Full-length ADAMTS13 Research Articles

Metalloprotease domain latency protects ADAMTS13 against broad-spectrum inhibitors of metalloproteases while maintaining activity toward VWF

BackgroundADAMTS13 is a circulating metalloprotease that cleaves von Willebrand factor (VWF) in a shear-dependent manner. ADAMTS13 is secreted as an active protease but has a long half-life, suggesting that it is resistant to circulating protease inhibitors. These zymogen-like properties indicate that ADAMTS13 exists as a latent protease that is activated by its substrate. ObjectivesTo investigate the mechanism of ADAMTS13 latency and resistance to metalloprotease inhibitors. MethodsProbe the active site of ADAMTS13 and variants using alpha-2 macroglobulin (A2M), tissue inhibitors of metalloproteases (TIMPs), and Marimastat. ResultsADAMTS13 and C-terminal deletion mutants are not inhibited by A2M, TIMPs, or Marimastat, but cleave FRETS-VWF73, suggesting that the metalloprotease domain is latent in the absence of substrate. Within the metalloprotease domain, mutating the gatekeeper triad (R193, D217, D252) or substituting the calcium-binding (R180-R193) or the variable (G236-S263) loops with corresponding features from ADAMTS5 did not sensitize MDTCS to inhibition. However, substituting the calcium-binding loop and an extended variable loop (G236-S263) corresponding to the S1–S1′ pockets with those from ADAMTS5, resulted in MDTCS-GVC5 inhibition by Marimastat, but not by A2M or TIMP3. Substituting the MD domains of ADAMTS5 into full-length ADAMTS13 resulted in a 50-fold reduction in activity compared with the substitution into MDTCS. However, both chimeras were susceptible to inhibition, suggesting that the closed conformation does not contribute to the latency of the metalloprotease domain. ConclusionThe metalloprotease domain protects ADAMTS13 from inhibitors and exists in a latent state that is partially maintained by loops flanking the S1 and S1′ specificity pockets.

A novel mechanism underlying allosteric regulation of ADAMTS-13 revealed by hydrogen−deuterium exchange plus mass spectrometry

BackgroundADAMTS-13, a plasma metalloprotease, cleaves von Willebrand factor. ADAMTS-13 activity appears to be regulated through allosteric inhibition by its distal C-terminus. ObjectivesThe objective of this study was to better understand how domain–domain interactions may affect ADAMTS-13 conformations and functions. MethodsWe performed deuterium–hydrogen exchange plus mass spectrometry to assess the number and rate of deuterium incorporation into various peptides of full-length ADAMTS-13 and its truncated variants. ResultsUnder physiological conditions, a bimodal distribution of deuterium incorporation was detected in the peptides from metalloprotease (217-230 and 282-304), cysteine-rich (446-482), and CUB (for complement C1r/C1s, Uegf, Bmp1) domains (1185-1214, 1313-1330, 1341-1347, 1358-1378, and 1393-1407) of full-length recombinant ADAMTS-13, but not of truncated variants. These results suggest that the full-length ADAMTS-13 undergoes conformational changes. On removal of the middle and distal C-terminal domains, the number and rate of deuterium incorporation were increased in the peptides from cysteine-rich (445-467, 467-482, and 495-503) and spacer domains (621-642 and 655-654) but decreased in the peptides from metalloprotease (115-124, 217-230, and 274-281). Moreover, most peptides, except for 217-230 and 1357-1376, exhibited a pD-dependent deuterium incorporation in the full-length ADAMTS-13, but not in the truncated variant (eg, MDTCS or T5C). These results further suggest that the bimodal deuterium incorporation observed in the peptides from the full-length ADAMTS-13 is the result of potential impact from the middle to distal C-terminal domains. Surface plasmon resonance revealed the direct binding interactions between the distal and proximal domains of ADAMTS-13. ConclusionOur results provide novel insight on how intramolecular interactions may affect conformations of ADAMTS-13, thus regulating its proteolytic functions.

The Mechanism of Antibody-Mediated Inhibition of ADAMTS13 in Immune Thrombotic Thrombocytopenic Purpura

Background and objective: Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially life-threatening microangiopathic disorder resulting from inhibitory immunoglobulin (Ig) G-type antibodies that target plasma metalloprotease ADAMTS13. This leads to the accumulation of ultra-large multimers of von Willebrand factor (VWF) that occludes small arterioles and capillaries and causes end-organ damage. The way inhibitory antibodies work to inhibit ADAMTS13 is an active area of investigation and may lead to a better tool for diagnosing and treating iTTP. To determine whether inhibitory antibodies prevent substrate binding to ADAMTS13, which would increase the substrate concentration at which velocity is the half maximal (K0.5); decrease the maximal enzyme velocity (Vmax) of ADAMTS13 by affecting catalytic turnover; or both, we developed an assay using a well-characterized inhibitor of ADAMTS13 to explore this important question. Method: A single chain fragment of the variable region (scFv4-20), derived from a human monoclonal antibody isolated from an iTTP patient by phage display with potent inhibitory effects on ADAMTS13 (Casina et al., PNAS. 2015; 112:9620-25), was expressed and purified. Recombinant full-length ADAMTS13 and native ADAMTS13 in normal human plasma (NHP) were titrated with a fluorescein-labeled surrogate substrate (FRETS-VWF73) in the presence of an increasing fixed concentration of scFv4-20. Titrations were analyzed both individually and applied to a global fit, which allowed for evaluation of the relative effects of the inhibitor on Vmax and K0.5. The relative effect of scFv4-20 on K0.5 was inferred from the global fit via the parameter α; values of α within an order of magnitude of unity suggest little to minimal effect of the inhibitor on K0.5. Results: When native ADAMTS13 in normal human plasma was titrated with FRETS-VWF73 in the presence of increasing concentrations of scFv4-20, the Vmax decreased with increasing concentration of scFv4-20, but the K0.5 did not show a significant change under the standard assay conditions (pH 6.0, 25 ˚C) (Fig. 1). Similar results were observed when acidic conditions closer to physiologically relevant pH and temperature were used (Table 1). Titration of recombinant ADAMTS13 with FRETS-VWF73 revealed similar results regarding the effect of scFv4-20 on the Vmax and K0.5 (Table 1) The parameter α was close to 1 when the titrations were analyzed by global fit, regardless of the reaction conditions or type of ADAMTS13 used, suggesting little to no effect of scFv4-20 on K0.5 (Table 1). Conclusions: The antibody-mediated inhibition of ADAMTS13 by the inhibitory scFv4-20 likely results primarily from reducing catalytic turnover of the enzyme rather than the binding of ADAMTS13 to VWF. Our ongoing study is to determine whether this is a general feature of inhibitory antibodies against ADAMTS13 or if other inhibitory mechanisms are also involved in the pathophysiology of iTTP. The assay developed and described here can be applicable to explore the way antibodies affect the activity of ADAMTS13 in this polyclonal autoimmune disease. Key words: Immune thrombotic thrombocytopenic purpura; ADAMTS13, inhibitory antibody Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Siglec-5 and Siglec-14 mediate the endocytosis of ADAMTS13

