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PLENARY PAPER
From the Department of Biochemistry, University of
Washington, Seattle.
von Willebrand factor (vWF) is synthesized in megakaryocytes and
endothelial cells as a very large multimer, but circulates in plasma as
a group of multimers ranging from 500 to 10 000 kd. An important
mechanism for depolymerization of the large multimers is the limited
proteolysis by a vWF-cleaving protease present in plasma. The absence
or inactivation of the vWF-cleaving protease results in the
accumulation of large multimers, which may cause thrombotic
thrombocytopenic purpura. The vWF-cleaving protease was first
described as a Ca++-dependent proteinase with an
apparent molecular weight of approximately 300 kd. Thus far, however,
it has not been isolated and characterized. In this study, the
purification of human vWF-cleaving protease from a commercial
preparation of factor VIII/vWF concentrate by means of several column
chromatographic steps, including 2 steps of heparin-Sepharose column,
is reported. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis analysis of the anion exchange and gel
filtration column fractions showed that the vWF-cleaving
protease activity corresponded to a protein band of 150 kd.
After reduction, it migrated with an apparent weight of 190 kd. The
amino terminal sequence of the 150-kd band was
AAGGIL(H)LE(L)L(D)AXG(P)X(V)XQ (single-letter amino acid
codes), with the tentative residues shown in parentheses. A
search of the human genome sequence identified the vWF-cleaving
protease as a new member of the ADAMTS (a disintegrin and
metalloproteinase with thrombospondin type I motif) family of
metalloproteinase. An active site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was located at 150 residues from the N
terminus of the protein.
(Blood. 2001;98:1662-1666) von Willebrand factor (vWF) plays an essential role
in platelet adhesion to damaged blood vessels by forming a bridge
between platelet surface glycoproteins and damaged subendothelium. vWF also plays an important role in hemostasis by binding and stabilizing coagulation factor VIII, protecting it from
proteolysis.1,2 vWF is composed of subunits of 2050 amino
acid residues with the following repeated motifs or domains:
D'-D3-A1-A2-A3-D4-B1-B2-B3-C1-C2.3,4 vWF precursor is
synthesized as a very large protein in endothelial cells5
and megakaryocytes6 and undergoes several
posttranslational events; these include removal of a signal peptide and
a large propeptide, the formation of intrachain and interchain
disulfide bonds, and glycosylation. Dimers are formed in the
endoplasmic reticulum through the formation of C-terminal interchain
disulfide bonds,7 and these dimers form multimers in the
Golgi apparatus by the formation of interdimer disulfide bonds at the
N-terminal region of the protein. This structure has been referred to
as a head-to-head and tail-to-tail mode.8
The mature vWF is synthesized and stored in endothelial
cells9,10 and then slowly released into the circulating
blood. Cultured human umbilical vein endothelial cells release only the fully polymerized form of vWF to the condition medium.11
Also, normal platelets contain exclusively the intact form of vWF in their granules.12 In plasma, however, vWF exists as a
mixture of disulfide-bonded multimers, with sizes ranging from dimers (500 kd) to highly polymerized forms as large as 10 000
kd.1,2 A size distribution of multimers is important for
normal hemostasis in that the larger multimers have a higher thrombotic
activity than smaller polymers.13 An excess of the very
large vWF multimers in circulation, however, can result in platelet
clumping, thrombosis, and thrombocytopenia.14 The large
polymerized forms of vWF undergo limited proteolytic processing after
their release into circulating blood. Accordingly, samples of
plasma-derived vWF give bands of 189, 140, and 170 kd after reduction,
in addition to the intact 225-kd band on sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The 140- and
170-kd bands result from the cleavage of a specific Y(842)-M(843)
(single-letter amino acid codes) peptide bond in the A2
domain.15 The cleavage site that generates the 189-kd band
has not been identified. Thus, partially cleaved vWF multimers are
composed of a variety of these fragments and intact chains, which are
linked by many disulfide bonds.15-17
Furlan et al18 and Tsai31 reported the
partial purification of the vWF-cleaving protease from human serum in
1996. The apparent molecular weight of the enzyme was approximately 300 kd as estimated by gel filtration. Also, the enzyme required divalent cations for catalytic activity. The enzyme was found to specifically cleave the Y(842)-M(843) bond to produce the 140- and 170-kd bands, which were the same as those present in plasma-derived vWF. EDTA and
ethyleneglycotetraacetic acid inhibited the protease activity, while serine protease or thioprotease inhibitors had no effect on the
enzyme. This suggested that the protease was a member of the
metalloproteinase family. In vitro, the protease cleaved vWF under
nonphysiological conditions, such as low ionic strength in the presence
of 1 to 1.5 M urea or guanidine. When plasma was placed under shear
stress, the very large multimers of vWF were converted to smaller
multimers, and the levels of the 170- and 140-kd fragments were
increased.19 Therefore, the protease appeared to cleave
vWF when it was partially denatured under shear stress.
