ADAMTS-13 conformation influences autoimmune recognition in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Patients with immune-mediated thrombotic thrombocytopenic purpura (iTTP) have anti-ADAMTS-13 immunoglobulin G (IgG) autoantibodies that enhance ADAMTS-13 clearance and/or inhibit its function. ADAMTS-13 normally circulates in a closed conformation, which is manifested by the interaction of the CUB domains with the central spacer domain. Disruption of the spacer-CUB interaction opens ADAMTS-13, which augments its proteolytic function but may also expose cryptic autoimmune epitopes that promote further autoantibody recognition. OBJECTIVES: To explore differences in autoantibody binding to ADAMTS-13 in its closed or open conformations in patients with iTTP and to correlate these differences with disease-related parameters. METHODS: We developed a novel assay to measure autoantibodies binding to closed and open ADAMTS-13. Autoantibody titer and IgG subclass binding to open or closed ADAMTS-13 were measured in 70 iTTP first presentation samples and correlated with clinical data, remission, and relapse. RESULTS: In 70 patients with iTTP, the mean autoantibody titer against open ADAMTS-13 was, on average, approximately 2-fold greater than that against closed ADAMTS-13, suggesting that ADAMTS-13 opening increases epitope exposure and immune complex formation. Autoantibody titer against closed/open ADAMTS-13 and IgG subclass did not correlate with ADAMTS-13 antigen at presentation. Two patients with iTTP and persistent autoantibodies lost specificity for closed ADAMTS-13 in remission. Recognition of closed/open ADAMTS-13 and autoantibody IgG subclass between the first and second iTTP episodes were very similar. CONCLUSION: ADAMTS-13 autoantibody binding is highly influenced by ADAMTS-13 conformation. Although this does not appear to modify the pathogenicity of autoantibodies, the autoantibody signature at relapse suggests that relapse represents re-emergence of the original autoimmune response rather than de novo presentation.

ADAMTS13 conformations and mechanism of inhibition in immune thrombotic thrombocytopenic purpura.

ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor, is crucial for normal hemostasis. Acquired autoantibody-mediated deficiency of plasma ADAMTS13 results in a potentially fatal blood disorder, immune thrombotic thrombocytopenic purpura (iTTP). Plasma ADAMTS13 protease appears to exist in multiple conformations. Under physiological conditions, plasma ADAMTS13 exists predominantly in its "closed" conformation (or latent form), which may be activated by lowering pH, ligand binding, and binding of an antibody against the distal domains of ADAMTS13. In patients with iTTP, polyclonal antibodies target at various domains of ADAMTS13. However, nearly all inhibitory antibodies bind the spacer domain, whereas antibodies that bind the distal C-terminal domains may activate ADAMTS13 through removing its allosteric inhibition. Additionally, the anti-C-terminal antibodies may alter the potency of inhibitory antibodies towards ADAMTS13 activity. This review summarizes some of the most recent knowledge about the ADAMTS13 conformation and its mechanism of inhibition by its autoantibodies.

Residues R1075, D1090, R1095, and C1130 Are Critical in ADAMTS13 TSP8-Spacer Interaction Predicted by Molecular Dynamics Simulation.

ADAMTS13 (A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats, member 13) cleaves von Willebrand Factor (VWF) multimers to limit the prothrombotic function of VWF. The deficiency of ADAMTS13 causes a lethal thrombotic microvascular disease, thrombotic thrombocytopenic purpura (TTP). ADAMTS13 circulates in a "closed" conformation with the distal domain associating the Spacer domain to avoid off-target proteolysis or recognition by auto-antibodies. However, the interactions of the distal TSP8 domain and the Spacer domain remain elusive. Here, we constructed the TSP8-Spacer complex by a combination of homology modelling and flexible docking. Molecular dynamics simulation was applied to map the binding sites on the TSP8 or Spacer domain. The results predicted that R1075, D1090, R1095, and C1130 on the TSP8 domain were key residues that interacted with the Spacer domain. R1075 and R1095 bound exosite-4 tightly, D1090 formed multiple hydrogen bonds and salt bridges with exosite-3, and C1130 interacted with both exosite-3 and exosite-4. Specific mutations of exosite-3 (R568K/F592Y/R660K/Y661F/Y665F) or the four key residues (R1075A/D1090A/R1095A/C1130A) impaired the binding of the TSP8 domain to the Spacer domain. These results shed new light on the understanding of the auto-inhibition of ADAMTS13.

