was evaluated in vitro. All TTP plasmas contained antibodies directed against the cysteine-rich spacer (cys-rich/spacer) domain of ADAMTS13. In the plasma of 3 patients, antibodies were detected that reacted exclusively with the cys-rich/spacer domain, underscoring the importance of this region for functional activity of ADAMTS13. In 64% of the plasmas, antibodies reacted with the 2 CUB domains, and in 56% they reacted with the isolated first thrombospondin type 1 (TSP-1) repeat and with the compound fragment consisting of the catalytic, the disintegrin-like, and the TSP1-1 domain. Less frequently, in 28% of the plasmas, antibodies reacted with the TSP1 repeats 2 to 8. Unexpectedly, antibodies reacted with the propeptide region in 20% of the plasmas. In conclusion, this study shows that even though anti-ADAMTS13 autoantibodies react with multiple domains of the protease, the cys-rich/spacer domain is consistently involved in antibody reactivity.     

Conformational activation of ADAMTS13

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a “closed” conformation of WT ADAMTS13 and suggested a more “open” conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB–spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.Von Willebrand factor (VWF) is a large, multidomain glycoprotein that recognizes vascular damage by binding to exposed collagen through its A3 domain (1–3). VWF tethered to collagen responds to shear forces by adapting its conformation (4). Under conditions of low shear, it is thought to adopt a globular conformation, whereas at high shear, it unfolds and reveals its binding site for the platelet GpIbα receptor on its A1 domain, thereby facilitating platelet recruitment to the site of vascular injury.VWF is stored before release into the plasma as multimers that can be as large as 20–40 mers (5–8). On release from the cell, the highest molecular weight multimers are the most hemostatically active. Indeed, “ultra-large” multimers present a potential hazard if their function is unregulated, because they can predispose to the formation of VWF-platelet microthrombi that can occlude small blood vessels, resulting in thrombotic thrombocytopenic purpura (TTP) (9).The metalloprotease ADAMTS13 is able to cleave the VWF A2 domain, dramatically reducing the multimeric size of VWF and its propensity to form platelet microthrombi (10, 11). Cleavage of VWF by ADAMTS13 is a multistep process. An initial positioning interaction occurs between the D4CK domains of globular VWF and ADAMTS13 (12, 13). As unfolding occurs, exposure of the VWF scissile bond, Y1605-M1606, within the A2 domain is controlled by structural elements contained within this domain (14–17). Progressive unfolding allows distinct functional exosites within its A2 domain to be exposed and engaged by complementary binding sites on the protease, spacer, and disintegrin-like domains of ADAMTS13 (18–22). Ultimately, docking of VWF scissile bond P1′, P1, and P3 residues into subsites on the protease position the scissile bond for cleavage (23). Thus, conformational changes in VWF are essential for its efficient cleavage by ADAMTS13.To explore the possible role of conformation on ADAMTS13 function (24, 25), we studied a recently described gain-of-function (GoF) variant of ADAMTS13 (26). Jian et al. (26) concluded that an ADAMTS13 GoF variant comprising composite spacer domain substitutions R568K/F592Y/R660K/Y661F/Y665F (hereinafter, GoF) had an approximate fourfold increased ability to cleave VWF substrates. A similar increase in activity on removal of the C-terminal domains from ADAMTS13 has been reported by others (27). Based on our investigation of the properties of ADAMTS13, the GoF variant, and their derivatives reported herein, we propose that ADAMTS13 normally adopts a globular conformation determined by interaction of its spacer and CUB domains and is unfolded during conformational activation.     

