Partial amino acid sequence of purified von Willebrand factor–cleaving protease

Gerritsen, Helena E.; Robles, Rodolfo; Lämmle, Bernhard; Furlan, Miha

doi:10.1182/blood.v98.6.1654

Cited by 355 publications

(269 citation statements)

References 39 publications

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“…7), confirming that domains C-terminal to the spacer domain are not required for proteolytic activity in vitro. If full-length recombinant and plasma ADAMTS13 (Ϸ170 kDa) have similar specific activity, the results suggest that the plasma concentration of active ADAMTS13 is Ϸ1.6 g/ml, which is similar to the value of 1 g/ml based upon the recovery of ADAMTS13 during purification from plasma (11). DISCUSSION The complex modular structure of many ADAMTS proteases is highly conserved among vertebrates, suggesting that their noncatalytic domains perform specific selectable functions.…”

Section: Fig 5 Adamts13 Variants Expressed In Cos-7 Cellssupporting

confidence: 57%

“…The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10,11), so named for the combination of a disintegrin-like and metalloprotease (reprolysin type), with thrombospondin type 1 motifs (12). The primary structure of the ADAMTS13 precursor was determined by cDNA cloning (13,14) and by positional cloning in families with inherited AD-AMTS13 deficiency (15).…”

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confidence: 99%

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Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Zheng

Nishio

Majerus

et al. 2003

Journal of Biological Chemistry

180

236

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ADAMTS13 consists of a reprolysin-type metalloprotease domain followed by a disintegrin domain, a thrombospondin type 1 motif (TSP1), Cys-rich and spacer domains, seven more TSP1 motifs, and two CUB domains. ADAMTS13 limits platelet accumulation in microvascular thrombi by cleaving the Tyr 1605 -Met 1606 bond in von Willebrand factor, and ADAMTS13 deficiency causes a lethal syndrome, thrombotic thrombocytopenic purpura. ADAMTS13 domains required for substrate recognition were localized by the characterization of recombinant deletion mutants. Constructs with C-terminal His 6 and V5 epitopes were expressed by transient transfection of COS-7 cells or in a baculovirus system. No association with extracellular matrix or cell surface was detected for any ADAMTS13 variant by immunofluorescence microscopy or chemical modification. Both plasma and recombinant full-length ADAMTS13 cleaved von Willebrand factor subunits into two fragments of 176 kDa and 140 kDa. Recombinant ADAMTS13 was divalent metal ion-dependent and was inhibited by IgG from a patient with idiopathic thrombotic thrombocytopenic purpura. ADAMTS13 that was truncated after the metalloprotease domain, the disintegrin domain, the first TSP1 repeat, or the Cys-rich domain was not able to cleave von Willebrand factor, whereas addition of the spacer region restored protease activity. Therefore, the spacer region is necessary for normal ADAMTS13 activity toward von Willebrand factor, and the more Cterminal TSP1 and CUB domains are dispensable in vitro.Thrombotic thrombocytopenic purpura (TTP) 1 is a syndrome characterized by microangiopathic hemolytic anemia and thrombocytopenia, and it may be accompanied by neurological dysfunction, renal failure, and fever (1-3). If untreated, the mortality can exceed 90%, but plasma-exchange therapy has reduced the mortality to less than 20% (4). Although the pathophysiology of TTP is not fully understood, a plausible model has been proposed in which the proteolysis of von Willebrand factor (VWF) plays a central role (5). VWF is a multimeric protein that binds receptors on the surface of platelets and in connective tissue, thereby mediating the adhesion of platelets to sites of vascular injury. If unchecked, the process can lead to microvascular thrombosis. A plasma VWF-cleaving protease has been described that acts on the Tyr 1605 -Met 1606 bond in the central A2 domain of the VWF subunit, and cleavage is stimulated by shear forces like those occurring at sites of thrombosis or by low concentrations of urea or guanidine (6, 7). This proteolytic reaction limits VWF-dependent platelet adhesion, and most adults with idiopathic TTP have an acquired autoantibody that inhibits the VWF-cleaving protease (8, 9). Therefore, therapeutic plasma exchange may ameliorate TTP by replacing the missing protease and removing the inhibitory antibody.The VWF-cleaving protease was recently purified and identified as a new member of the ADAMTS family of metalloproteases (10, 11), so named for the combination of a disintegrin-like and metalloprote...

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Section: Fig 5 Adamts13 Variants Expressed In Cos-7 Cellssupporting

confidence: 57%

mentioning

confidence: 99%

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Zheng

Nishio

Majerus

et al. 2003

Journal of Biological Chemistry

180

236

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“…15,16 Most recently, this enzyme has been purified as a single-chain glycoprotein with a molecular mass of ෂ150 kDa by two different groups of investigators. 17,18 Northern blotting based on the N-terminal amino acid sequence of this purified enzyme revealed that the full-length (4.6 kb) mRNA is only expressed in liver, and cDNA sequencing identified this enzyme as a new member of the metalloproteases belonging to the ADAMTS (a disintegrin-like domain, and metalloproteinase, with thrombospondin type-1 motif) family, designated ADAMTS13. [18][19][20] The deduced amino acid residue number of this enzyme is 1427, and its gene is located on chromosome 9q34.…”

Section: And Jones Et Almentioning

confidence: 99%

Impaired activity of plasma von Willebrand factor-cleaving protease may predict the occurrence of hepatic veno-occlusive disease after stem cell transplantation

