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ADAMTS proteases typically employ some combination of ancillary C-terminal disintegrin-like, thrombospondin-1, cysteine-rich, and spacer domains to bind substrates and facilitate proteolysis by an N-terminal metalloprotease domain. We constructed chimeric proteases and substrates to examine the role of C-terminal domains of ADAMTS13 and ADAMTS5 in the recognition of their physiological cleavage sites in von Willebrand factor (VWF) and aggrecan, respectively. ADAMTS5 cleaves Glu(373)-Ala(374) and Glu(1480)-Gly(1481) bonds in bovine aggrecan but does not cleave VWF. Conversely, ADAMTS13 cleaves the Tyr(1605)-Met(1606) bond of VWF, which is exposed by fluid shear stress but cannot cleave aggrecan. Replacing the thrombospondin-1/cysteine-rich/spacer domains of ADAMTS5 with those of ADAMTS13 conferred the ability to cleave the Glu(1615)-Ile(1616) bond of VWF domain A2 in peptide substrates or VWF multimers that had been sheared; native (unsheared) VWF multimers were resistant. Thus, by recombining exosites, we engineered ADAMTS5 to cleave a new bond in VWF, preserving physiological regulation by fluid shear stress. The results demonstrate that noncatalytic thrombospondin-1/cysteine-rich/spacer domains are principal modifiers of substrate recognition and cleavage by both ADAMTS5 and ADAMTS13. Noncatalytic domains may perform similar functions in other ADAMTS family members.

The metalloprotease ADAMTS13 efficiently cleaves only the Tyr1605-Met1606 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 α-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 Gln1624 and Arg1668, and together these exosite interactions increase the rate of substrate cleavage by at least approximately 300-fold. Internal deletion of Gln1624-Arg1641 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 Tyr1605-Met1606 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.

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr 1605 –Met 1606 scissile bond in the VWF A2 domain depends on a Glu 1660 –Arg 1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme–substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage ≈20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.

A novel, simple and highly sensitive amplified fluorescence label-free immunosensor by using p-acid-encapsulated silica nanomaterials has been developed for the first time. 4,4'-(2,5-Dimethoxy-1,4-phenylene)bis(ethyne-2,1-diyl)dibenzoic acid (p-acid) and p-acid-encapsulated silica were prepared, and characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet visible spectroscopy (UV-vis) and fluorescence spectroscopy. In layer-by-layer self-assembling processes using (3-aminopropyl)triethoxysilane, p-acid@SiO(2) was assembled on the glass substrate. Antibody was immobilized on the surface of p-acid@SiO(2) with N,N'-carbonyldiimidazole. The functional nanomaterials present good analytical properties with a calibration range of 0.1-100 ng mL(-1), and allow the detection of carcinoembryonic antigen (CEA) at a concentration as low as 0.04 ng mL(-1). What is important is that the as-synthesized p-acid@SiO(2) nanomaterials could be further extended for the detection of other biomarkers or biocompounds.

Abstract 3298 Introduction: Thrombotic thrombocytopenic purpura (TTP) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and microvascular thrombosis. TTP is usually due to acquired, autoimmune deficiency of ADAMTS13, a metalloprotease that cleaves von Willebrand factor (VWF) and inhibits the growth of platelet thrombi. Most patients respond to treatment with plasma exchange, but inhibitory autoantibodies and persistent ADAMTS13 deficiency are associated with an increased risk of refractory or relapsing disease. Therefore, ADAMTS13 activity and inhibitor assays can be useful for diagnosis, prognosis, and monitoring the response to therapy. ADAMTS13 assays currently use the fluorogenic substrate FRETS-VWF73, which absorbs/emits at 340 nm/430 nm. These spectral properties make FRETS-VWF73 subject to interference from plasma proteins, hemoglobin and bilirubin. To avoid this problem, plasma is diluted at least 1:20, which reduces assay sensitivity to 5% of normal ADAMTS13 levels and prevents the detection of some clinically relevant inhibitors. We have addressed these limitations by developing FRETS-rVWF71, a recombinant fluorogenic substrate with chromophores that absorb/emit in the near infrared. Methods: The substrate polypeptide corresponds to VWF residues Gln1599-Arg1668, with mutation N1610C to introduce a reactive thiol and K1617R to remove an amino group that competes with the N-terminus for chemical modification. This peptide was expressed in E. coli as a thioredoxin-(His6)-fusion protein, purified by Ni2+-NTA chromatography, and digested with TEV protease to remove the thioredoxin. After modification at Cys1610 with DyLight 633-maleimide (abs 638 nm, em 658 nm) and at the N-terminus with IRDye QC-1 N-hydroxysuccinimidyl ester (abs 500–800 nm), the substrate FRETS-rVWF71 was purified by C18-HPLC. Assays were performed with 1 μM FRETS-rVWF71 under physiological buffer conditions (50 mM HEPES, pH 7.4, 150 mM NaCl, 10 mM CaCl2) to facilitate the assay of samples containing up to 95% plasma (Figure, panel A). Inhibitor assays were performed by preincubating equal volumes of pooled normal plasma and serially diluted patient plasma, followed by addition of an equal volume of buffer containing FRETS-rVWF71. Product generation was monitored in a fluorescence microplate reader with 635 nm excitation and 660 nm emission filters. Results: Serum and matched samples of plasma anticoagulated with citrate or heparin had equivalent ADAMTS13 activity that was stable indefinitely at −20°C. Bilirubin (>20 mg/dL) did not inhibit ADAMTS13 activity. As reported, hemoglobin was a weak inhibitor (EC50 approximately 1g/dL). Neither bilirubin nor hemoglobin interfered with product detection. Healthy donors (Li+-heparin plasma, n = 96) had a mean ADAMTS13 activity of 107.1 ± 18% (SD). Intra-assay and inter-assay coefficients of variation (CV) were Conclusions: The use of chromophores that absorb/emit in the near infrared avoids interference from blood proteins, bilirubin or hemoglobin. The combination of brighter chromophores and compatibility with undiluted plasma makes ADAMTS13 activity assays with FRETS-rVWF71 substantially more sensitive than with FRETS-VWF73. Higher sensitivity allows discrimination between very low levels of activity that may influence the risk of relapse in congenital or acquired TTP. Optimized detection of ADAMTS13 inhibitors will facilitate the monitoring of antibody responses to therapy and should help to determine why some patients with acquired TTP relapse and others do not. Disclosures: No relevant conflicts of interest to declare.