BackgroundThe plasma metalloprotease ADAMTS13 regulates the thrombotic activity of the von Willebrand factor (VWF). ADAMTS13 is highly glycosylated and its carbohydrate chains are capped with sialic acid (SA). Thus, ADAMTS13 may interact with carbohydrate- and/or SA-binding plasma membrane receptors that are involved in the clearance of various plasma proteins. We have investigated ADAMTS13 endocytosis via Siglecs, which were originally identified as SA-binding immunoreceptor family proteins expressed on leukocytes and are also known as endocytic receptors. Materials and methodsEndocytic internalization of fluorescently labeled ADAMTS13 into HEK293 cells expressing Siglecs was examined via fluorescence microscopy. In vitro binding of ADAMTS13 to the extracellular region of Siglec-5 was examined. Plasma ADAMTS13 activity in human Siglec-5-expressing mice was measured. Results and conclusionsSiglec-5- and Siglec-14-expressing cells internalized not only full-length ADAMTS13 (FL) but also the truncated form (MDTCS) at least partly in an SA-independent manner. Replacement of the V-set domain of Siglec-14 with that of Siglec-3 abrogated the internalization of ADAMTS13. ADAMTS13 directly bound to the extracellular region of Siglec-5 in vitro. Expression of Siglec-5 in the mouse liver resulted in a significant decrease in plasma ADAMTS13 activity. These results suggest that Siglec-5 and Siglec-14, which have nearly identical ligand-binding domains, may contribute to the regulation of plasma ADAMTS13 levels as endocytic receptors for ADAMTS13.

Hydrogen-Deuterium Exchange Plus Mass Spectrometry (HX-MS) Reveals a Novel Mechanism Underlying the pH-Dependent Regulation of ADAMTS13 Activity

Background: ADAMTS13 is a metalloprotease that is essential for the cleavage of von Willebrand factor (VWF). Deficiency of plasma ADAMTS13 activity causes a potentially fatal blood disorder, thrombotic thrombocytopenic purpura. Previous studies demonstrated a two-fold increase in the proteolytic activity of ADAMTS13 towards the VWF73 substrate by deleting the distal C-terminal domains, decreasing the pH, or binding a monoclonal antibody to the C-terminal domains. This suggests an allosteric interaction between the distal and proximal domains of ADAMTS13 modulated by pH.Objectives: To identify peptide regions within a recombinant full-length ADAMTS13 (rADAMTS13) responsible for the pH-modulated allosteric mechanism.Methods: To identify regions in the rADAMTS13 that undergo conformational changes in response to a change in pH, we performed hydrogen deuterium exchange plus mass spectrometry (HX-MS) at three different pH conditions, pH 6, pH 7, and pH 8. Because the rate of hydrogen and deuterium exchange is dependent upon the pH in a logarithmic fashion, a single time point from each of these pH conditions were compared. For instance, 6000 s was used for pH 6, 600 s for pH 7, and 60 s for pH 8. Any differences in the deuterium incorporation in these conditions in a given peptide should be contributed to conformational changes within the protein effecting accessibility and not simply the rate of hydrogen and deuterium exchange.Results: Purified rADAMTS13 and a variant truncated after the spacer domain (rMDTCS) were incubated with deuterium for various times (0- 6000 s). After quenching the reaction by lowering pH to 2.5 and temperature to 0 °C, the rADAMTS13 and rMDTCS were digested by pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Upon comparing the mass spectra of these three conditions, the peptides with mass spectra shift greater than 1 m/z unit as a result of conformational changes from the pH change were identified in the metalloprotease, cysteine-rich, spacer, and CUB-2 domains. In the metalloprotease, three peptide regions were identified (i.e. residues 110 -131, 181-197, and 217-230) with 19, 6, and 1 overlapping peptides, respectively. Within the cysteine rich domain, there were two identified peptide regions, 468 - 480 and 502 - 503. Within the spacer domain, there was only one identified peptide region 629-630, with 5 overlapping peptides. Finally, In CUB-2, there were two peptide regions, 1327-1340 and 1366-1377, with 2 overlapping peptides each (see Figure). All of these regions, with only the exception of residues 217-230, were more accessible to deuterium at pH 6 than at pH 8.To determine if these results are contingent upon the whole protein being present, and possible intramolecular protein interactions, the HX experiment was repeated using two ADAMTS13 variants (i.e. rMDTCS and rT5C). The rT5C contains the C-terminal TSP1 5-8 repeats and CUB domains. Because of smaller size, more overlapping peptides were recovered, which allows for narrowing down the residues central to the conformational change. Two small areas were narrowed down to several peptides, residues 120-123, with 6 overlapping peptides, and residue 503 within the cysteine rich with 3 overlapping peptides. Curiously, both regions within the CUB-2 domain showed little change with pH, despite 6 overlapping peptides in the residues 1327 -1340 and residues 1366-1377 (see Figure), suggesting the necessity of the proximal domains of the protein to modulate the conformation as a function of pH.Finally, the regions within the rADAMTS13 that exhibit a pH dependent conformation change are similar to the regions we reported showing bimodal behavior previously. Regions within the metalloprotease, the cysteine rich, and CUB-2 are all shared in residues 217-230, 468-480, and 1327-1328, respectively (see Figure).Conclusions: Our results, along with the direct binding experiments using a surface plasmon resonance assay, suggest that the N- and C-terminus of ADAMTS13 interact with each other at multiple discrete sites, which mediates the pH-dependent regulation of ADAMTS13 conformation and function. [Display omitted] DisclosuresZheng:Alexion: Speakers Bureau; Ablynx: Consultancy.

Modifying ADAMTS13 to modulate binding of pathogenic autoantibodies of patients with acquired thrombotic thrombocytopenic purpura.

Antibodies that develop in patients with immune thrombotic thrombocytopenic purpura commonly target the spacer epitope R568/F592/R660/Y661/Y665 (RFRYY) of ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In this study we present a detailed contribution of each residue in this epitope for autoantibody binding. Different panels of mutations were introduced to create a large collection of full-length ADAMTS13 variants comprising conservative (Y← →F), semi-conservative (Y/F→L), non-conservative (Y/F→N) or alanine (Y/F/R→A) substitutions. Previously reported gain-of-function (KYKFF) and truncated ‘MDTCS’ variants were also included. Sera from 18 patients were screened against all variants. Conservative mutations of the aromatic residues did not reduce the binding of autoantibodies. Moderate resistance was achieved by replacing R568 and R660 by lysines or alanines. Semi-conservative mutations of aromatic residues showed a moderate effectiveness in autoantibody resistance. Non-conservative asparagine or alanine mutations of aromatic residues were the most effective. In the mixtures of autoantibodies from the majority (89%) of patients screened, autoantibodies targeting the spacer RFRYY epitope were preponderant compared to other epitopes. Reductions in ADAMTS13 proteolytic activity were observed for all full-length mutant variants, in varying degrees. The greatest reductions in activity were observed in the most autoantibodyresistant variants (15-35% residual activity in a FRETS-VWF73 assay). Among these, a triple-alanine mutant . RARAA . showed activity in a von Willebrand factor multimer assay. This study shows that non-conservative and alanine modifications of residues within the exosite-3 spacer RFRYY epitope in full-length ADAMTS13 resist the binding of autoantibodies from patients with immune thrombotic thrombocytopenic purpura, while retaining residual proteolytic activity. Our study provides a framework for the design of autoantibody-resistant ADAMTS13 variants for further therapeutic development.