The activity of the intrinsically water-soluble enzyme ADAMTS13 correlates with the membrane state when bound to a phospholipid bilayer.

Membrane-associated enzymes have been found to behave differently qualitatively and quantitatively in terms of activity. These findings were highly debated in the 1970s and many general correlations and reaction specific models have been proposed, reviewed, and discarded. However, new biological applications brought up the need for clarification and elucidation. To address literature shortcomings, we chose the intrinsically water-soluble enzyme a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) and large unilamellar vesicles with a relative broad phase transition. We here present activity measurements of ADAMTS13 in the freely dissolved state and the membrane associated state for phosphocholine lipids with different acyl-chain lengths (13:0, 14:0 and 15:0) and thus main phase transition temperatures. While the freely dissolved enzyme shows a simple Arrhenius behavior, the activity of membrane associated ADAMTS13 in addition shows a peak. This peak temperature correlates with the main phase transition temperature of the used lipids. These findings support an alternative theory of catalysis. This theory predicts a correlation of the membrane associated activity and the heat capacity, as both are susceptibilities of the same surface Gibb's free energy, since the enzyme is attached to the membrane.

Incidence, diagnosis, and outcome of immune-mediated thrombotic thrombocytopenic purpura: A nationwide survey by the Spanish registry of thrombotic thrombocytopenic purpura.

BACKGROUND: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare disease characterized by the presence of anti-ADAMTS13 autoantibodies. Achieving accurate information on incidence and customary disease management is important to provide appropriate diagnostic and therapeutic resources. The aim of this study was to determine the incidence and outcomes of iTTP in Spain. STUDY DESIGN AND METHODS: A cross-sectional survey was carried out among Spanish hospitals, focused on iTTP patients ≥16 years old attended between 2015 and 2017, and those at follow-up before that interval. Incidence, prevalence, mortality, refractoriness, exacerbations, treatment complications, relapses, and sequelae were estimated. RESULTS: Forty-two hospitals covering roughly 20 million inhabitants answered the survey and reported 203 episodes (138 newly-diagnosed and 65 relapses), of which 193 (95.1%) were treated. Incidence was 2.67 (95% CI 1.90-3.45) patients per million inhabitants per year and prevalence 21.44 (95% CI% 19.10-23.73) patients per million inhabitants. At diagnosis, ADAMTS13 activity and anti-ADAMTS13 autoantibody were measured in 97% and 84.3% of reported episodes, respectively. Fifteen patients (7.4%) died as a direct consequence of iTTP, 6 of them before receiving any iTTP-specific treatment. Thirty-one (16.1%) of the 193 treated episodes were refractory to plasma exchange and corticosteroids, and 51 (26.4%) suffered at least one exacerbation. CONCLUSION: iTTP incidence and prevalence were somewhat higher than those documented in neighboring countries. Together with data on treatments and outcomes, this information will allow us to better estimate what is needed to improve diagnosis and prognosis of iTTP patients in Spain.

N-glycan-mediated shielding of ADAMTS13 prevents binding of pathogenic autoantibodies in immune-mediated TTP.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is an autoimmune disorder caused by the development of autoantibodies targeting different domains of ADAMTS13. Profiling studies have shown that residues R568, F592, R660, Y661, and Y665 within exosite-3 of the spacer domain provide an immunodominant region of ADAMTS13 for pathogenic autoantibodies that develop in patients with iTTP. Modification of these 5 core residues with the goal of reducing autoantibody binding revealed a significant tradeoff between autoantibody resistance and proteolytic activity. Here, we employed structural bioinformatics to identify a larger epitope landscape on the ADAMTS13 spacer domain. Models of spacer-antibody complexes predicted that residues R568, L591, F592, K608, M609, R636, L637, R639, R660, Y661, Y665, and L668 contribute to an expanded epitope within the spacer domain. Based on bioinformatics-guided predictions, we designed a panel of N-glycan insertions in this expanded epitope to reduce the binding of spacer domain autoantibodies. One N-glycan variant (NGLY3-ADAMTS13, containing a K608N substitution) showed strongly reduced reactivity with TTP patient sera (28%) as compared with WT-ADAMTS13 (100%). Insertion of an N-glycan at amino acid position 608 did not interfere with processing of von Willebrand factor, positioning the resulting NGLY3-ADAMTS13 variant as a potential novel therapeutic option for treatment of iTTP.