Recently, a number of patients have been reported who have a complete
absence of the vWF-cleaving protease activity, and this correlated with
thrombotic thrombocytopenic purpura. Some patients developed antibodies
of the immunoglobulin (Ig)-G subclass to the protease, while others
carry familial deficiencies of the protease.20-23 These
results showed that the vWF-cleaving protease plays an essential role
in generating the normal size distribution of vWF multimers in plasma.
Despite the importance of this protease, it has not been identified and
characterized. In this study, the vWF-cleaving protease has been
purified to apparent homogeneity from a commercial factor VIII/vWF
concentrate and its N-terminal sequence determined. Computer searches
of the human genome sequence identified the protease as a new member of
the metalloproteinase family.
Materials
Assay of vWF-cleaving protease activity
Purification of vWF-cleaving protease from commercial factor VIII/vWF concentrates The frozen commercial factor VIII/vWF concentrate (380 mL) was thawed overnight at 4°C. CaCl2 (final, 20 mM) and thrombin (60 µg) were added, and incubated overnight at 4°C. Fibrin clots were removed by centrifugation at 16 000g for 15 minutes. Ammonium sulfate was added to the supernatant (total protein, 354 mg) to 33% saturation and stirred for 20 minutes. The precipitate obtained by centrifugation was suspended in approximately 50 mL of 50 mM Tris-HCl (pH 8.5), treated with DFP (final, 0.1 mM) and IAA (final, 5 mM) for 1 hour, and then dialyzed overnight against 2 L of 50 mM Tris-HCl (pH 7.5)/0.6 M ammonium sulfate. Gelatinous precipitates that appeared during dialysis were removed, and the sample was applied to a butyl-Sepharose column (1.2 × 6 cm) at a flow rate of 6 mL/10 min at ambient temperature. After the column was washed with the same buffer, adsorbed proteins were eluted with 50 mM Tris-HCl (pH 7.5). The eluted fractions were pooled, and ammonium sulfate was added to 40% saturation. The precipitates were collected by centrifugation and dissolved in approximately 5 mL of 50 mM Tris-HCl (pH 8.5). The sample (160 mg) was treated for 1 hour with DFP (final, 1 mM) and IAA (5 mM) and applied to a Sephacryl-500 column (2.5 × 60 cm) at a flow rate of 5 mL/12 min at 4°C. The proteins were eluted with 50 mM Tris-HCl (pH 8.0)/0.2 M NaCl and appeared in a single peak. The major portion of the peak was pooled and dialyzed overnight against 1 L of 50 mM MES-NaOH (pH 6.6)/0.1 M NaCl with one buffer change. The sample (114 mg) was then applied to a heparin-Sepharose column (1.4 × 9 cm) at a flow rate of 0.8 mL/min, and nonadsorbed fractions were pooled and concentrated to approximately 40 mL, followed by dialysis against 2 L of 50 mM MES-NaOH (pH 6.6). The sample (13 mg) was then applied to a second heparin-Sepharose column (1.4 × 3 cm). After the column was washed successively with 50 mM MES-NaOH (pH 6.6) and 50 mM MES-NaOH (pH 6.6)/25 mM NaCl, adsorbed proteins were eluted by a 50-mL linear NaCl gradient (25 mM and 225 mM NaCl). Fractions with protease activity were pooled and dialyzed against 1 L of 50 mM Tris-HCl (pH 8.0)/40 mM NaCl with one buffer change. The sample (3.6 mg) was then applied at a flow rate of 0.5 mL/min to a Waters diethylaminoethanol (DEAE) column (1.0 × 6 cm) attached to a Waters Protein Purification System (Milford, MA). After washing with 50 mM Tris-HCl (pH 8.0)/40 mM NaCl, proteins were eluted by a 40-mL salt gradient (40 mM and 640 mM NaCl) in the buffer. A portion of the active fraction from the DEAE column was applied to Superose-6 column (0.9 × 30 cm) and run with 50 mM Bis-Tris (pH 7.2)/0.15 M NaCl at a flow rate of 0.5 mL/min. Fractions of 0.5 mL were collected.Sequence and other analysis After SDS-PAGE, the protein bands were transferred to Immobilon-P membrane, which was stained briefly with Coomassie Blue G. After destaining, the 150-kd band was excised and subjected to sequence analysis. Automated Edman degradation was carried out in an Applied Biosystems 477A Protein Sequencer connected to 120 A Applied Biosystems Analyzer (Foster City, CA). SDS-PAGE was performed on 6% Laemmli gels, which were then stained with Blue stain reagent (Pierce, Rockford, IL) or silver stain reagent (Bio-Rad, Hercules, CA) for proteins and periodic acid-Schiff reagent28 for glycoprotein. Protein concentrations were estimated by absorbance assuming