Disruption of ADAMTS13-Autoantibody Complexes with an ADAMTS13 Spacer Domain As a Potential Therapeutic Strategy for Immune Thrombotic Thrombocytopenic Purpura

Background: Immune thrombotic thrombocytopenic purpura (iTTP) is primarily caused by acquired immunoglobulin (Ig) G against ADAMTS13, a member of the A Disintegrin and Metalloprotease with Thrombospondin Type 1 repeats family, which specifically cleaves von Willebrand factor (VWF). Such proteolytic cleavage is essential for normal hemostasis. An inability to cleave VWF due to IgG autoantibody-mediated inhibition of plasma ADAMTS13 activity results in an accumulation of ultra large VWF multimers and disseminated microvascular thromboses, a pathognomonic feature of TTP. Previous studies have demonstrated that the spacer domain of ADAMTS13 is the primary target of IgG autoantibody; also, spacer domain is important for recognition of VWF under various conditions. Objective: In the present study, we explored if an isolated spacer domain can compete or reverse IgG autoantibody-mediated inhibition of ADAMTS13 activity, an important question to be addressed for developing a new strategy to treat iTTP. Methods:A recombinant ADAMTS13-spacer domain was expressed and purified to homogeneity; additionally, a single-chain fragment of the variable region of anti-ADAMTS13 IgG (i.e. scFv4-20) was isolated via phage display from a patient with iTTP, which was shown to specifically target the spacer domain and potently inhibit plasma ADAMTS13 activity with sub-nanomolar affinity. A functional assay with a fluorescein-labeled VWF73 substrate was used to determine the ADAMTS13 activity. Results: First, when a purified recombinant spacer domain (0-1.0 µM) was pre-incubated with scFv4-20 at various concentrations, followed by an addition of normal human plasma (NHP) that provides wild-type ADAMTS13 and a fluorescein-labeled recombinant VWF73 (F-rVWF73) substrate, we demonstrated that the inhibitory activity of scFv4-20 towards plasma ADAMTS13 decreased in a concentration-dependent manner. As a control, recombinant Cys-rich domain had no effect on blocking the scFv4-20-mediated inhibition of plasma ADAMTS13 activity under the same conditions. When scFv4-20 was pre-incubated with plasma ADAMTS13, recombinant spacer domain could also reverse the scFv4-20-mediated inhibition of ADAMTS13 activity but to a lesser degree. At the final concentration of 1.0 µM, recombinant spacer domain was able to increase ADAMTS13 activity by 2.1-fold when the recombinant spacer domain was added prior to the formation of ADAMTS13-antibody complexes but only by 1.5-fold if the recombinant spacer domain was added after the ADAMTS13 and antibody complexes were formed. Conclusion: Our results demonstrate that autoantibody-mediated inhibition of plasma ADAMTS13 is reversible and can be blocked by a molecular mimic that is the primary antibody-binding region of the full-length ADAMTS13 protein. The data suggest that it may be feasible to explore the development of agents capable of displacing inhibitory antibodies against ADAMTS13 in patients with iTTP. Disclosures Zheng: Alexion: Speakers Bureau; Ablynx/Sanofi: Consultancy, Speakers Bureau; Shire/Takeda: Research Funding; Clotsolution: Other: Co-Founder.

Effect of Recombinant Protein in the Spacer Domain on ADAMTS13 Activity

To obtain the recombinant protein of spacer domain in von Willebrand factor cleaving protease (ADAMTS13), and further study its biological function in ADAMTS13. The prokaryotic expression vector was constructed by using the template of plasmid with full-length ADAMTS13, and then transfected into E coli., following the induction of IPTG with the low temperature (30 ℃). The recombinant protein was purified with Ni-NTA agarose column by gradient imidazole. The purity and immune activity of purified products were identified with SDS-PAGE and Western blot respectively. By Adding the recombinant protein to the plasma of immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients, the activity of ADAMTS13 was tested. The prokaryotic expression vector was successfully constructed and the protein of spacer domain with the high purity was obtained. Western blot showed that the recombinant fragment could both react with monoclonal antibody against 6×His and polyclonal sheep IgG against ADAMTS13 (Gln34-Trp688). The protein formed a main lane at the position of 15 kDa with SDS-PAGE. It was demonstrated that the recombinant protein could efficiently elevate the ADAMTS13 activity in plasma of iTTP patients to reach normal level by functional experiment. The recombinant protein has high purity and immune activity, which provides the experimental basis for further research on mechanism of iTTP involved in spacer domain.

Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

ABSTRACTA 3-month-old male infant developed an extremely severe episode of atypical hemolytic uremic syndrome (aHUS) associated with partial deficiencies of full-length complement factor H (FH; ∼15% of infant normal) and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) (39% of normal) and autoantibodies reactive with both proteins. His FH and ADAMTS13 genes were normal, indicating that the partial deficiencies were acquired, probably as the result of autoantibodies against full-length FH and ADAMTS13. The child also had a homozygous deletion of the complement factor H–related (CFHR)3–CFHR1 portion in the complement factor H (CFH) gene cluster. He therefore had deficiency of CFHR proteins and autoantibody-positive hemolytic uremic syndrome (DEAP-HUS) with an unusual early onset associated with a partial deficiency of ADAMTS13 and an anti-ADAMTS13 autoantibody. His clinical episode of aHUS responded to plasma infusion and subsequent treatment with mycophenolate and rituximab. We believe that this is the first report of DEAP-HUS in an infant with partial deficiencies in both ADAMTS13 and full-length FH acquired in association with autoantibodies to both proteins.