Co(III)-NTA Mediated Antigen Immobilization on a Fiber Optic-SPR Biosensor for Detection of Autoantibodies in Autoimmune Diseases: Application in Immune-Mediated Thrombotic Thrombocytopenic Purpura.

Autoantibodies are key biomarkers in clinical diagnosis of autoimmune diseases routinely detected by enzyme-linked immunosorbent assays (ELISAs). However, the complexity of these assays is limiting their use in routine diagnostics. Fiber optic-surface plasmon resonance (FO-SPR) can overcome these limitations, but improved surface chemistries are still needed to guarantee detection of autoantibodies in complex matrices. In this paper, we describe the development of an FO-SPR immunoassay for the detection of autoantibodies in plasma samples from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients. Hereto, hexahistidine-tagged recombinant ADAMTS13 (rADAMTS13-His6) was immobilized on nitrilotriacetic acid (NTA)-coated FO probes chelated by cobalt (Co(III)) and exposed to anti-ADAMTS13 autoantibodies. Initial studies were performed to optimize rADAMTS13-His6 immobilization and to confirm the specificity of the immunoassay for detection of anti-ADAMTS13 autoantibodies with FO-SPR. The performance of the immunoassay was then evaluated by comparing Co(III)- and nickel (Ni(II))-NTA stabilized surfaces, confirming the stable immobilization of the antigen in Co(III)-NTA-functionalized FO probes. A calibration curve was prepared with a dilution series of a cloned human anti-ADAMTS13 autoantibody in ADAMTS13-depleted plasma resulting in an average interassay coefficient of variation of 7.1% and a limit of detection of 0.24 ng/mL. Finally, the FO-SPR immunoassay was validated using seven iTTP patient plasma samples, resulting in an excellent correlation with an in-house-developed ELISA (r = 0.973). In summary, the specificity and high sensitivity in combination with a short time-to-result (2.5 h compared to 4-5 h for a regular ELISA) make the FO-SPR immunoassay a powerful assay for routine diagnosis of iTTP and with extension for any other autoimmune disease.

Tryptophan 387 and 390 residues in ADAMTS13 are crucial to the ability of vascular tube formation and cell migration of endothelial cells.

A disintegrin and metalloproteinase with thrombospondin motifs 13 (ADAMTS13) was mainly generated and secreted from endothelial cells (ECs). Our previous study showed that tryptophan (Trp) residues at 387 and 390 in ADAMTS13 are required for its secretion and enzymatic activity. However, the effects on its host cell as well as the potential mechanism have not been clear. The aim of the study was to examine the effects of Trp residues 387 and 390 of ADAMTS13 on the biological processes of ECs. Herein, Trp was substituted with alanine in ADAMTS13 to generate ADAMTS13 mutants at 387 (W387A), 390 (W390A), and double mutants at 387 and 390 (2WA), respectively. We found that substitution mutation impaired vascular endothelial growth factor (VEGF) secretion and the downstream JAK1/STAT3 activation, the binding ability to Von Willebrand factor, cell proliferation, migration, and vascular tube formation. Overall, our study concluded that Trp387 and Trp390 of ADAMTS13 play vital roles in the biological function of ECs.

Mechanochemistry of von Willebrand factor.

Von Willebrand factor (VWF), a blood multimeric protein with a very high molecular weight, plays a crucial role in the primary haemostasis, the physiological process characterized by the adhesion of blood platelets to the injured vessel wall. Hydrodynamic forces are responsible for extensive conformational transitions in the VWF multimers that change their structure from a globular form to a stretched linear conformation. This feature makes this protein particularly prone to be investigated by mechanochemistry, the branch of the biophysical chemistry devoted to investigating the effects of shear forces on protein conformation. This review describes the structural elements of the VWF molecule involved in the biochemical response to shear forces. The stretched VWF conformation favors the interaction with the platelet GpIb and at the same time with ADAMTS-13, the zinc-protease that cleaves VWF in the A2 domain, limiting its prothrombotic capacity. The shear-induced conformational transitions favor also a process of self-aggregation, responsible for the formation of a spider-web like network, particularly efficient in the trapping process of flowing platelets. The investigation of the biophysical effects of shear forces on VWF conformation contributes to unraveling the molecular mechanisms of many types of thrombotic and haemorrhagic syndromes.