Allosteric activation of ADAMTS13 by von Willebrand factor

The metalloprotease ADAMTS13 cleaves von Willebrand factor (VWF) within endovascular platelet aggregates, and ADAMTS13 deficiency causes fatal microvascular thrombosis. The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains of ADAMTS13 recognize a cryptic site in VWF that is exposed by tensile force. Another seven T and two complement C1r/C1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains of uncertain function are C-terminal to the MDTCS domains. We find that the distal T8-CUB2 domains markedly inhibit substrate cleavage, and binding of VWF or monoclonal antibodies to distal ADAMTS13 domains relieves this autoinhibition. Small angle X-ray scattering data indicate that distal T-CUB domains interact with proximal MDTCS domains. Thus, ADAMTS13 is regulated by substrate-induced allosteric activation, which may optimize VWF cleavage under fluid shear stress in vivo. Distal domains of other ADAMTS proteases may have similar allosteric properties.After vascular injury, platelets adhere to von Willebrand factor (VWF) multimers bound to endothelial cell surfaces and connective tissue. The force of flowing blood on a growing platelet-rich thrombus stretches the central A2 domain of VWF and exposes a Tyr¹⁶⁰⁵-Met¹⁶⁰⁶ cleavage site for ADAMTS13 (Fig. 1A) (1–5), a metalloprotease that severs VWF and releases adherent platelets. Deficiency of ADAMTS13 disrupts this feedback regulatory mechanism and causes thrombotic thrombocytopenic purpura (TTP), which is characterized by life-threatening microvascular thrombosis (3, 6, 7).Open in a separate windowFig. 1.Activation of ADAMTS13 by autoantibodies from a patient with TTP or by low pH. (A) Structure of ADAMTS13. (B) Fluorogenic substrates terminate at VWF residues indicated by arrows. Each substrate has Lys¹⁶¹⁷ replaced with Arg, N-terminal Gly modified with IRDye QC-1 (QC1), and Asn¹⁶¹⁰ replaced by Cys and modified with DyLight 633 (DyL) (22). The arrowhead indicates the cleaved Tyr-Met bond. Secondary structure elements of the VWF A2 domain (11) are indicated below and segments that interact with specific ADAMTS13 domains (13) are indicated above the sequence. (C) BCW49 plasma activated ADAMTS13 with a titer of 9.6 U at pH 7.4 (orange squares), but not at pH 6.0 (orange circle). BCW49 plasma did not activate MDTCS at pH 6 (blue circle) or pH 7.4 (blue circle). (D) Rates of VWF71 cleavage were determined as a function of pH for ADAMTS13 (orange circles) and MDTCS (blue circles). Error bars indicate 95% confidence intervals and if not shown are smaller than the symbols.The recognition and cleavage of VWF is a formidable challenge. VWF and ADAMTS13 occur at ∼10 µg/mL and ∼1 µg/mL, respectively, compared with total plasma protein of ∼80,000 µg/mL. ADAMTS13 is constitutively active and has no known inhibitors in vivo. Nevertheless, VWF is the only identified ADAMTS13 substrate, and VWF is resistant to cleavage until subjected to fluid shear stress (8), adsorbed on a surface (9), or treated with denaturants (8, 10). This specificity depends on structural features of both ADAMTS13 and VWF that have not been characterized fully.The proximal metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains domains of ADAMTS13 bind to cryptic sites that are uncovered by unfolding VWF domain A2 (11-15) (Fig. 1B), and these interactions are required for efficient cleavage of VWF or peptide substrates. More distal ADAMTS13 domains bind to sites in or near VWF domain D4 that are always available (16–18). Deletion of distal ADAMTS13 domains impairs the cleavage of VWF multimers in vitro (16, 19) and increases VWF-dependent microvascular thrombosis in vivo (20) but accelerates the cleavage of peptide substrates (12, 13). In addition, ADAMTS13 cleaves guanidine hydrochloride-treated VWF multimers with an apparent K_m of ∼15 nM (21), which is 100-fold lower than the K_m of ∼1.6–1.7 µM for peptide substrates that are based on the sequence of VWF domain A2 (12, 14). These striking differences suggest that distal T or complement c1r/c1s, sea urchin epidermal growth factor, and bone morphogenetic protein (CUB) domains regulate ADAMTS13 activity. We have now shown that these distal domains inhibit ADAMTS13, and binding to VWF relieves this autoinhibition.     

Apical sorting of ADAMTS13 in vascular endothelial cells and Madin-Darby canine kidney cells depends on the CUB domains and their association with lipid rafts

ADAMTS13 biosynthesis appeared to occur mainly in hepatic stellate cells, but detection of ADAMTS13 mRNA in many other tissues suggests that vascular endothelium may also produce ADAMTS13. We showed that ADAMTS13 mRNA and protein were detectable in human umbilical vein endothelial cells, aortic endothelial cells, and endothelium-derived cell line (ECV304). ADAMTS13 in cell lysate or serum-free conditioned medium cleaved von Willebrand factor (VWF) specifically. ADAMTS13 and VWF were localized to the distinct compartments of endothelial cells. Moreover, ADAMTS13 was preferentially sorted into apical domain of ECV304 and Madin-Darby canine kidney (MDCK) cells. Apical sorting of ADAMTS13 depended on the CUB domains and their association with lipid rafts. A mutation in the second CUB domain of ADAMTS13 (4143-4144insA), naturally occurring in patients with inherited thrombotic thrombocytopenic purpura, resulted in a significant reduction of ADAMTS13 secretion and a reversal of its polarity in MDCK cells. These data demonstrated that ADAMTS13 is synthesized and secreted from endothelial cells; the apically secreted ADAMTS13 from endothelial cells may contribute significantly to plasma ADAMTS13 proteases. The data also suggest a critical role of the CUB domains and a novel cargo-selective mechanism for apical sorting of a soluble ADAMTS protease in polarized cells.     

