Cleavage of ultralarge multimers of von Willebrand factor by C-terminal–truncated mutants of ADAMTS-13 under flow
http://www.100md.com
《血液学杂志》
the Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, Houston, TX
the Hematology Division, Department of Medicine, Washington University, St Louis, MO.
Abstract
A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.
Introduction
Thrombotic thrombocytopenic purpura (TTP) is caused by widespread microvascular thrombi rich in platelets and von Willebrand factor (VWF).1,2 The disorder is associated with the deficiency of the VWF-cleaving metalloprotease, a disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13), which cleaves the Y842/M843 peptide bond in the VWF A2 domain,3-6 converting the ultralarge (UL) and hyperactive VWF multimers newly released from endothelial cells to smaller and less active forms.7 ADAMTS-13 deficiency, caused either by congenital defects of the ADAMTS-13 gene or the presence of autoantibodies against the metalloprotease,8 results in the accumulation of ULVWF multimers in plasma and systemic thrombosis in the microvasculature.1 ADAMTS-13 activity is mostly measured under static conditions that require denaturing the VWF substrate. We recently showed that freshly released ULVWF multimers are anchored to the endothelial surface and form long platelet-decorated strings under venous and arterial shear stresses.9,10 These ULVWF strings are cleaved within seconds to minutes by normal plasma, but not plasma from patients with TTP,9 suggesting that ULVWF proteolysis in vivo may occur on the surfaces of endothelial cells and is regulated by fluid shear stress.
ADAMTS-13 contains multiple domains characteristic of the ADAMTS family of metalloproteases. experiments performed under static conditions suggest that many of these domains are dispensable for protease activity, as recombinant ADAMTS-13 mutants lacking much of the C-terminal region, including 7 thrombospondin-1 (TSP-1) motifs and 2 complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, can still cleave plasma VWF.11,12 However, more than 50% of mutations identified in patients with congenital TTP were located in the deleted regions. One possibility for such discrepancy is that the C-terminal regions are required for ADAMTS-13 to function in vivo under flow conditions. To examine this possibility and further investigate the relationship between ADAMTS-13 structure and function, we examined the activity of 2 ADAMTS-13 truncation mutants under flow.
Study design
ADAMTS-13 activity was measured under flow conditions by a previously described method.9 Briefly, human umbilical vein endothelial cells (HUVeCs) were stimulated with 25 μM histamine and then perfused with washed platelets suspended in either buffer or citrated plasma at 2.5 dyne/cm2 of shear stress. Platelets tethered to newly released ULVWF multimers to form long and platelet-decorated strings on endothelial cells in the absence of ADAMTS-13, whereas perfusion of platelets in normal plasma produced no strings because of their cleavage by plasma ADAMTS-13. Using this system, we examined the ability of 2 truncation mutants to cleave ULVWF strings. The mutants are described previously (Figure 1A)11 and named in this study as MDTCS, which represents the domains remained in the mutant (metalloprotease, disintegrin, the first TSP-1 motif, Cys-rich, and spacer), lacking TSP-1 motifs second through eighth and the C-terminal CUB domains, and MDTC, which also lacks the spacer. Wild-type (WT) ADAMTS-13 and the 2 mutants were transiently expressed in Hela cells and purified through the C-terminal His-tag using a Y-per 6xHis Fusion Protein Purification Kit (Pierce Chemicals, Rockford, IL). We have also tested WT and the mutants expressed in sf9 insect cells.11 expression of the recombinant polypeptides was verified by immunoblotting using an ADAMTS-13 antibody (Bethyl Laboratories, Houston, TX) and a myc-tag antibody (Sigma, St Louis, MO). Protein concentrations were determined using a commercial kit (Pierce Chemicals). WT and ADAMTS-13 mutants suspended in either Tyrode buffer or ADAMTS-13-depleted plasma were perfused over stimulated HUVeCs. ULVWF proteolysis was determined after 2-minute perfusion.
Human blood was used for the studies presented in the manuscript for platelets and ADAMTS-13. The use of human blood has been approved by the institutional review board of the Baylor College of Medicine. All donors signed informed consent in accordance with the Declaration of Helsinki before blood was drawn. ADAMTS-13-depleted plasma was prepared from the plasma of a patient with acquired TTP with a high-titer ADAMTS-13 autoantibody. Briefly, plasma was first incubated overnight with 500 μL protein A/G beads (Pierce Chemicals) at 4°C and then centrifuged at 3000g for 10 minutes at 4°C to remove the beads. The plasma was then incubated with a human ADAMTS-13 antibody (previously purified from the patient) and then with protein A/G beads. ADAMTS-13 activity in plasma thus prepared was undetectable under static and flow conditions, but was restored by the addition of either normal plasma or recombinant ADAMTS-13 (data not shown).