A mechano-reactive coarse-grained model of the blood-clotting agent von Willebrand factor.

The von Willebrand Factor (vWF) is a large blood glycoprotein that aids in hemostasis. Within each vWF monomer, the A2 domain hosts a cleavage site for enzyme ADAMTS13, which regulates the size of vWF multimers. This cleavage site can only be exposed when an A2 domain unfolds, and the unfolding reaction energy landscape is highly sensitive to the force conditions on the domain. Based on previous optical tweezer experimental results, we advance here a new activated A2 monomer model (AA2MM) for coarse-grained modeling of vWF that accurately represents the force-based probabilistic change between the unfolded/refolded states. A system of springs is employed to mimic the complex mechanical response of vWF monomers subject to pulling forces. AA2MM was validated by comparing monomer scale simulation results to data from prior pulling experiments on vWF monomer fragments. The model was further validated by comparing multimer scale Brownian dynamics simulation results to experiments using microfluidic chamber microscopy to visualize tethered vWF proteins subject to flow. The A2 domain unfolding reaction was studied in bulk flow simulations (pure shear and elongation flow), giving evidence that elongational flow drives the vWF size regulation process in blood. The mechanoreactive, coarse-grained AA2MM accurately describes the complex mechanical coupling between human blood flow conditions and vWF protein reactivity.

Identification of cysteine thiol-based linkages in ADAMTS13 in support of a non-proteolytic regulation of von Willebrand factor.

BACKGROUND: ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) to regulate its function. Additionally, ADAMTS13 is thought to regulate lateral association of VWF multimers to form fibrillar structures through its free thiols. OBJECTIVE: The purpose of the present study is to obtain direct evidence for ADAMTS13 to engage in thiol/disulfide exchange reactions. METHODS: Covalent complexes between ADAMTS13 and VWF were determined by agarose gel electrophoresis under nonreducing conditions. Free thiols in ADAMST13 were identified by a reversed phase high-performance liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry system. RESULTS: We demonstrate formation of covalent linkage between ADAMTS13 and VWF, which is time, concentration, temperature, and shear dependent. This interaction is independent of proteolytic activity of ADAMTS13 but depends on the C-terminal domains comprising the fifth through eighth thrombospondin type 1 repeats and C1r/C1s, Uegf, Bmp1 (CUB) domains. The interaction can be blocked by thiol-reactive agents, indicating that association is accomplished through disulfide bridge formation. Several partially reduced free thiols are identified in ADAMTS13, with cysteines 1254 and 1275 being the most prominent, although a point mutation (C1275S) in ADAMTS13 does not alter its ability to form covalent linkages with VWF. This suggests functionally relevant disulfide plasticity in ADAMTS13. Interestingly, ADAMTS13 also forms homo-oligomers under the same conditions as required for the generation of hetero-oligomeric complexes of ADAMTS13 and VWF. CONCLUSIONS: Our results suggest that a dynamic network of free thiols in ADAMTS13 undergoing intra- and inter-molecular redox reactions may add another layer of regulation to VWF function under various conditions.

Exosites in Hypervariable Loops of ADAMTS Spacer Domains control Substrate Recognition and Proteolysis.

ADAMTS (A Disintegrin-like and Metalloproteinase domain with Thrombospondin type 1 Motif)-1, -4 and -5 share the abilities to cleave large aggregating proteoglycans including versican and aggrecan. These activities are highly relevant to cardiovascular disease and osteoarthritis and during development. Here, using purified recombinant ADAMTS-1, -4 and -5, we quantify, compare, and define the molecular basis of their versicanase activity. A novel sandwich-ELISA detecting the major versican cleavage fragment was used to determine, for the first time, kinetic constants for versican proteolysis. ADAMTS-5 (kcat/Km 35 × 105 M-1 s-1) is a more potent (~18-fold) versicanase than ADAMTS-4 (kcat/Km 1.86 × 105 M-1 sec-1), whereas ADAMTS-1 versicanase activity is comparatively low. Deletion of the spacer domain reduced versicanase activity of ADAMTS-5 19-fold and that of ADAMTS-4 167-fold. Co-deletion of the ADAMTS-5 cysteine-rich domain further reduced versicanase activity to a total 153-fold reduction. Substitution of two hypervariable loops in the spacer domain of ADAMTS-5 (residues 739-744 and 837-844) and ADAMTS-4 (residues 717-724 and 788-795) with those of ADAMTS-13, which does not cleave proteoglycans, caused spacer-dependent reductions in versicanase activities. Our results demonstrate that these loops contain exosites critical for interaction with and processing of versican. The hypervariable loops of ADAMTS-5 are shown to be important also for its aggrecanase activity. Together with previous work on ADAMTS-13 our results suggest that the spacer domain hypervariable loops may exercise significant control of ADAMTS proteolytic activity as a general principle. Identification of specific exosites also provides targets for selective inhibitors.