Distal Carboxyl-Terminal Domains of ADAMTS13 Determine Its Substrate Specificity

Background: ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF), thereby regulating normal hemostasis. While VWF is the only substrate identified to date, more evidence is emerging to indicate that ADAMTS13 may exert direct thrombolytic activity that cannot be fully explained by its known VWF-cleaving activity. We hypothesize that ADAMTS13 may directly interact with the fibrinolytic system to accelerate fibrinolysis; the distal carboxyl-terminal domains of ADAMTS13 may be important in determining its substrate specificity.Objective: To determine the effect of full-length ADAMTS13 (FL-A13) and various carboxyl-terminally truncated ADAMTS13 variants in plasminogen activation and ultimate fibrinolysis.Methods: Using biochemical and biophysical approaches to assess the proteolytic cleavage of plasminogen and fibrinogen/fibrin by ADAMTS13 and variants and binding interactions between plasminogen/fibrinogen and ADAMTS13 and variants.Results: Coomassie blue staining and Western Blot assays demonstrated that a carboxyl terminally truncated ADAMTS13 variant (MT7) that was truncated after the 7th TSP1 repeat cleaved both Glu-plasminogen (Glu-Plg) and Lys-plasminogen (Lys-Plg) to form plasmin in a concentration and time-dependent manner (Fig. 1). MT8 (truncated after the 8th TSP1) and MS (truncated after the spacer domain) also cleaved both types of plasminogen but with much lower efficiency. Interestingly, full-length ADAMTS13 (FL-A13) did not show proteolytic activity towards plasminogen. The plasmin formed by MT7 is biologically active in proteolysis of fibrinogen in a time- and dose-dependent manner. The fibrinogen degradation product pattern, generated by MT7 and other carboxyl terminally truncated ADAMTS13 variants in the presence of plasminogen, was similar to those by tissue-plasminogen activation (t-PA). In addition, the plasmin formed by MT7 cleaved the peptidyl substrate S-2251 in a dose dependent manner. The MT7 and other carboxyl terminally truncated variants exhibited no direct proteolytic activity towards fibrinogen. When added to the preformed clot, MT7 was able to accelerate the lysis of the fibrin clot, initially slowly, but then rapidly as time progressed. The kinetics of fibrinolysis by MT7 was different from that of the t-PA control. Direct binding interactions between the carboxyl terminally truncated ADAMTS13 variants and plasminogen were then determined by surface plasmon resonance. MT7 bound to both types of plasminogen with similar affinities (KD of ~7 nM). However, FL-A13 did not bind to either Glu-Plg or Lys-Plg immobilized on a CM5 chip. Interestingly, when FL-A13 was immobilized on a CM5 chip, it bound to Lys- Plg but not Glu- Plg, but with 20-fold lower affinity compared to MT7. These results suggest that conformational change induced by ligand binding, proteolysis, or truncation may be necessary to expose ADAMTS13 binding site for plasminogen and alter its substrate recognition and specificity.Conclusion: We are able to identify a novel substrate for ADAMTS13 - plasminogen. The carboxyl terminal domains of ADAMTS13 appear to regulate the substrate specificity. These findings not only shed new light on the allosteric regulation of ADAMTS13 function but also provide rationale for the use of ADAMTS13 variants for a broader spectrum of thrombotic disorders including thrombotic thrombocytopenic purpura, ischemic cerebral stroke, myocardial infarction, as well as pulmonary embolism and deep vein thrombosis. [Display omitted] DisclosuresZheng:Ablynx: Consultancy; Alexion: Research Funding.

The Hunt for the "Minimal" Structure of a Functional ADAMTS13: Study of Deletion Mutations of ADAMTS13 By Small-Angle X-Ray Scattering

Introduction: ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by 7 T domains and 2 CUB domains. ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willebrand factor (VWF), which inhibits the growth of platelet-rich thrombi. When subjected to tensile stress in solution, bound to platelets, or on endothelial cell surfaces, VWF interacts with multiple exosites on ADAMTS13, changing the conformation of both proteins. These close contacts enhance the specific interaction between ADAMTS13 and VWF in vivo. Interactions between VWF and proximal MDTCS domains of ADAMTS13 have been investigated extensively. ADAMTS13 distal domains T8-CUB2 are required to bind the D4 domain of VWF, and the CUB domains promote the cleavage of platelet-decorated VWF strings. Using mutagenesis, small-angle X-ray scattering (SAXS) and enzyme kinetics, we have shown that ADAMTS13 adopts a folded conformation with distal T8-CUB2 domains close to the proximal MDTCS domains and a hinge point between the T4 and T5 domains. We have used internal deletions of T domains to identify a possible "minimal" structure for a functional and stably folded ADAMTS13.Methods: Recombinant human hADAMTS13, pigeon pADAMTS13, and various T domain deletion mutations were produced in T-Rex 293 cell lines and purified to homogeneity. Similar inactive variants of each protein were prepared with the mutation E225Q, which abolishes catalytic activity but does not affect protein folding. SAXS data were collected at the SIBYLS beamline (Lawrence Berkeley National Laboratory) for ADAMTS13 and deletion variants. The radius of gyration (Rg) and maximum particle size (Dmax) were calculated from scattering profiles using DATGNOM. Ab initio envelopes were generated from scattering profiles using DAMMIN. A molecular model of ADAMTS13 was built from crystal structures of ADAMTS4 MD domains (2rjp) and ADAMTS13 DTCS domains (3ghm), and using HHpred to model distal T domains, CUB domains, and linkers after T4 and T8. ADAMTS13 activity assays were performed at pH 6.0 and pH 7.4 with the fluorogenic substrate FRETS-rVWF71. Monoclonal antibodies (Mabs) against different distal domains of ADAMTS13 and recombinant VWF D4 domain were added to assess allosteric activation.Results: Rg and Dmax of ADAMTS13 variants are shown in Table 1. For Del4L, with deletion of linker region between T4 and T5, values of Rg and Dmax are ~5% smaller than for full length ADAMTS13. Deletion of both T4 and the linker region (Del4pL), reduced Rg and Dmax ~10%. Deletions of distal domains (Del7, Del8, Del8L, and Del8pL) that are spatially close to the proximal domains increased Rg and Dmax ~14%, which is consistent with partial unfolding of the condensed full length ADAMTS13 structure. Del2to8, which lacks all distal T domains between S and CUBs, aggregated in solution. Del2to7, which retains only T8, has Rg and Dmax 15% and 17% smaller than ADAMTS13, respectively. The kcat values for all variants at pH 7.4 are consistent with autoinhibition of the proximal MDTCS domains by any remaining distal domains. The additional of Mabs recognizing T7, T8 and CUBs slightly (~1.5 fold) activated individual deletion mutants. Del2to7, Del2to8, and Del3to6 had similar activities when compared with ADAMTS13. Though Del2to7 and Del3to6 could be activated by Mabs ~3 fold, only Del3to6 could be activated slightly ~1.3-fold by D4 domain, compared to 1.8-fold activation of hADAMTS13. pADAMTS13 is structurally similar to human Del3to6, and was activated by D4 domain ~4 fold but not by Mabs. Fitting to the ab initio envelopes show a folded conformation for hADAMTS13, Del7, Del8 and Del8L (Figure 1), consistent with a hinge in the flexible linker after T4. pADAMTS13 and Del2to7 had more compact envelopes, consistent with removal of most of the folded segment present in full length hADAMTS13.Conclusions: Deletion of individual distal T domains does not relieve the autoinhibition of ADAMTS13. Del3to6 is the only human deletion mutant examined that can be activated by both Mabs and VWF D4 domain, similar to hADAMTS13. pADAMTS13 is structurally similar to Del3to6, retains normal activation by VWF D4, and shows a SAXS envelope consistent with a truncated version of hADAMTS13. Our findings suggest that Del3to6 and pADAMTS13 represent the "minimal" structure of allosterically-regulated, functional ADAMTS13. [Display omitted] [Display omitted] DisclosuresSadler:Ablynx: Consultancy; 23andMe: Consultancy; BioMarin: Consultancy.