Purification of vWF-cleaving protease A commercial preparation of factor VIII/vWF concentrate was used as the starting material, since it contained a substantial amount of the protease activity with far less protein contamination than plasma. Fibrinogen was initially removed by thrombin addition because it interfered with the chromatographic resolution and was abundant in the starting material. The defibrinated sample was then fractionated by ammonium sulfate precipitation (0% to 33% saturation) followed by chromatography on a hydrophobic interaction column with the adoption of the conditions described by Furlan et al.18 The protease activity was adsorbed and eluted from the column as reported by these authors. Substantial purification was then achieved by successive heparin-Sepharose column chromatography. In the first heparin-Sepharose column, the sample was applied in the presence of 0.1 M NaCl. Under these conditions, the activity did not bind to the column, whereas approximately 90% of contaminant proteins were bound and removed. The nonadsorbed fraction was then applied to a second heparin-Sepharose column in the absence of salt, where it was bound and then eluted by a salt gradient. After the second heparin column, the protease was further purified by DEAE and gel filtration column chromatography.The second heparin column fraction was applied to a DEAE column, and
the proteins were eluted in a single peak with an ascending shoulder
(Figure 1A). By SDS-PAGE analysis, a
discrete 150-kd band appeared to coincide with the ascending shoulder
area, trailing into the major protein peak area. A number of large
proteins were present in the shoulder area, and several distinct bands
with high molecular weights appeared in the major peak. A small protein band was also seen in the major peak area (Figure 1B). The protease activity was assayed by immunoblotting analysis to detect proteolytic fragments of frag III, which extends from S(1) to E(1365) in vWF. The
cleavage at Y(842)-M(843) bond generates 2 fragments of 140 kd and 65 kd. The 140-kd fragment, which originates from the N terminus, is
identical to the 140-kd fragment produced from plasma vWF. The 65-kd
fragment results from the C-terminal portion of frag III. The protease
activity was detected in fractions 40 to 54 and was highest in
fractions 42 and 46. This activity correlated with the intensity of the
150-kd protein band. With these fractions, the intensity of the 190-kd
band (frag III) decreased and that of the 140-kd and 65-kd bands
increased when compared with the control. vWF antigen was detected in
the major peak, including fractions 48 through 54. In these fractions,
vWF was also cleaved by the protease, resulting in the generation of a
170-kd fragment that migrated as a band below intact frag III. Trailing
of both the 150-kd protein and the protease activity into the major
peak indicated that the 150-kd protease binds to and comigrates with larger proteins. In the following gel filtration column, the larger proteins were completely separated from the 150-kd protease, which eluted in the fractions 26 through 30 (Figure
2 A-B). vWF protease activity was
detected in fractions 24 through 30, with a higher activity in
fractions 28 through 30 (Figure 2C). This was coincident with the
intensity of the 150-kd band. Results obtained in the DEAE and gel
filtration columns confirmed that the 150-kd protein was the
vWF-cleaving protease.