Abstract 2211 The von Willebrand factor (VWF) cleaving protease, ADAMTS13, and the aggrecanase, ADAMTS5, have a modular structure that includes metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), cysteine-rich (C), and spacer (S) domains. Both enzymes utilize some combination of DTCS domains to bind substrates and facilitate proteolysis, thereby maintaining the homeostatic balance between the synthesis and degradation of their substrates. We constructed chimeric metalloproteinases and substrates to examine the activity of ADAMTS13 and ADAMTS5 towards their respective physiological cleavage sites at VWF Tyr1605-Met1606 and aggrecan interglobular domain (IGD) Glu373-Ala374 (as shown in table). Cleavage rates were determined by ELISA, and C-terminal products were sequenced to confirm specificity. ADAMTS13 truncated after the S domain (MDTCS13) cleaved recombinant VWF Asp1596-Arg1668 (VWF73) much more rapidly than did constructs MDT13/CS5 and MD13/TCS5, in which distal ADAMTS13 domains were replaced by those of ADAMTS5. Similarly, replacement of the C-terminal 52 residues of VWF73 by aggrecan IGD residues Glu394-Gly458 (VWF/IGD) markedly impaired cleavage by MDTCS13, MDT13/CS5, and MD13/TCS5.Therefore, optimal cleavage of VWF73 depends on interactions between ADAMTS13 TCS domains and C-terminal sites of VWF73. In contrast, MDTCS5 was inactive toward VWF73 or VWF/IGD, whereas MD5/TCS13 efficiently cleaved VWF73 (at Glu1615-Ile1616) but not VWF/IGD. Thus, the TCS domains of ADAMTS13 interact with the Lys1617-Arg1668 segment of VWF73. In the context of ADAMTS13, this interaction accelerates the physiological cleavage of VWF. In the context of the chimeric MD5/TCS13, this interaction allows the ADAMTS5 active site to recognize and cleave a new site in VWF73, which is otherwise resistant to ADAMTS5. As expected, MDTCS5 readily cleaved recombinant IGD residues Thr331-Gly458 (IGD). Replacing the C-terminal 65 residues of IGD with VWF Lys1617-Arg1668 (IGD/VWF) minimally altered the rate of cleavage by MDTCS5, and replacement of the ADAMTS5 TCS domains by those of ADAMTS13 (MD5/TCS13) had little effect on the cleavage of either IGD or IGD/VWF. MDTCS5 and MD5/TCS13 also cleaved the same Glu373-Ala374 site in purified aggrecan with similar efficiency (not shown in table), indicating that recognition of the major cleavage site in the IGD domain does not depend strongly on specific TCS domains. In contrast, the other major site of aggrecan proteolysis by MDTCS5, at Glu1480-Ala1481, was completely resistant to MD5/TCS13, indicating that the TCS domains of ADAMTS13 cannot substitute for those of ADAMTS5. These results show that non-catalytic domains, particularly TCS domains, are principal modifiers of physiological substrate recognition and cleavage by ADAMTS5 and ADAMTS13, and these domains may have a similar function in other members of the ADAMTS family. Values (mean ± SD) for kcat/Km (×105 M-1s-1) were calculated from the initial rate of substrate cleavage determined by ELISA of products. Cleavage of aggrecan at the Glu1480-Ala1481 bond was analyzed by Western blotting with a site-specific antibody. N/A (no activity) indicates no significant cleavage identified by Western blotting or ELISA. Disclosures: No relevant conflicts of interest to declare.