Domain-specific mechanical modulation of VWF-ADAMTS13 interaction.

Hemodynamic forces activate the Von Willebrand factor (VWF) and facilitate its cleavage by a disintegrin and metalloprotease with thrombospondin motifs-13 (ADAMTS13), reducing the adhesive activity of VWF. Biochemical assays have mapped the binding sites on both molecules. However, these assays require incubation of two molecules for a period beyond the time allowed in flowing blood. We used a single-molecule technique to examine these rapid, transient, and mechanically modulated molecular interactions in short times under forces to mimic what happens in circulation. Wild-type ADAMTS13 and two truncation variants that either lacked the C-terminal thrombospondin motif-7 to the CUB domain (MP-TSP6) or contained only the two CUB domains (CUB) were characterized for interactions with coiled VWF, flow-elongated VWF, and a VWF A1A2A3 tridomain. These interactions exhibited distinctive patterns of calcium dependency, binding affinity, and force-regulated lifetime. The results suggest that 1) ADAMTS13 binds coiled VWF primarily through CUB in a calcium-dependent manner via a site(s) outside A1A2A3, 2) ADAMTS13 binds flow-extended VWF predominantly through MP-TSP6 via a site(s) different from the one(s) at A1A2A3; and 3) ADAMTS13 binds A1A2A3 through MP-TSP6 in a Ca2+-dependent manner to autoinhibit another Ca2+-independent binding site on CUB. These data reveal that multiple sites on both molecules are involved in mechanically modulated VWF-ADAMTS13 interaction.

Temperature-dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation.

BACKGROUND: The catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion-depleting environment has not been explored. OBJECTIVES: To address the stability of ADAMTS13 in citrated human plasma. METHODS: ADAMTS13 activity was measured using the FRETS-VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Higher-order structural changes were analyzed using Fourier-transformed infrared spectroscopy and dynamic light scattering. RESULTS: ADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time-dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC-ICP-MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+ . Concomitant higher-order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less-ordered protein structures. CONCLUSIONS: Zn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.

von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain.

von Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues ("Lock-VWF") reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain ("ΔA1 proteins") suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

Exploring the "minimal" structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering.

Human ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)-platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58-amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a "minimal" structure for allosterically regulated ADAMTS13.

Long-ranged Protein-glycan Interactions Stabilize von Willebrand Factor A2 Domain from Mechanical Unfolding.

von Willebrand Factor (vWF) is a large multimeric protein that binds to platelets and collagen in blood clotting. vWF A2 domain hosts a proteolytic site for ADAMTS13 (A Disintegrin and Metalloprotease with a ThromboSpondin type 1 motif, member 13) to regulate the size of vWF multimers. This regulation process is highly sensitive to force conditions and protein-glycan interactions as the process occurs in flowing blood. There are two sites on A2 domain (N1515 and N1574) bearing various N-linked glycan structures. In this study, we used molecular dynamics (MD) simulation to study the force-induced unfolding of A2 domain with and without a single N-linked glycan type on each site. The sequential pullout of ß-strands was used to represent a characteristic unfolding sequence of A2. This unfolding sequence varied due to protein-glycan interactions. The force-extension and total energy-extension profiles also show differences in magnitude but similar characteristic shapes between the systems with and without glycans. Systems with N-linked glycans encountered higher energy barriers for full unfolding and even for unfolding up to the point of ADAMTS13 cleavage site exposure. Interestingly, there is not much difference observed for A2 domain structure itself with and without glycans from standard MD simulations, suggesting roles of N-glycans in A2 unfolding through long-ranged protein-glycan interactions.

Preemptive rituximab prevents long-term relapses in immune-mediated thrombotic thrombocytopenic purpura.