One or 2 faint bands below the 150-kd band were also detected in the gel filtration column fractions (Figure 2B). Since these bands were not detected in the DEAE column fractions, it appeared likely they were generated from the 150-kd protein after its separation from the larger proteins. The 150-kd protein was stable, and no accompanying bands were observed in earlier steps, probably because it was protected by the presence of other proteins. A large 280-kd protein, which eluted in the first peak of the gel filtration column, was reactive to anti-vWF antibodies (Figure 2C), indicating that the protease binds to vWF. The binding affinity was not strong, however, since the 2 proteins were readily separated by gel filtration in the presence of 0.15 M NaCl. When an excess amount of vWF was present, the protease activity was completely bound to vWF: the protease coeluted with large proteins from a Sephacryl-500 column used in the early stage of purification. The protease migrated as 150-kd band without reduction, but it migrated
more slowly after reduction (Figure 3,
lane 1) with molecular weight estimated to be 190 kd. This value was
consistent with the elution position from the Superose 6-column: the
150-kd protein eluted between fibrinogen (330 kd) and IgG (150 kd) as shown in Figure 2A. The vWF-cleaving protease was positive by carbohydrate staining (Figure 3, lane 2). These data show that vWF-cleaving protease is a single-chain glycoprotein with an apparent molecular weight of 190 kd.
Furlan et al18 first found vWF-cleaving protease activity in plasma and characterized its enzymatic properties. The enzyme properties of our preparation are consistent with their observations: cation-dependency, fragmentation, and apparent molecular weight (300 kd by gel filtration in an earlier stage of purification). Unique assay (dialysis of vWF/protease against 1 M urea in buffer of low ionic strength) was required to detect the enzyme activity. In addition, we used exactly the same conditions for the butyl-Sepharose column and obtained the same results. Thus, we are confident that the purified protease reported here is the same as the one that Furlan et al18 described. N-terminal amino acid sequence of vWF-cleaving protease and computer search of the human genome The 150-kd band from the DEAE column fraction was subjected to sequence analysis, and an N-terminal sequence of AAGGIL(H)LE(L)L(D)AXG(P)X(V)XQ (single-letter amino acid codes) was obtained. Residues in the parentheses were tentatively identified, while X represents uncertain residues. A search of the human genome sequences with this N-terminal amino acid sequence of vWF-cleaving protease indicated that the positively identified residues in the protein were identical to a sequence translated from human chromosome 9q34. Furthermore, the tentatively identified amino acids in positions 7, 10, 12, 16, 18, and 20 were confirmed in the genomic sequence. A probe sequence of 97 bases from chromosome 9q34 encompassing the aligned region was then used to search the human-expressed sequence tags (EST) database. One entry that was identified (GenBank gi 3841324) was 592 bases in length and overlapped with the probe sequence. Translation of the composite genomic and EST sequence showed that it encoded a novel sequence that was homologous to the protease domain of metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motif) subfamily.29 Specifically, it contained the sequence HEIGHSFGLEHD (single-letter amino acid code), 150 residues from the N-terminal AAGG sequence (Figure 4). This matched the consensus extended active site sequence of metalloproteinases in this family.30 This sequence also showed the presence of a precursor or pro piece with a typical furin-processing site.