Identification of Monoclonal Antibodies against von Willebrand Factor Cleaving Protease (ADAMTS13) and Their Function

To construct and identify the monoclonal antibodies against von willebrand factor cleaving protease(ADAMTS13), and to study their biological function. BALB/c mice were immunized by purified recombinant ADAMTS13 truncated eukaryotic protein (ADAMTS13-T7). Murine anti-human ADAMTS13 monoclonal antibodies (McAbs) were constructed by standard hybridoma technology and identified by ELISA. The recognition of McAbs with full-length recombinant ADAMTS13 protein was identified by Western blot. In function assay, the influence of McAbs on the proteolysis of vWF by ADAMTS13 was observed. A group of 6 murine anti-ADAMTS13 McAbs was obtained with the clone number 1G11, 2F11, 6G3, 9E1, 10A8 and 10B4. In ELISA, the highest titers of 1G11 and 2F11 were observed, both of which showed a higher affinity to ADAMTS13-T7 than full-length ADAMTS13. The Western blot demonstrated that the 6 McAbs all could recognize ADAMTS13, among which 1G11 and 2F11 showed stronger reaction with ADAMTS13. In addition, under the denatured conditions, 1G11 and 2F11 could inhibit hydrolysis of vWF by ADAMTS13, and that was stronger with the increasing of McAbs concentration. McAbs against ADAMTS13 have been gained, two of which are inhibitory antibodies.

ADAMTS13 Reactive CD4+ T Cells Targeting CUB-2 Domain Derived Peptides in Autoimmune Thrombotic Thrombocytopenic Purpura

Acquired thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder that results from the development of auto-antibodies against ADAMTS13. Recent research suggests that HLA-DRB1*11 provides a risk factor for the development of acquired TTP. Previous work in our department identified ADAMTS13 derived peptides that are presented on MHC class II (Sorvillo et al. 2013). Pulsing of dendritic cells from HLA typed healthy donors showed preferential presentation of the CUB-2 domain derived peptides "FINVAPHAR" and "ASYILIRD" on HLA-DRB1*11 and HLA DRB1*03 respectively. In this study we screened peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with acquired TTP for reactive CD4+ T cells against the previously identified peptides.The presence of CD4+ T cells recognizing FINVAPHAR or the ASYILIRD peptide was addressed by flow cytometry. PBMCs were stimulated for 24 hours and up regulation of CD40L was visualized on CD4+ T cells. Stimulation with Staphylococcus aureus enterotoxin B (SEB) and overlapping peptides from cytomegalovirus (CMV) pp65 protein were used as controls. Several samples derived from patients in the acute phase of the disease were analyzed in this study.A high frequency of FINVAPHAR responsive CD4+ T cells was identified by monitoring the up regulation of CD40L on CD4+ T cells in a sample from a HLA-DRB1*1104 patient. Four percent of the CD4+ T cells were activated in response to the FINVAPHAR peptide. A control peptide in which the anchor-residues of the FINVAPHAR peptide were modified did not induce activation of CD4+ T cells. Incubation with full length ADAMTS13 also induced activation of CD4+ T cells although to a lesser extent. No FINVAPHAR reactive CD4+ T cells were observed after stimulation in HLA-DRB1*1104 positive healthy individuals.PBMC samples from HLA-DRB1*0301 positive patients were screened for CD4+ T cells reactive with the ASYILIRD peptide. ASYILIRD positive CD4+ T cells were detected in samples of 2 DRB1*0301 positive patients. In one patient 0.5% reactive CD4+ T cells were detected after stimulation with the ASYILIRD peptide. In samples derived from a second patient 1.5% of CD4+ T cells reactive against the ASYILIRD peptide were detected. Stimulation with a control peptide in which the anchor residues were modified did not result in activation of CD4+ T cells in both patients. Stimulation with SEB and CMV derived peptides resulted in activation of CD4+ T cells in the samples analyzed. No CD40L+ CD4+ T cells were observed after incubation with full length ADAMTS13, this may be due to insufficient processing of ADAMTS13 under our experimental conditions.Taken together our data provide the first evidence for the presence of ADAMTS13 reactive CD4+ T cells in acquired TTP. Based on our results we propose that FINVAPHAR and ASYILIRD CD4+ T cells are involved in the onset and/or relapse of acquired TTP. DisclosuresNo relevant conflicts of interest to declare.

A Folded ADAMTS13 Conformation Identified By Small-Angle X-Ray Scattering Can Account for Allosteric Regulation By Distal Thrombospondin-1 and CUB Domains