Preemptive rituximab infusions prevent relapses in immune thrombotic thrombocytopenic purpura (iTTP) by maintaining normal ADAMTS13 activity. However, the long-term outcome of these patients and the potential adverse events of this strategy need to be determined. We report the long-term outcome of 92 patients with iTTP in clinical remission who received preemptive rituximab after identification of severe ADAMTS13 deficiency (activity <10%) during the follow-up. Thirty-seven patients had >1 iTTP episode, and the median cumulative relapse incidence before preemptive rituximab was 0.33 episode per year (interquartile range [IQR], 0.23-0.66). After preemptive rituximab, the median cumulative relapse incidence in the whole population decreased to 0 episodes per year (IQR, 0-1.32; P < .001). After preemptive rituximab, ADAMTS13 activity recovery was sustained in 34 patients (37%) during a follow-up of 31.5 months (IQR, 18-65), and severe ADAMTS13 deficiency recurred in 45 patients (49%) after the initial improvement. ADAMTS13 activity usually improved with additional courses of preemptive rituximab. In 13 patients (14%), ADAMTS13 activity remained undetectable after the first rituximab course, but retreatment was efficient in 6 of 10 cases. In total, 14 patients (15%) clinically relapsed, and 19 patients (20.7%) experienced benign adverse effects. Preemptive rituximab treatment was associated with a change in ADAMTS13 conformation in respondent patients. Finally, in the group of 23 historical patients with iTTP and persistently undetectable ADAMTS13 activity, 74% clinically relapsed after a 7-year follow-up (IQR, 5-11). In conclusion, persistently undetectable ADAMTS13 activity in iTTP during remission is associated with a higher relapse rate. Preemptive rituximab reduces clinical relapses by maintaining a detectable ADAMTS13 activity with an advantageous risk-benefit balance.

Identification of a crucial tryptophan residue in ADAMTS13 required for its secretion and enzymatic activity.

Functional deficiency of A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)13 causally assists in the pathology of thrombotic thrombocytopenic purpura (TTP). Previous study showed that the WXXW motif is very important to the enzymatic activity of ADAMTS13. However, the functional role for a single amino acid residue within WXXW motif is still undetermined. Here, Trp390 residue within WXXW motif was substituted with Ala to generate Trp390Ala (W390A) mutant ADAMTS13. We found that W390A mutant ADAMTS13 had impaired binding affinity to its substrate Von Willebrand factor (VWF). Moreover, W390A mutant retarded ADAMTS13 secretion, leading to its deposition in endoplasmic reticulum. Compared with the wild type ADAMTS13, W390A mutant also had a decreased cleavage activity for multimeric VWF under both static and shear stress conditions. These data indicate that Trp390 residue within the WXXW motif is required for ADAMTS13 secretion and enzymatic activity, which provide insight into understanding the pathological basis of TTP and opens new avenues for exploring potential treatment strategies.

Prediction of spacer-α6 complex: a novel insight into binding of ADAMTS13 with A2 domain of von Willebrand factor under forces.

Force-regulated cleavage of A2 domain of von Willebrand factor (vWF) by ADAMTS13 is a key event in preventing thrombotic thrombocytopenic purpura (TTP). Recognition and cleavage depend on cooperative and modular contacts between several ADAMTS13 subdomains and discrete segments of vWF A2 domain. Spacer domain of ADAMTS13 contains an important exosite interacting with α6 helix of unfold A2 domain, but it remains unclear whether stretching of α6 regulates binding to spacer. To understand the molecular mechanism underlying the interactions between spacer and α6 under stretching, we successfully predicted spacer-α6 complex by a novel computer strategy combined the steered molecular dynamics (SMD) and flexible docking techniques. This strategy included three steps: (1) constant-velocity SMD simulation of α6; (2) zero-velocity SMD simulations of α6, and (3) flexible dockings of α6 to spacer. In our spacer-α6 complex model, 13 key residues, six in α6 and seven in spacer, were identified. Our data demonstrated a biphasic extension-regulated binding of α6 to spacer. The binding strength of the complex increased with α6 extension until it reaches its optimum of 0.25 nm, and then decreased as α6 extension further increased, meaning that spacer is in favor to binding with a partially extended α6, which may contribute to the optimal contact and proteolysis. Changes of interface area and intermolecular salt bridge may serve as the molecular basis for this characteristic. These findings provide a novel insight into mechano-chemical regulation on interaction between ADAMTS13 and vWF A2 domain under forces.