The EST sequence was then used as a probe sequence in a subsequent search, which identified an entry in the GenBank database (GenBank gi 12735207) that overlaps with the 3' end of the EST sequence for 140 nucleotides. This entry (2272 bases) contains an open reading frame derived from chromosome 9 (C9ORF8) predicted by computer analysis of the human genomic sequence in the 9q34 region. Analysis of this predicted open reading frame shows it encodes a disintegrinlike sequence, followed by 3 thrombospondin type I motifs. All overlapping sequences align with the human chromosome 9q34 contig sequence in sequential order in 18 apparent exons. The composite sequence represents an incomplete complementary DNA (cDNA) without the 5' end coding for the signal peptide and the N-terminal pro region and the 3' end coding for additional C-terminal thrombospondin motifs. Other inconsistencies, including base variations and reading frame shifts, are also present. These analyses show that the vWF-cleaving protease is a new metalloproteinase that belongs to the ADAMTS subfamily. The gene encoding this enzyme, which has been designated HGNC, is located in chromosome 9q34. Experiments to clarify the cDNA sequence and identify the 5' and 3' regions are in progress. In the accompanying paper (see Gerritsen et al,32 page 1654) the authors also purified vWF-cleaving protease to apparent homogeneity. The amino-terminal sequence was essentially identical to that reported in this paper.
We are grateful to Dr Earl W. Davie for his valuable advice and encouragement throughout this work. We also thank Dr Ling Xie for technical assistance and Jeff E. Harris for assistance in manuscript preparation.
Submitted April 23, 2001; accepted May 29, 2001.
Supported by grant HL16919 from the National Institutes of Health.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kazuo Fujikawa, Department of Biochemistry, Box 357350, University of Washington, Seattle, WA 98195; e-mail: fujikawa{at}u.washington.edu.
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T. Ono, J. Mimuro, S. Madoiwa, K. Soejima, Y. Kashiwakura, A. Ishiwata, K. Takano, T. Ohmori, and Y. Sakata Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure Blood, January 15, 2006; 107(2): 528 - 534. [Abstract] [Full Text] [PDF]
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 K. Soejima, H. Nakamura, M. Hirashima, W. Morikawa, C. Nozaki, and T. Nakagaki Analysis on the Molecular Species and Concentration of Circulating ADAMTS13 in Blood J. Biochem., January 1, 2006; 139(1): 147 - 154. [Abstract] [Full Text] [PDF]
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 J. E. Sadler Thrombotic Thrombocytopenic Purpura: A Moving Target Hematology, January 1, 2006; 2006(1): 415 - 420. [Abstract] [Full Text] [PDF]
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B. Plaimauer, J. Fuhrmann, G. Mohr, W. Wernhart, K. Bruno, S. Ferrari, C. Konetschny, G. Antoine, M. Rieger, and F. Scheiflinger Modulation of ADAMTS13 secretion and specific activity by a combination of common amino acid polymorphisms and a missense mutation Blood, January 1, 2006; 107(1): 118 - 125. [Abstract] [Full Text] [PDF]
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L. H. Nolasco, N. A. Turner, A. Bernardo, Z. Tao, T. G. Cleary, J.-f. Dong, and J. L. Moake Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers Blood, December 15, 2005; 106(13): 4199 - 4209. [Abstract] [Full Text] [PDF]
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J. J. J. Hulstein, P. G. de Groot, K. Silence, A. Veyradier, R. Fijnheer, and P. J. Lenting A novel nanobody that detects the gain-of-function phenotype of von Willebrand factor in ADAMTS13 deficiency and von Willebrand disease type 2B Blood, November 1, 2005; 106(9): 3035 - 3042. [Abstract] [Full Text] [PDF]