ADAMTS13 reduces the size of plasma von Willebrand factor (VWF) multimers by cleaving the Y1605-M1606 bond within the central A2 domain of the VWF subunit. The C-terminus of VWF domain A2 is known to bind an exosite in the Spacer domain of ADAMTS13. However, ADAMTS13 cleaves smaller substrates that lack this C-terminal binding segment of the A2 domain, which suggests that additional sites in domain A2 interact with additional exosites on ADAMTS13. To identify these recognition sites, we constructed a series of recombinant substrates and analyzed their cleavage by ADAMTS13 variants (Table 1). Products were characterized by Western blotting and with a quantitative MALDI-MS assay. The progressive deletion of C-terminal amino acids of VWF domain A2 progressively reduced the rate of cleavage by ADAMTS13. In addition, the removal of 9 amino acids at the N-terminus of VWF73 (VWF64n) abolished cleavage. Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the first TSP1 domain (construct MDT) cleaved VWF64 ∼9-fold slower than did ADAMTS13 truncated after the Cys-rich domain (construct MDTC). Removal of 18 amino acids from the C-terminus of VWF64 (VWF46) also decreased the rate of cleavage by MDTC ∼9-fold, suggesting that the Cys-rich domain interacts with the segment I1642-R1659. Further C-terminal deletions of the substrate (VWF35, VWF28) had little effect on the rate of cleavage by enzymes MDTC or MDT. Comparing substrates VWF73 and VWF73d3, internal deletion of the region missing from VWF35 and VWF28 (Q1624 to R1641) had only a small effect ( Specificity Constants (kcat/Km) for Substate Cleavage by ADAMTS13 Variants Substrate VWF Sequence MDT MDTC ADAMTS13 Values (×105M−1s−1) are based on at least two independent experiments. No entry: not done. VWF73 D1596-R1668 1.0 7.5 VWF64 D1596-R1659 0.1 0.9 0.5 VWF46 D1596-R1641 0.04 0.1 VWF35 D1596-V1630 0.05 0.1 VWF28 D1596-I1623 0.02 VWF64n Y1605-R1668 Inactive Inactive Inactive VWF73d1 D1596-R1641+E1660-R1668 0.12 0.21 0.24 VWF73d2 D1596-R1641+D1653-R1668 0.16 0.36 0.84 VWF73d3 D1596-I1623+I1642-R1668 0.11 1.5 2.5 VWF73d4 D1596-T1608+Q1624-R1668 Inactive Inactive Inactive

ADAMTS13 is a plasma metalloproteinase that limits platelet-rich thrombi by cleaving von Willebrand factor (VWF) at the Tyr1605-Met1606 bond in the A2 domain. A minimal substrate that consists of GST linked to VWF residues Asp1596-Arg1668 with a C-terminal 6×His tag (GST-VWF73) is cleaved rapidly by plasma ADAMTS13. Further removal of Glu1660-Arg1668, which comprises a predicted C-terminal α-helix (GST-VWF64) markedly reduced the rate of cleavage, suggesting this helix comprises an exosite for substrate recognition. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of GST-VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. After removal of the GST moiety by proteolysis, the kinetic constants for cleavage of VWF73 and VWF64 were determined for several recombinant ADAMTS13 variants using a quantitative MALDI-MS assay (Table). Comparison of the specificity constants (kcat/Km) shows that ADAMTS13 truncated after the spacer domain (construct MDTCS) cleaved VWF73 ~20-fold faster than did a similar enzyme without the spacer domain (construct MDTC). In contrast, both MDTCS and MDTC cleaved VWF64 slowly at a rate similar to the cleavage of VWF73 by MDTC. Most of the variation in cleavage rates was explained by differences in Km, suggesting that the spacer domain recognizes an exosite at the C-terminus of VWF73 that is missing from VWF64. Cleavage of VWF73 yields a C-terminal product (cVWF63). Purified cVWF63 (7.5 μM) inhibited MDTCS activity toward VWF73 or VWF64 (1.5 μM) by »90%, but did not inhibit MDTC, suggesting that the exosite of VWF73 or cVWF63 interacts directly with the spacer domain. Moreover, cVWF63 inhibited the cleavage of multimeric VWF by full-length ADAMTS13 and MDTCS up to 80%, but did not inhibit MDTC. The selective effects of deleting the ADAMTS13 spacer domain and Glu1660-Arg1668 of the VWF domain A2 suggest that the C-terminal exosite of the VWF A2 domain accelerates substrate cleavage by binding specifically to the ADAMTS13 spacer domain. Table Kinetics studies of VWF73 and VWF64 cleavage by ADAMTS13 and its truncations VWF73 VWF64 Enzyme K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) K m (μM) k cat (s−1) k cat /K m (×105 M−1 s−1) FL-ADAMTS13 1.7±0.4 1.3±0.1 7.5±2.0 37.7±12.8 1.9±0.4 0.5±0.3 MDTCS 0.8±0.2 1.7±0.1 20.5±6.6 5.5±1.4 0.6±0.1 1.0±0.3 MDTC 16.0±4.5 1.8±0.3 1.1±0.5 17.9±6.3 1.6±0.3 0.9±0.5