Introduction: ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by 7 T domains and 2 CUB domains. ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willerbrand factor (VWF), which inhibits the growth of platelet-rich thrombi. When subjected to tensile stress in solution, bound to platelets, or on endothelial cell surfaces, VWF interacts with multiple exosites on ADAMTS13, changing the conformation of both proteins. These close contacts enhance the highly specific interaction between ADAMTS13 and VWF in vivo. Interactions between VWF and proximal MDTCS domains of ADAMTS13 have been investigated extensively. ADAMTS13 distal domains T8-CUB2 are required to bind the D4 domain of VWF, and the CUB domains promote the cleavage of platelet-decorated VWF strings. However, the functional relationship between distal and proximal domains is not understood. Using mutagenesis, small-angle X-ray scattering (SAXS) and enzyme kinetics, we now have shown that distal T8-CUB domains interact with and inhibit the proximal MDTCS domains.Methods: Recombinant ADAMTS13, MDTCS, and variants truncated after each distal T domain (MT2, MT3ÉMT8), and VWF D4 domain were produced in T-Rex 293 cell lines and purified to homogeneity. Similar inactive variants of each protein were prepared with the mutation E225Q, which abolishes catalytic activity but does not affect protein folding (MT2Q, MT3QÉMT8Q). SAXS data were collected at the SIBYLS beamline (Lawrence Berkeley National Laboratory) for ADAMTS13 (E225Q) and truncated variants. The radius of gyration (Rg) and maximum particle size (Dmax) were calculated from scattering profiles using DATGNOM. Ab initio envelopes were generated from scattering profiles using DAMMIN. A molecular model of ADAMTS13 was built from crystal structures of ADAMTS4 MD domains (2rjp) and ADAMTS13 DTCS domains (3ghm), and using HHpred to model distal T domains, CUB domains, and linkers after T4 and T8. Plasma VWF was purified from Facteur Willebrand-LFB concentrate on Superdex 200. ADAMTS13 activity assays were performed at pH 6 and pH 7.4 with fluorogenic substrate FRETS-rVWF71.Results: Rg and Dmax progressively increased for variants MDTCS, MT2, MT3, MT4 and MT5, but did not increase proportionately for MT7, MT8 and ADAMTS13 despite a 50% increase in mass (Table 1). This behavior is consistent with a folded conformation. Fitting ADAMTS13 into ab initio envelopes for these variants requires a bend after domain T4, which is followed by a flexible linker that could serve as a hinge. As a consequence, distal T8-CUB2 domains are positioned near the proximal MDTCS domains (Figure 1). At pH 7.4, variants MDTCS through MT7 had similar activity toward FRETS-rVWF71. Full length ADAMTS13 was only ~10% as active at pH 7.4, but was ~7-fold more active at pH 6. MT8 had intermediate activity at pH 7.4 and was not activated by lowering the pH to 6. Adding VWF or recombinant VWF D4 increased the cleavage of FRETS-rVWF71 by full-length ADAMTS13 up to 4-fold at pH 7.4, but had no effect at pH 6 and did not change the activity of MT8 or MDTCS.Conclusions: ADAMTS13 adopts a folded conformation with distal T8-CUB2 domains close to the proximal MDTCS domains and a hinge point between the T4 and T5 domains. The T8-CUB domains inhibit ADAMTS13 activity, and this autoinhibition can be relieved by low pH or by binding to VWF. Thus, VWF functionally serves as both an ADAMTS13 activator and substrate. This allosteric mechanism would localize and concentrate ADAMTS13 activity on VWF multimers at sites of thrombosis.Table 1Properties of ADAMTS13 VariantsVariantMass (kDa)Rg(Angstrom)aDmax(Angstrom)bkcat, pH 6 (min-1)ckcat, pH 7.4 (min-1)cMDTCS6745.2 ± 1.1148.3 ± 2.09.86 ± 0.0410.94 ± 0.01MT27347.8 ± 2.8154.8 ± 6.19.19 ± 0.2410.23 ± 0.07MT37954.3 ± 4.8179.5 ± 10.19.69 ± 0.279.69 ± 0.27MT49058.7 ± 3.7193.1 ± 15.97.85 ± 0.049.80 ± 0.27MT59663.4 ± 3.0210.1 ± 8.97.23 ± 0.239.43 ± 0.22MT710963.8 ± 2.0210.5 ± 11.58.34 ± 0.258.90 ± 0.10MT812164.7 ± 2.5206.2 ± 2.53.01 ± 0.022.44 ± 0.01ADAMTS1314767.2 ± 1.9228.9 ± 6.27.15 ± 0.201.05 ± 0.11aCalculated using the program AutoRg, errors are SD. bCalculated using the program DATGNOM, errors are SD. cErrors are SE [Display omitted] DisclosuresSadler:XO1 Limited: Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Band Therapeutics: Consultancy; Baxter HealthCare: Consultancy, Honoraria.

Domain Specificity and Mechanism of Action of Anti-ADAMTS13 Antibodies at Presentation and Relapse in Acquired Thrombotic Thrombocytopenic Purpura (TTP)

Acquired thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease due to autoantibodies (Auto-Ab) against ADAMTS13, resulting in a severe deficiency in enzyme activity (<10%). Little is known about the pathogenicity of anti-ADAMTS13 IgG directed against different domains of the enzyme, or how domain specificity alters between presentation, remission or TTP relapse. We developed novel assays to explore domain specificity and inhibitory potential of anti-ADAMTS13 IgG using samples from the UK TTP Registry.Full-length ADAMTS13 and ADAMTS13 fragments (MD, MDTC and MDTCS - N-terminal domains; TSR2-8 and CUB1/2 - C-terminal domains) were expressed in HEK293T cells and purified. Microtitre plates were coated with ADAMTS13 or ADAMTS13 fragments, incubated with TTP plasmas and bound IgG detected. Domain specificity was further analysed by preincubation of patient samples with ADAMTS13 fragments, assessing the ability of fragments to compete for full-length ADAMTS13 binding on the plate. From a cohort of 92 acquired TTP patients at presentation, 100% (92/92) contained auto-Ab that recognised ADAMTS13 N-terminal domains (MDTCS). Of these, 39% (36/92) had auto-Ab recognising the N-terminal domains alone, 22% (20/92) also had anti-TSR2-8, and 39% (36/92) had anti-CUB1/2 auto-Abs. There was no correlation between auto-Ab domain specificity and mortality/disease severity (anti-ADAMTS13 IgG titre, plasma exchanges to remission, platelet count and cardiac involvement).There was no evidence for auto-Ab recognising MD or MDTC domains in 25 patients tested, suggesting immunoreactivity for MDTCS was primarily mediated through the spacer domain. Consistent with this, TTP IgG inhibited proteolysis of VWF115 (a recombinant VWF A2 domain fragment), but failed to inhibit VWF106 proteolysis (VWF A2 domain fragment lacking the spacer domain binding exosite).To examine the inhibitory potency of anti-ADAMTS13 auto-Ab, IgG isolated from TTP patient samples were titrated in reactions measuring MDTCS-mediated proteolysis of FRETS-VWF73. Twenty eight out of 43 presentation samples tested had inhibitory IgG (IC50 ranging from <0.2-5μM total IgG). Addition of excess of MDTC to 19/19 of these samples did not alter the IC50, confirming inhibition in these samples is mediated solely by anti-spacer antibodies. Intriguingly, 15/43 patients had no evidence of inhibitory auto-Ab, suggesting the pathogenic mechanism in these cases may be enhanced clearance of ADAMTS13. We therefore determined ADAMTS13 antigen concentrations by ELISA. All patients at presentation exhibited markedly reduced ADAMTS13 antigen levels (n=91, median 58ng/ml (range 0-450) vs. normal n=67, median 951ng/ml (515-1829); p<0.0001). Interestingly, patients with predominantly C terminal auto-Ab and no evidence of inhibitory auto-Ab had significantly lower ADAMTS13 antigen levels at first presentation than the rest of the cohort (median 2ng/ml (0-141) vs. 57ng/ml (0-356); p=0.006).Cases where rituximab failed to normalise ADAMTS13 activity despite clinical remission were associated with the persistence of inhibitory anti-N-terminal auto-Ab. Two patients with persistently high anti-ADAMTS13 IgG but normal ADAMTS13 activity and antigen levels during remission had non-pathogenic anti-TSR2-8 antibodies that seemingly had no effect upon ADAMTS13 function or clearance. Nine out of 16 patients exhibited altered auto-Ab domain specificity upon relapse, with either loss or development of anti-C-terminal auto-Ab.In conclusion, from our functional studies ADAMTS13 inhibition appears to be mediated exclusively by anti-spacer antibodies, with no evidence for the presence of auto-Ab that recognise/inhibit other N terminal domains. Importantly, we demonstrate ADAMTS13 antigen depletion is a crucial pathogenic mechanism in acquired TTP, accounting for a large proportion of the reduced ADAMTS13 activity detected in patient samples. Our results suggest that auto-Ab directed against the C-terminal domains of ADAMTS13 may be particularly important in mediating this clearance. There is continual development of the autoimmune response during treatment of TTP patients that can result in altered domain specificity at relapse. DisclosuresNo relevant conflicts of interest to declare.