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 J. Ai, P. Smith, S. Wang, P. Zhang, and X. L. Zheng The Proximal Carboxyl-terminal Domains of ADAMTS13 Determine Substrate Specificity and Are All Required for Cleavage of von Willebrand Factor J. Biol. Chem., August 19, 2005; 280(33): 29428 - 29434. [Abstract] [Full Text] [PDF]
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G. G. Levy, D. G. Motto, and D. Ginsburg ADAMTS13 turns 3 Blood, July 1, 2005; 106(1): 11 - 17. [Abstract] [Full Text] [PDF]
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R. De Cristofaro, F. Peyvandi, R. Palla, S. Lavoretano, R. Lombardi, G. Merati, F. Romitelli, E. Di Stasio, and P. M. Mannucci Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13 J. Biol. Chem., June 17, 2005; 280(24): 23295 - 23302. [Abstract] [Full Text] [PDF]
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 M-A von Mach, A Eich, L S Weilemann, and T Munzel Subacute coronary stent thrombosis in a patient developing clopidogrel associated thrombotic thrombocytopenic purpura Heart, February 1, 2005; 91(2): e14 - e14. [Abstract] [Full Text] [PDF]
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T. Uchida, H. Wada, M. Mizutani, M. Iwashita, H. Ishihara, T. Shibano, M. Suzuki, Y. Matsubara, K. Soejima, M. Matsumoto, et al. Identification of novel mutations in ADAMTS13 in an adult patient with congenital thrombotic thrombocytopenic purpura Blood, October 1, 2004; 104(7): 2081 - 2083. [Abstract] [Full Text] [PDF]
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K. Nishio, P. J. Anderson, X. L. Zheng, and J. E. Sadler Binding of platelet glycoprotein Ib{alpha} to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS13 PNAS, July 20, 2004; 101(29): 10578 - 10583. [Abstract] [Full Text] [PDF]
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F. Banno, K. Kaminaka, K. Soejima, K. Kokame, and T. Miyata Identification of Strain-specific Variants of Mouse Adamts13 Gene Encoding von Willebrand Factor-cleaving Protease J. Biol. Chem., July 16, 2004; 279(29): 30896 - 30903. [Abstract] [Full Text] [PDF]
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A. Bernardo, C. Ball, L. Nolasco, J. F. Moake, and J.-f. Dong Effects of inflammatory cytokines on the release and cleavage of the endothelial cell-derived ultralarge von Willebrand factor multimers under flow Blood, July 1, 2004; 104(1): 100 - 106. [Abstract] [Full Text] [PDF]
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C. Klaus, B. Plaimauer, J.-D. Studt, F. Dorner, B. Lammle, P. M. Mannucci, and F. Scheiflinger Epitope mapping of ADAMTS13 autoantibodies in acquired thrombotic thrombocytopenic purpura Blood, June 15, 2004; 103(12): 4514 - 4519. [Abstract] [Full Text] [PDF]
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X. L. Zheng, R. M. Kaufman, L. T. Goodnough, and J. E. Sadler Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and nonidiopathic thrombotic thrombocytopenic purpura Blood, June 1, 2004; 103(11): 4043 - 4049. [Abstract] [Full Text] [PDF]
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J.-D. Studt, J. A. K. Hovinga, R. Radonic, V. Gasparovic, D. Ivanovic, M. Merkler, U. Wirthmueller, C. Dahinden, M. Furlan, and B. Lammle Familial acquired thrombotic thrombocytopenic purpura: ADAMTS13 inhibitory autoantibodies in identical twins Blood, June 1, 2004; 103(11): 4195 - 4197. [Abstract] [Full Text] [PDF]
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J. E. Pimanda, T. Ganderton, A. Maekawa, C. L. Yap, J. Lawler, G. Kershaw, C. N. Chesterman, and P. J. Hogg Role of Thrombospondin-1 in Control of von Willebrand Factor Multimer Size in Mice J. Biol. Chem., May 14, 2004; 279(20): 21439 - 21448. [Abstract] [Full Text] [PDF]
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 S. Porter, S. D. Scott, E. M. Sassoon, M. R. Williams, J. L. Jones, A. C. Girling, R. Y. Ball, and D. R. Edwards Dysregulated Expression of Adamalysin-Thrombospondin Genes in Human Breast Carcinoma Clin. Cancer Res., April 1, 2004; 10(7): 2429 - 2440. [Abstract] [Full Text] [PDF]