ADAMTS13 is a member of the A Disintergrin And Metalloprotease with ThromboSpondin type I repeat family, and it cleaves the Tyr1605-Met1606 bond in the von Willebrand factor (VWF) central A2 domain, thereby decreasing platelet adhesion mediated by VWF. Recently a minimal substrate for ADAMTS13 was characterized that consists of GST linked to Asp1596-Arg1668 with a C-terminal 6×His tag (VWF73). Further removal of 9 amino acids that comprise a predicted C-terminal α-helix (VWF64) appeared to eliminate cleavage by plasma ADAMTS13, suggesting a critical role for this helix in substrate recognition. We obtained similar results, but VWF64 was cleaved significantly at long reaction times. For example, plasma ADAMTS13 (0.3 nM, one-tenth of normal plasma) cleaved 50% of 65 nM VWF73 in 2 hours and 50% of 62 nM VWF64 in 24 hours. Similar results were obtained in either 50 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM CaCl2, 0.1 μM ZnCl2, or 5 mM Tris-HCl, pH 8.0, 10 mM BaCl2. By amino acid sequencing, ADAMTS13 was shown to cleave the Tyr1605-Met1606 bond of VWF64. Truncation of ADAMTS13 after certain structural domains had different effects on substrate cleavage. Using 3 nM enzyme for 30 min, VWF73 was cleaved ~2-fold faster by ADAMTS13 truncated after the spacer domain (MDTCS) than by ADAMTS13 truncated after the cysteine-rich domain (MDTC). Conversely, VWF64 was cleaved ~3-fold faster by MDTC than by MDTCS. Also, in 30 min MDTCS cleaved 70% of VWF73 and

von Willebrand factor-cleaving protease/ADAMTS13 is a plasma metalloproteinase that degrades unusually large von Willebrand factor multimers (UL-vWF) derived from endothelial cells (ECs) and megakaryocytes (MCs) into small peptides circulating in blood. It is documented that transcript mRNAs of the protease are present in many human tissues; however, the protein expression of ADAMTS13 remains to be elucidated. In the present work, the gene of metalloproteinase domain of human ADAMTS13 was cloned into the multiclone site of pET28a(+). After induced by IPTG, the recombinant protein was purified using a Ni-NTA column and the Bal b/c mice were immunized with the protein. Screened with ELISA, three monoclonal antibodies against the metalloproteinase domain of ADAMTS13 were obtained and two of them, SZ-112 and SZ-113, were further evaluated. Both of them belonged to IgG1 subclass. The quantity of them in ascites were 4 mg/ml, and their titers were as high as 1×10−5. The data of competitive ELISA showed that SZ-112 and SZ-113 recognized different epitopes of the recombinant protein. Western blot results demonstrated that SZ-112 not only reacted against the recombinant protein, but also recognized the full-length recombinant ADAMTS13 protein that expressed in CHO cell line (the vectors containing the ADAMTS13 cDNA sequences were provided by Prof. Sadler JE). The immunoprecipitation results showed that the two antibodies could react to an approximately 200 KDa protein in platelet lysate. Then, the expression panels of ADAMTS13 in human normal tissues were investigated using immunohistochemistry with the monoclonal antibodies. And the protease was found to be present in many kinds of tissues such as liver, spleen, ovary, prostate, bladder, small intestine, thyroid and thymus with significantly positive staining. The protease was also present in lung, uterus, large intestine and heart but stained weakly. We did not found the protease in brain. In most of these organs, the protease was expressed in epithelium of the tissues. While in liver, spleen and thymus, it was mainly presented in a subgroup of the solid tissue cells. Moreover, the preliminary results showed that the expression of ADAMTS13 slightly decreased in liver tissues of patients suffering form hepatitis type B and cirrhosis. In conclusion, our data indicated that two novel monoclonal antibodies against the metalloproteinase domain of human ADAMTS13 were successfully prepared, and the expression of ADAMTS13 in different tissue and specific locality might be associated with the regulation and function of the protease, which would contribute to the further research of the deficiency mechanism of the proteases in some disorders.