Molecular biology of ADAMTS13 and diagnostic utility of ADAMTS13 proteolytic activity and inhibitor assays

ADAMTS13, a reprolysin-like metalloprotease, limits platelet-rich thrombus formation in the small arteries by cleaving von Willebrand factor (vWF) at the Tyr1605-Met1606 peptide bond. Deficiency of plasma ADAMTS13 activity, due to either an inherited or an acquired etiology, may lead to a potentially lethal syndrome, thrombotic thrombocytopenic purpura (TTP). Molecular cloning and characterization of the ADAMTS13 gene have provided further insight into the structure-function relationships, biosynthesis, and regulation of the ADAMTS13 protease, in addition to understanding the pathogenesis of TTP and perhaps other thrombotic disorders. ADAMTS13 consists of a short propeptide, a typical reprolysin-like metalloprotease domain, followed by a disintegrin-like domain, first thrombospondin type 1 (TSP1) repeat, Cys-rich domain, and spacer domain. The carboxyl terminus of ADAMTS13 has seven more TSP1 repeats and two CUB domains. ADAMTS13 is synthesized mainly in hepatic stellate cells, but also in vascular endothelial cells. Recognition and cleavage of vWF require the proximal carboxyl terminal domains, but not the middle and distal carboxyl terminal domains. Cleavage of vWF appears to be modulated by shear force, binding to platelet or platelet glycoprotein-1balpha, heparin, inflammatory cytokine (interleukin-6), and chloride ion. At the site of thrombus formation, the ADAMTS13 may be inactivated by thrombin, plasmin, and factor Xa. Having a sensitive and specific assay for ADAMTS13 activity is not only critical to understand the basic biology of ADAMTS13 protease, but also to facilitate a more timely and accurate clinical diagnosis of TTP, and to initiate potentially life-saving plasma exchange therapy. Although many assays have been developed and tested for clinical applications, the fluorescent resonance energy transfer-vWF73 assay appears to be the simplest and most promising assay to date.     

ADAMTS13 cleavage efficiency is altered by mutagenic and, to a lesser extent, polymorphic sequence changes in the A1 and A2 domains of von Willebrand factor

The multimeric plasma protein von Willebrand factor (VWF) is regulated in size by its protease, ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13). Y1605-M1606 cleavage site mutations and single nucleotide polymorphisms (SNPs) in the VWF A1 and A2 domains were examined for alteration in ADAMTS13-mediated cleavage of VWF. Recombinant human full-length VWF (rVWF) was digested with recombinant human ADAMTS13 (rADAMTS13) using a dialysis membrane method with 1.5 mol/l urea, and analyzed via multimer migration distance. The glutathione-S-transferase (GST) and histidine-tagged construct, E1554-R1668 of VWF (VWF115) was assayed via enzyme-linked immunosorbent assay: VWF115 was bound to anti-GST coated plates, digested with rADAMTS13, and intact VWF115 detected via horseradish peroxidase-labelled anti-histidine tag antibody. All alterations examined in the Y1605-M1606 cleavage site greatly reduced the cleavability of VWF by ADAMTS13 in the rVWF assay. Greatest cleavage resistance in both assays was observed in Y1605A/M1606A. In contrast, Y1605H and M1606L show a loss of cleavability only in the rVWF assay, suggesting that an aromatic ring at 1605 is critical for ADAMTS13 recognition. Additionally, under our rVWF assay conditions, the G1643S polymorphism showed increased cleavage, suggesting a Type 2A VWD phenotype, while D1472H, Q1571H and P1601T showed slightly decreased ADAMTS13 cleavage. Our two complementary assay conditions show that A-domain changes in VWF alter ADAMTS13-mediated proteolysis.     