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A. Padilla, J. L. Moake, A. Bernardo, C. Ball, Y. Wang, M. Arya, L. Nolasco, N. Turner, M. C. Berndt, B. Anvari, et al. P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface Blood, March 15, 2004; 103(6): 2150 - 2156. [Abstract] [Full Text] [PDF]
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M. Matsumoto, K. Kokame, K. Soejima, M. Miura, S. Hayashi, Y. Fujii, A. Iwai, E. Ito, Y. Tsuji, M. Takeda-Shitaka, et al. Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome Blood, February 15, 2004; 103(4): 1305 - 1310. [Abstract] [Full Text] [PDF]
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 A. Zakarija, N. Bandarenko, D. K. Pandey, A. Auerbach, D. W. Raisch, B. Kim, H. C. Kwaan, J. M. McKoy, B. P. Schmitt, C. J. Davidson, et al. Clopidogrel-Associated TTP: An Update of Pharmacovigilance Efforts Conducted by Independent Researchers, Pharmaceutical Suppliers, and the Food and Drug Administration Stroke, February 1, 2004; 35(2): 533 - 537. [Abstract] [Full Text] [PDF]
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K. Kokame, M. Matsumoto, Y. Fujimura, and T. Miyata VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13 Blood, January 15, 2004; 103(2): 607 - 612. [Abstract] [Full Text] [PDF]
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J. E. Sadler, J. L. Moake, T. Miyata, and J. N. George Recent Advances in Thrombotic Thrombocytopenic Purpura Hematology, January 1, 2004; 2004(1): 407 - 423. [Abstract] [Full Text] [PDF]
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E. M. Majerus, X. Zheng, E. A. Tuley, and J. E. Sadler Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity J. Biol. Chem., November 21, 2003; 278(47): 46643 - 46648. [Abstract] [Full Text] [PDF]
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K. Soejima, M. Matsumoto, K. Kokame, H. Yagi, H. Ishizashi, H. Maeda, C. Nozaki, T. Miyata, Y. Fujimura, and T. Nakagaki ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage Blood, November 1, 2003; 102(9): 3232 - 3237. [Abstract] [Full Text] [PDF]
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J.-f. Dong, J. L. Moake, A. Bernardo, K. Fujikawa, C. Ball, L. Nolasco, J. A. Lopez, and M. A. Cruz ADAMTS-13 Metalloprotease Interacts with the Endothelial Cell-derived Ultra-large von Willebrand Factor J. Biol. Chem., August 8, 2003; 278(32): 29633 - 29639. [Abstract] [Full Text] [PDF]
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X. Zheng, K. Nishio, E. M. Majerus, and J. E. Sadler Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13 J. Biol. Chem., August 8, 2003; 278(32): 30136 - 30141. [Abstract] [Full Text] [PDF]
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 H.-M. Tsai Advances in the Pathogenesis, Diagnosis, and Treatment of Thrombotic Thrombocytopenic Purpura J. Am. Soc. Nephrol., April 1, 2003; 14(4): 1072 - 1081. [Abstract] [Full Text] [PDF]
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H.-M. Tsai Platelet Activation and the Formation of the Platelet Plug: Deficiency of ADAMTS13 Causes Thrombotic Thrombocytopenic Purpura Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 388 - 396. [Abstract] [Full Text] [PDF]
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R. Schneppenheim, U. Budde, F. Oyen, D. Angerhaus, V. Aumann, E. Drewke, W. Hassenpflug, J. Haberle, K. Kentouche, E. Kohne, et al. von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP Blood, March 1, 2003; 101(5): 1845 - 1850. [Abstract] [Full Text] [PDF]
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 X. Zheng, A. M. Pallera, L. T. Goodnough, J. E. Sadler, and M. A. Blinder Remission of Chronic Thrombotic Thrombocytopenic Purpura after Treatment with Cyclophosphamide and Rituximab Ann Intern Med, January 21, 2003; 138(2): 105 - 108. [Abstract] [Full Text] [PDF]