Autoantibody Epitope Mapping By Hydrogen-Deuterium Exchange Mass Spectrometry at Nearly Single Amino Acid Residue Resolution Reveals Novel Exosites on ADAMTS13 Critical for Substrate Recognition and Mechanism of Autoimmune Thrombotic Thrombocytopenic Purpura

Acquired thrombotic thrombocytopenic purpura (TTP), a potentially fatal arterial thrombotic disorder, is primarily caused by autoantibodies that bind and inhibit plasma von Willebrand factor (VWF)-cleaving metalloprotease (ADAMTS13) activity. However, the mechanisms underlying autoantibody-mediated inhibition of ADAMTS13 activity and acquired TTP are not fully understood. By a hydrogen-deuterium exchange coupled with mass spectrometry (HX-MS) technique, we found that human monoclonal anti-ADAMTS13 antibodies, the single chain variable region fragments (scFvs)4-20, 4-16, and 3-1 that were, isolated by phage display from two patients with acquired TTP, predominantly bound to a discontinuous and conformational epitope in the spacer domain of ADAMTS13 with a subtle difference. The epitope for scFvs4-20 and 4-16 comprises five small flexible loops, including a previously described motif A (or exosite 3, R659-E664), motif B (exosite 4, L632-R639), and several other outlying residues (F592, Y658, and Y665), while scFv3-1 bound all other residues except for those in motif A. Site-directed mutagenesis and biochemical analysis demonstrated that both motifs A and B were found to be critical for recognition and proteolysis of VWF73 and multimeric VWF. Deletion of motif A or motif B in full-length ADAMTS13 abolished the binding of scFvs4-20 and 4-16 but not 3-1 (which did not bind motif A). Our findings demonstrate the powerful use of HX-MS for mapping antibody epitopes at nearly single amino acid resolution. This provides a new way to reveal mechanisms of autoantibody-mediated inhibition of plasma ADAMTS13 activity and acquired TTP. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

ADAMTS13 Expressed In Platelets Offers Systemic Protection Against Arterial Thrombosis and Therapeutic Benefits In Murine Models Of Thrombotic Thrombocytopenic Purpura

ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) and inhibits arterial thrombosis and inflammatory response. Deficiency of plasma ADAMTS13, due hereditary mutations in ADAMTS13 gene or autoantibodies against ADAMTS13 protease, results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective therapy available to date. To develop novel therapeutics against TTP, we tested a hypothesis that recombinant ADAMTS13, expressed specifically in platelets, may offer systemic protection against arterial thrombosis and therefore provide therapeutic benefit for TTP. To test this hypothesis, we first generated transgenic mice (JAX B6SJL) carrying a human full-length ADAMTS13 gene under a murine platelet glycoprotein 1b (CD41) promoter. The transgenic mice were then bred with Adamts13-/- (CAST/Ei) mice for &gt;4 generations. By Western blotting and activity assay, we show that a full-length human ADAMTS13 protein (∼195 kDa) and its proteolytic activity toward a FRETS-hVWF73 peptide are detected in platelet lysate obtained from transgenic (rA13-PltTG) mice but not in Adamts13-/- mice or wild-type mice. Little to no ADAMTS13 activity was detected in plasma in transgenic mice, suggesting the expressed human ADAMTS13 is stored inside platelets. ADAMTS13 was releasable upon stimulation with thrombin (1 U/ml), collagen (10 µg/ml), and AYPGKF (0.5 mM). More significantly, rA13-PltTG mice had higher baseline platelet count than Adamts13-/- mice, but exhibited a dramatically reduced rate of thrombus formation in mesenteric arterioles after oxidative injury with 10% ferric chloride as compared with Adamts13-/-mice and wild-type mice. Finally, rA13-PltTG mice were protected from Shigatoxin-2 (Stx-2) or murine recombinant VWF (mVWF)-induced “TTP-like” syndrome, as demonstrated by fewer rA13-PltTG mice having thrombocytopenia (defined by a 40% drop in platelet count from the baseline after challenge with Stx-2 or mVWF), faster and more complete recovery of thrombocytopenia, and significantly higher survival rate than Adamts13-/- mice. In summary, we have generated transgenic mice expressing human ADAMTS13 in platelets. Platelet ADAMTS13 is releasable upon stimulation by agonists and biologically functional in proteolytic cleavage of VWF in vitro. The platelet-derived ADAMTS13 offers systemic protection against arterial thrombosis after oxidative injury and provide a therapeutic benefit to murine models of TTP, resulting from hereditary ADAMTS13 deficiency. We are now in the process testing the efficacy of this strategy as a novel therapeutic for acquired TTP with inhibitors. Disclosures: No relevant conflicts of interest to declare.

ADAM28 Metalloprotease Domain Is Sufficient For Proteolytic Cleavage Of Von Willlebrand Factor

ADAM28, a member of ADAM (A Disintegrin and Metalloprotease) family, consists of two isoforms: a membrane-type form and a secreted form. Both ADAM28 isoforms are expressed in normal T and B lymphocytes of peripheral blood and lymphatic organs and certain malignant tumor cells. ADAM28 exhibits catalytic activity toward a few substrates including insulin-like growth factor binding protein-3 (IGFBP-3) and von Willebrand factor (VWF). ADAM28 is also overexpressed in human non-small cell lung carcinomas and breast carcinomas, predominantly by maligant cells, and the expression levels correlate with malignant cell proliferation and lymph node metastasis, presumably mediated by the cleavage of VWF. However, the structural components of ADAM28 required for proteolytic cleavage of VWF are not known. In this study, we constructed and expressed a soluble murine ADAM28 lacking the transmembrane domain and cytoplasmic tail (ΔTMA28) and various other carboxyl-terminal truncated ADAM28 variants containing a metalloprotease domain (M), plus a disintegrin domain (MD), and a Cys-rich domain (MDC) in COS-7 cells. All constructs were tagged at their carboxyl termini with a 3xflag epitope. Conditioned medium from transfected cells was collected and concentrated by a filtration technique. Protein concentrations of each construct in the concentrated conditioned medium were determined by a semi-quantitative Western blotting using a monoclonal anti-Flag IgG. A carboxyl-terminal truncated ADAMTS13 fragment (MDTCS) containing a 3xFlag epitope was used for a calibration. The proteolytic cleavage of a murine recombinant VWF (mVWF) by murine recombinant ADAM28 variants was carried out at 37 °C in a buffer containing 1 µM ZnCl2 and 5 mM CaCl2 in the presence or in the absence of guanidine-HCl at neutral pH 7.5. A full-length murine recombinant ADAMTS13 (mFL-A13) was used as a positive control and the conditioned medium derived from untransfected cells was used for a negative control. We show that mVWF that is pre-denatured with 1.0 M guanidine-HCl (at 37 °C for 2 hours) was efficiently cleaved by the constructs M, MD, MDC, and ΔTMA28 at the concentration of 50-100 nM after 18 hours of incubation. The proteolytic cleavage of VWF by ADAMTS28 variants results in dramatically reduced immune reactivity with rabbit anti-VWF IgG and multimer size as demonstrated by SDS-agarose gel (1%) electrophoresis and Western blotting. Paradoxically, this cleavage significantly increases the VWF-collagen binding activity despite of smaller VWF multimer size, suggesting that ADAM28-mediated proteolysis of VWF exposes its high-affinity collagen binding site. The cleavage efficiency of mVWF by all ADAM28 variants is comparable to that by mFL-A13 under the same conditions. The cleavage of native mVWF by ADAM28 is much less than that of pre-denatured VWF. EDTA (10 mM) added to the reaction completely abolishes the proteolytic cleavage of mVWF by all mADAM28 variants. We conclude that a metalloprotease domain of mADAM28 is sufficient for proteolytic cleavage of mVWF. The carboxyl terminal ancillary domains of mADAM28 appear to be dispensable for recognition and cleavage of mVWF. Our findings suggest a different mechanism underlying the proteolysis of VWF by a ADAM family protease. Further investigation into the biological activity of ADAM28 variants under more physiological conditions may shed new light on the mechanism of ADAM and ADAMTS proteolysis. The findings may provide a potential novel therapy for arterial thrombosis and acquired thrombotic thrombocytopenic purpura due to autoantibody inhibition because ADAM28 activity is not expected to be inhibited by ADAMTS13-specific autoantibodies. Disclosures:No relevant conflicts of interest to declare.