Extensive contacts between ADAMTS13 exosites and von Willebrand factor domain A2 contribute to substrate specificity

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr(1605)-Met(1606) bond in the central A2 domain of multimeric von Willebrand factor (VWF), even though VWF constitutes only 0.02% of plasma proteins. This remarkable specificity depends in part on binding of the noncatalytic ADAMTS13 spacer domain to the C-terminal alpha-helix of VWF domain A2. By kinetic analysis of recombinant ADAMTS13 constructs, we show that the first thrombospondin-1, Cys-rich, and spacer domains of ADAMTS13 interact with segments of VWF domain A2 between Gln(1624) and Arg(1668), and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln(1624)-Arg(1641) minimally affected the rate of cleavage, indicating that ADAMTS13 does not require a specific distance between the scissile bond and auxiliary substrate binding sites. Smaller deletions of the P2-P9 or the P4'-P18' residues on either side of the Tyr(1605)-Met(1606) bond abolished cleavage, indicating that the metalloprotease domain interacts with additional residues flanking the cleavage site. Thus, specific recognition of VWF depends on cooperative, modular contacts between several ADAMTS13 domains and discrete segments of VWF domain A2.     

ADAMTS13 and TTP

Thrombotic thrombocytopenic purpura (TTP) has been a mysterious and deadly disease that often could be treated effectively by plasma exchange, but without real understanding of the underlying pathophysiology. Recent advances now suggest that deficiency of a specific von Willebrand factor (VWF) cleaving protease promotes tissue injury in TTP. VWF multimers participate in the formation of platelet thrombi. Proteolytic cleavage of VWF multimers normally limits platelet thrombus growth, and failure to cleave VWF appears to encourage microvascular thrombosis. The VWF cleaving protease proves to be a new member of the ADAMTS family of metalloproteases, designated ADAMTS13. Autoantibodies that inhibit ADAMTS13 cause sporadic TTP, and mutations in the ADAMTS13 gene cause an autosomal recessive form of chronic relapsing TTP. Further studies of ADAMTS13 seem likely to change our approach to the diagnosis and treatment of TTP and other thrombotic microangiopathies.     

N-Glycans of ADAMTS13 modulate its secretion and von Willebrand factor cleaving activity

Severe deficiency of ADAMTS13, a plasma metalloprotease, leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains 10 putative N-glycosylation sites in or near its metalloprotease sequence, spacer region, thrombospondin type 1 repeat no. 4 (TSR no. 4), and CUB domains. Tunicamycin treatment markedly decreased the secretion of ADAMTS13 into the culture medium of transfected cells. Nevertheless, the protease was efficiently secreted from N-acetylglucosaminyltransferase I-deficient Lec1 Chinese hamster ovary cells, indicating that N-glycosylation in the endoplasmic reticulum, but not the conversion of oligomannose to complex N-glycans in the Golgi complex, is important for secretion. However, ADAMTS13 with oligomannose N-glycans cleaved its substrate, von Willebrand factor (VWF) multimers, less effectively, with a higher K(m) but similar k(cat) value. In mutagenesis analysis, decreased secretion and VWF cleaving activity was observed with the N146Q and N828Q mutants, while decreased secretion only was observed with the N552Q mutant of ADAMTS13. Enzymatic removal of N-glycans from ADAMTS13 did not affect its VWF cleaving activity. Thus, N-glycosylation is necessary for efficient secretion of ADAMTS13, while conversion of the N-glycans from oligomannose to complex type in the Golgi complex enhances the proteolytic activity of the protease toward VWF multimers. After its secretion, ADAMTS13 does not require N-glycans for its VWF cleaving activity.     

Crystal structures of the noncatalytic domains of ADAMTS13 reveal multiple discontinuous exosites for von Willebrand factor

ADAMTS13 specifically cleaves plasma von Willebrand factor (VWF) and thereby controls VWF-mediated platelet thrombus formation. Severe deficiencies in ADAMTS13 can cause life-threatening thrombotic thrombocytopenic purpura. Here, we determined 2 crystal structures of ADAMTS13-DTCS (residues 287–685), an exosite-containing human ADAMTS13 fragment, at 2.6-Å and 2.8-Å resolution. The structures revealed folding similarities between the disintegrin-like (D) domain and the N-terminal portion of the cysteine-rich domain (designated the C_A domain). The spacer (S) domain forms a globular functional unit with a 10-stranded β-sandwich fold that has multiple interaction sites with the C_A domain. We expressed 25 structure-based mutants of ADAMTS13-MDTCS (residues 75–685) and measured their enzymatic activity. We identified 3 VWF-binding exosites on the linearly aligned discontinuous surfaces of the D, C_A, and S domains traversing the W-shaped molecule. Since the MDTCS domains are conserved among ADAMTS family proteins, the structural framework of the multiple enzyme-substrate interactions identified in the ADAMTS13-VWF system provides the basis for a common substrate recognition mode in this class of proteinases.     