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J.-f. Dong, J. L. Moake, L. Nolasco, A. Bernardo, W. Arceneaux, C. N. Shrimpton, A. J. Schade, L. V. McIntire, K. Fujikawa, and J. A. Lopez ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions Blood, December 1, 2002; 100(12): 4033 - 4039. [Abstract] [Full Text] [PDF]
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B. Plaimauer, K. Zimmermann, D. Volkel, G. Antoine, R. Kerschbaumer, P. Jenab, M. Furlan, H. Gerritsen, B. Lammle, H. P. Schwarz, et al. Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13) Blood, November 15, 2002; 100(10): 3626 - 3632. [Abstract] [Full Text] [PDF]
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C. R. Flannery, W. Zeng, C. Corcoran, L. A. Collins-Racie, P. S. Chockalingam, T. Hebert, S. A. Mackie, T. McDonagh, T. K. Crawford, K. N. Tomkinson, et al. Autocatalytic Cleavage of ADAMTS-4 (Aggrecanase-1) Reveals Multiple Glycosaminoglycan-binding Sites J. Biol. Chem., November 1, 2002; 277(45): 42775 - 42780. [Abstract] [Full Text] [PDF]
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J. E. Sadler A new name in thrombosis, ADAMTS13 PNAS, September 3, 2002; 99(18): 11552 - 11554. [Full Text] [PDF]
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K. Kokame, M. Matsumoto, K. Soejima, H. Yagi, H. Ishizashi, M. Funato, H. Tamai, M. Konno, K. Kamide, Y. Kawano, et al. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity PNAS, September 3, 2002; 99(18): 11902 - 11907. [Abstract] [Full Text] [PDF]
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 J. L. Moake Thrombotic Microangiopathies N. Engl. J. Med., August 22, 2002; 347(8): 589 - 600. [Full Text] [PDF]
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G. Remuzzi, M. Galbusera, M. Noris, M. T. Canciani, E. Daina, E. Bresin, S. Contaretti, J. Caprioli, S. Gamba, P. Ruggenenti, et al. von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome Blood, July 18, 2002; 100(3): 778 - 785. [Abstract] [Full Text] [PDF]
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V. Bianchi, R. Robles, L. Alberio, M. Furlan, and B. Lammle Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura Blood, June 28, 2002; 100(2): 710 - 713. [Abstract] [Full Text] [PDF]
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M. Arya, B. Anvari, G. M. Romo, M. A. Cruz, J.-F. Dong, L. V. McIntire, J. L. Moake, and J. A. Lopez Ultralarge multimers of von Willebrand factor form spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers Blood, May 13, 2002; 99(11): 3971 - 3977. [Abstract] [Full Text] [PDF]
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J. N. George, J. E. Sadler, and B. Lammle Platelets: Thrombotic Thrombocytopenic Purpura Hematology, January 1, 2002; 2002(1): 315 - 334. [Abstract] [Full Text]
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H. E. Gerritsen, R. Robles, B. Lammle, and M. Furlan Partial amino acid sequence of purified von Willebrand factor-cleaving protease Blood, September 15, 2001; 98(6): 1654 - 1661. [Abstract] [Full Text] [PDF]
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X. Zheng, D. Chung, T. K. Takayama, E. M. Majerus, J. E. Sadler, and K. Fujikawa Structure of von Willebrand Factor-cleaving Protease (ADAMTS13), a Metalloprotease Involved in Thrombotic Thrombocytopenic Purpura J. Biol. Chem., October 26, 2001; 276(44): 41059 - 41063. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
20°C. Diisopropyl fluorophosphate (DFP) and iodoacetamide (IAA) were purchased from Sigma (St Louis, MO). Superose-6, Superdex-200, butyl-Sepharose 4 Fast Flow, Q Sepharose, and
Sephacryl-500 were obtained from Pharmacia (Piscataway, NJ). Heparin-Sepharose was prepared as described earlier.