Yoshioka

Kawa

et al. 2002

Bone Marrow Transplant

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Summary:Hepatic veno-occlusive disease (VOD) is a life-threatening complication after stem cell transplantation (SCT), characterized by thrombus formation in hepatic venules leading to a symptom triad of hyperbilirubinemia, hepatomegaly, and ascites. Multifactorial defects in the hemostatic system may contribute to its pathogenesis, but its remains to be investigated. Unusually large VWF multimers (UL-VWFMs), produced in and released from vascular endothelial cells, are most biologically active in the interaction with platelets under a high shear stress. UL-VWFMs are cleaved and degraded into smaller VWFMs by a specific liver producing plasma protease, termed VWF-cleaving protease (VWF-CPase), which has recently been identified as a metalloprotease solely produced in liver, termed ADAMTS13. Herein, we studied the correlation between plasma VWF-CPase activity and UL-VWFMs in 21 patients who received SCT, seven patients with VOD and 14 patients without VOD. In non-VOD patients, activities (mean ؎ 1s.d.) of VWF-CPase were 78 ؎ 17% of the control before the conditioning regimen, 76 ؎ 18% on day 0, 64 ؎ 19% on day 7, 57 ؎ 23% on day 14, 68 ؎ 13% on day 21 and 79 ؎ 19% on day 28 after SCT. The respective values in VOD patients were 32 ؎ 19%, 27 ؎ 15%, 18 ؎ 11%, 22 ؎ 18%, 26 ؎ 22% and 12 ؎ 4%. Thus, VWF-CPase activity was significantly reduced in VOD patients, even before the conditioning regimen, and such a difference was not found in other laboratory tests. However, despite such a clear difference, UL-VWFMs were present in plasmas of both patient groups, together with the increase of VWF antigen and ristocetin cofactor activity. These results indicate that the measurement of this enzyme activity is extremely useful in predicting the occurrence of VOD prior to a demonstration of its direct involvement in its pathogenesis.

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“…The specific protease activity of recombinant mADAMTS13 (3 U/mlC8 nmol/l ¼ 375 U/nmol) was similar to the presumed specific activity of human plasma ADAMTS13 (1 U/mlC5 nmol/l ¼ 200 U/nmol, assuming plasma ADAMTS13 level ¼ 1 mg/ml, or 5 nmol/ l). 22 These estimates need to be confirmed in the future by direct analysis of purified proteins. We also determined the ADAMTS13 activity level in the heparin-anticoagulated plasma of FVB/N mice by using the same hVWF substrate and found that the mean (7s.d.)…”

Section: Cloning Of Mouse Full-length Adamts13mentioning

confidence: 98%

ADAMTS13 is expressed in hepatic stellate cells

Zhou

Inada

Lee

et al. 2005

Laboratory Investigation

180

126

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ADAMTS13 is a circulating zinc metalloprotease that cleaves the hemostatic glycoprotein von Willebrand factor (VWF) in a shear-dependent manner. Deficiency in ADAMTS13, owing to genetic mutations or autoimmune inhibitors, causes thrombotic thrombocytopenic purpura (TPP). Northern blot analysis has shown that ADAMTS13 is expressed primarily in the liver. By using real-time RT-PCR, we confirmed that in mice the liver had the highest level of the ADAMTS13 transcript. To identify the liver cell-type-specific origin of ADAMTS13, we used in situ hybridization techniques to investigate the pattern of ADAMTS13 expression in the liver; analyzed the ADAMTS13 proteolytic activity in the culture media of fractionated liver cells; and confirmed ADAMTS13 expression with RT-PCR analysis and cloning of the mouse ADAMTS13 gene. The results revealed that ADAMTS13 was expressed primarily in cell fractions enriched in hepatic stellate cells. The mouse ADAMTS13 cloned from primary hepatic stellate cells was similar to its human counterpart in digesting VWF and was susceptible to suppression by EDTA or the IgG inhibitors of patients with TTP. Since hepatic stellate cells are believed to play a major role in the development of hepatic fibrosis and cirrhosis, the identification of the liver cell-type expressing ADAMTS13 will have important implications for understanding pathophysiological mechanisms regulating ADAMTS13 expression. The von Willebrand factor (VWF) is a multimeric glycoprotein that mediates adhesion and aggregation of platelets at sites of vascular injury. A metalloprotease cleaves VWF at the Tyr1605-Met1606 bond, generating homodimers of the 176 and 140 kDa fragments. 1,2 The VWF-cleaving protease is essential for preventing platelet aggregation in the circulation, and deficiency of the protease is associated with thrombotic thrombocytopenic purpura (TTP), a disease characterized by the development of VWF-platelet-rich thrombi in the arterioles and capillaries. 3 Recent studies identified this protease as ADAMTS13, a member of the reprolysin-type zinc metalloprotease family. [4][5][6] The human ADAMTS13 gene, spanning 37 kb on human chromosome 9q34, comprises 29 exons that encode a polypeptide of 1427-amino-acid residues and possibly several splicing isoforms. Although it shares with other members of the ADAMTS family a common domain architecture consisting of metalloprotease, disintegrin-like sequence, thrombospondin type 1 repeat, cysteine-rich and spacer regions, ADAMTS13 exhibits several distinct features, such as an RGDS sequence in the spacer domain and two copies of CUB domains at the carboxyl terminus. Substitution of the D residue in the RGDS sequence does not appear to diminish the proteolytic activity of ADAMTS13. 7 Unlike other ADAMTS proteases, pro-ADAMTS13 is proteolytically active. 8 These unique features of ADAMTS13 are consistent with

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Partial amino acid sequence of purified von Willebrand factor–cleaving protease

Cited by 355 publications

References 39 publications

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13

Impaired activity of plasma von Willebrand factor-cleaving protease may predict the occurrence of hepatic veno-occlusive disease after stem cell transplantation

ADAMTS13 is expressed in hepatic stellate cells

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