Crystal Structure of a Proteolytic Fragment of ADAMTS13 Reveals Alternative Disulfide Pairings in the Cysteine-Rich Domain

Abstract 3363Proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 depends on its non-catalytic domain (or exosite) interactions. These exosites, particularly the residues between Tyr659 and Tyr665 and their adjacent residues Arg568 and Phe592 in the ADAMTS13 spacer domain, are the major targets of anti-ADAMTS13 autoantibodies in patients with acquired TTP. In an effort to further determine the potential exosite interactions between ADAMTS13 and VWF, we carried out crystal screening of a recombinant full-length ADAMTS13 purified from stably transfected Chinese hamster ovary cells. After over 900 conditions were tested, we obtained multiple well-formed and diffractable crystals. Upon X-ray diffraction, we discovered that only a proteolytic fragment of full-length ADAMTS13 (Met432-Arg670) had been consistently crystallized. Here, we report the structure of this non-catalytic ADAMTS13 fragment at 2.2Å. The fragment encompasses 8 residues from the first TSP-1 repeat, an entire cysteine-rich domain (CA and CB), and a majority of the spacer domain. While recombinant ADAMTS13 in the present study is fully glycosylated, the overall structure agrees very well with the analogous portion of the ADAMTS13-DTCS structure previously reported (RMSD = 0.02Å, 1817 atoms, 232 residues). A region within the CA domain does vary slightly from that in the ADAMTS13-DTCS (RMSD = 0.08Å, 379 atoms, 51 residues). As with the ADAMTS13-DTCS structure, our structure shows 3 intermolecular disulfide bonds in CA, in addition to 2 free cysteine residues. In this region, our structure shows differences in the disulfide bonding pairing, and a slight shift in the α4 helix from Gly479-Cys483. Specifically, our structure reveals an intermolecular disulfide bond between the residues Cys483 and Cys527 and Cys508 and Cys522. These disulfide pair patterns are different from those reported in the ADAMTS13-DTCS structure, in which disulfide bonds are formed between the residues Cys483 and Cys522 (4.6Å apart in our structure) and the residues Cys508 and Cys527 (3.8Å apart in our structure). Our results indicate that, despite the difference in conditions under which crystals of ADAMTS13 fragments are formed, the overall structure of the Cys-rich and spacer domains are quite similar. The difference in the disulfide bond pattering may provide a novel insight into the mechanism regarding the ADAMTS13 exosite interaction with VWF. Our ongoing work is aimed to determine the complex formation between ADAMTS13 and its substrate or autoantibodies. Disclosures:Rottensteiner:Baxter Innovations GmbH: Employment.

Ectopic Expression of ADAMTS13 in Platelets As a Novel Therapeutic Approach for Arterial Thrombosis

Abstract 491Megakaryocytes and platelets have been shown to produce ADAMTS13 and its only known substrate, von Willebrand factor (VWF). However, the role of platelet expression of ADMTS13 in modulation of thrombus formation is not known. Previous studies have shown that platelet-targeted delivery of clotting factor VIII corrects bleeding phenotype in hemophilia A mice despite of inhibitors. These results suggest that platelet-delivery of ADAMTS13 may also be efficacious for anti-arterial thrombosis and perhaps for treatment of acquired idiopathic thrombotic thrombocytopenic purpura (TTP) with inhibitors. In the present study, transgenic mice (JAX B6SJL/F1 hybrid) carrying a human full-length ADAMTS13 gene under a platelet glycoprotein 1b alpha promoter were generated. The mice were crossed with Adamts13−/− and TTP-sensitive mice (CAST/Ei) for 4 generations. Plasma and platelet ADAMTS13 protein and proteolytic activity were determined. By Western blotting and the cleavage of a fluorescein-labeled VWF73 substrate, we were able to show that human ADAMTS13 protein (∼195 kDa) and activity were present in the platelet lysate of transgenic (A13-PltTG) mice, but not in adamts13−/− mice or wild-type mice. No proteolytic activity was detected in plasma of the transgenic mice. The platelet ADAMTS13 protein was releasable upon stimulation with various concentrations of thrombin (0.1–0.5 U/ml) and collagen (2.5–10 μg/ml). The released ADAMTS13 and VWF (as a positive control) were primarily associated with platelet membrane, demonstrated by surface biotinylation. However, a small fraction of the released ADAMTS13 and VWF proteins were detected in the releasate after stimulation. Moreover, the A13-PltTG mice exhibited systemic anti-thrombotic activity, which attenuated the rate of thrombus formation in the mesenteric arterioles induced by a topical application of 10% ferric chloride. The rate of arterial thrombus formation in the transgenic mice was significantly lower than that in Adamts13−/−mice and wild-type mice in the same genetic background. We conclude that we have generated transgenic mice overexpressing human ADAMTS13 metalloprotease in platelets. The platelet expressed ADAMTS13 is releasable upon stimulation by agonists. The platelet derived ADAMTS13 is biologically functional in cleaving VWF in vitro and in vivo, which attenuate systemic arterial thrombosis after oxidative injury. Our ongoing effort is to determine the efficacy of platelet delivered ADAMTS13 as a potential novel therapeutic for acquired TTP patients with inhibitors. Disclosures:No relevant conflicts of interest to declare.