Platelet-VWF complexes are preferred substrates of ADAMTS13 under fluid shear stress

Endothelial cells secrete prothrombotic ultralarge von Willebrand factor (VWF) multimers, and the metalloprotease ADAMTS13 cleaves them into smaller, less dangerous multimers. This reaction is stimulated by tensile force applied to the VWF substrate, which may occur on cell surfaces or in the circulating blood. The cleavage of soluble VWF by ADAMTS13 was accelerated dramatically by a combination of platelets and fluid shear stress applied in a cone-plate viscometer. Platelet-dependent cleavage of VWF was blocked by an anti-GPIbalpha monoclonal antibody or by a recombinant soluble fragment of GPIbalpha that prevents platelet-VWF binding. Multimeric gel analysis showed that shear and platelet-dependent cleavage consumed large VWF multimers. Therefore, ADAMTS13 preferentially acts on platelet-VWF complexes under fluid shear stress. This reaction is likely to account for a majority of VWF proteolysis after secretion and to determine the steady-state size distribution of circulating VWF multimers in vivo.     

Cleavage of ultralarge multimers of von Willebrand factor by C-terminal-truncated mutants of ADAMTS-13 under flow

A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.     

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.     

Changes in von Willebrand factor-cleaving protease (ADAMTS13) activity after infusion of desmopressin

von Willebrand factor-cleaving protease (ADAMTS13) cleaves von Willebrand factor (VWF) and regulates its physiologic function. To investigate the relation between ADAMTS13 activity and VWF, we compared ADAMTS13 activity with the VWF-related parameters VWF antigen (VWF:Ag), VWF collagen-binding activity (VWF:CBA), VWF-propeptide, proVWF, and VWF multimeric composition in 10 healthy volunteers and 3 patients with type 1 von Willebrand disease before and after infusing 0.3 microg/kg desmopressin. The VWF-related parameters in the volunteers increased 60 minutes after start of infusion by 3.7-fold for VWF:Ag, 7.2-fold for propeptide, and 2.2-fold for VWF:CBA. Unusually large VWF multimers and traces of proVWF appeared. The ADAMTS13 activity decreased to about half the initial value. After 24 hours values returned to baseline. Patients with type 1 von Willebrand disease showed similar results. We conclude that the inverse correlation between ADAMTS13 and VWF-related parameters suggests a consumption of ADAMTS13 after the desmopressin-induced release of higher multimers of VWF.     

Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers

Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.     

Platelet factor 4 inhibits ADAMTS13 activity and regulates the multimeric distribution of von Willebrand factor

The efficiency of von Willebrand factor (VWF) in thrombus formation is related to its multimeric size, which is controlled by the protease ADAMTS13. However, it is not clear what regulates ADAMTS13 activity. In this study, we investigated whether PF4 could bind to VWF and inhibit ADAMTS13 activity. We found that PF4 binds to VWF and protects against ADAMTS13 activity. We also found that VWF-PF4 complexes circulate in patients with thrombotic thrombocytopenic purpura (TTP). Our data provides the first evidence that PF4 may have a novel role in regulating VWF multimers during primary haemostasis and thrombosis.     

Acquired TTP: ADAMTS13 meets the immune system

The majority of the patients affected by acquired thrombotic thrombocytopenic purpura (TTP) develop autoantibodies directed towards ADAMTS13 that interfere with its von Willebrand Factor (VWF) processing activity. B cell responses have been shown to primarily target the spacer domain of ADAMTS13 thereby prohibiting the binding of ADAMTS13 to the VWF A2 domain. In this review we summarize recent knowledge gained on the immune recognition and processing of ADAMTS13 by antigen-presenting cells (APCs). HLA-DRB1*11 has been identified as a risk factor for acquired TTP. Analysis of MHC class II/peptide complexes of ADAMTS13 pulsed dendritic cells have shown that the CUB2 domain derived peptide FINVAPHAR is preferentially presented on HLA-DRB1*11. Based on these findings we propose a model for the initiation of the autoimmune reactivity against ADAMTS13 in previously healthy individuals. We hypothesize that mimicry between a pathogen-derived peptide and the CUB2 derived FINVAPHAR-peptide might contribute to the onset of acquired TTP.