Results and discussion
ADAMTS-13 activity was defined as the number of ULVWF strings present in 20 continuous view fields (x 400). There were 78 ± 24 strings counted when the Tyrode buffer containing platelets was perfused and defined as 0% activity, whereas 0 to 7 strings were detected when recombinant ADAMTS-13 (670 nM) was perfused and defined as 100% activity. At the equal molar concentration, the MDTCS mutant demonstrated 100% cleavage under these conditions, whereas the MDTC mutant cleaved only 26.5 ± 2.5% of strings, significantly less than the percentage cleaved by MDTCS (n = 96, P < .001, Figure 1B). ADAMTS-13 activities at the maximal concentration of 670 nM were comparable between the WT and the MDTCS mutant, but the latter exhibited significantly greater activities at lower concentrations (Figure 1C). Similar results were obtained using recombinant ADAMTS-13 expressed in Hela and sf9 cells (data not shown), suggesting that difference in activity is an intrinsic property of the mutant. This result is consistent with that obtained under static condition,11,12 suggesting that the C-terminal domains are not critical for, but may regulate, the enzymatic activity under flow conditions. This is different from recent studies that the C-terminus CUB domains either directly bind to VWF13,14 or regulate the affinity of VWF-ADAMTS-13 interaction.15 These results suggest that the C-terminal regions (especially CUB domains) may serve as or be a part of the docking site for ULVWF, a critical step for cleaving endothelial cell–anchored ULVWF under flow. The reason for such a discrepancy is unclear, but may suggest that there are either multiple docking sites on ADAMTS-13 or new sites created by the truncations. Additional studies to solve the discrepancy are critical for understanding how ADAMTS-13 cleaves ULVWF under flow conditions.
One potential problem with the studies described in the previous section is that plasma, in which ADAMTS-13 functions in vivo, was not present. We therefore tested the activity of WT and 2 mutants in ADAMTS-13-depleted plasma, which presumably contains any potential ADAMTS-13 modifiers. As shown in Figure 2A, both WT and the MDTCS mutant fully restored the ability of ADAMTS-13-depleted plasma to cleave ULVWF strings at concentrations comparable with those used in buffer. To our surprise, the presence of plasma increased the cleavage of ULVWF strings by the MDTC mutant (800 nM) from 26.5 ± 3.5% in buffer to 54.8 ± 6.8% in plasma (Figure 2A, Student t test, n = 9, P < .05), even though activity was not detected at lower concentrations (400 and 200 nM). To investigate whether plasma also regulates the activity of WT ADAMTS-13, we then measured ULVWF cleavage by various concentrations of WT ADAMTS-13 in the presence and absence of plasma. We found that WT ADAMTS-13 at 84 nM cleaved 19.5 ± 4.3% more strings in the presence of plasma than in its absence (Figure 2B, Student t test, n = 4, P < .05).
In summary, we show that the truncation mutant that lacks the second to eighth TSP-1 motifs and the CUB domains (MDTCS) is active under flow conditions, consistent with observations under static conditions.11,12 Furthermore, the mutant appears to be more active than WT ADAMTS-13 (Figure 1C), suggesting that the C-terminal domain may regulate the enzyme activity. This hypothesis is supported by a study showing that the activity and substrate specificity of ADAMTS4 is regulated by the C-terminal domains, including the TSP-1 motif.16,17 In contrast to the hyperactivity of the MDCTS mutant, truncation to further remove the spacer domain (MDTC) rendered the mutant severely deficient in this system.18 More importantly, the metalloprotease is significantly more active in the presence of plasma (Figure 2), suggesting that ADAMTS-13 activity may be regulated by plasma factors. The nature and identity of these cofactors remains to be investigated, but a recent study shows that glycoprotein 1b (GP Ib), the platelet VWF receptor, enhances ADAMTS-13 activity.19 Glycocalicin, the soluble extracellular portion of GP Ib that normally circulates in plasma in various amounts, may therefore be one of such cofactors. Studying how ADAMTS-13 is regulated by plasma factors is important not only for understanding the ADAMTS-13 function in vivo, but also for identifying new factors that affect ULVWF proteolysis and contribute to bleeding or thrombotic phenotypes.
Footnotes
Prepublished online as Blood First edition Paper, March 17, 2005; DOI 10.1182/blood-2004-11-4188.
Supported by National Institutes of Health (NIH) grants HL64796, 1-P50-HL65967, and HL18673. J.-F.D is an established Investigator of the American Heart Association.
Z.T. and Y.W. contributed equally to this work.
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.
References
Moake JL, Rudy CK, Troll JH, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N engl J Med. 1982;307: 1432-1435.
Moake JL. Thrombotic thrombocytopenic purpura: the systemic clumping "plague." Annu Rev Med. 2002;53: 75-88.
Gerritsen He, Robles R, Lammle B, Furlan M. Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood. 2001;98: 1654-1661.
Furlan M, Robles R, Lammle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood. 1996;87: 4223-4234.
Dent JA, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J Clin Invest. 1991;88: 774-782.
Furlan M. Von Willebrand factor: molecular size and functional activity. Ann Hematol. 1996;72: 341-348.
Moake JL. Thrombotic microangiopathies. N engl J Med. 2002;347: 589-600.
Levy GG, Nichols WC, Lian eC, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001;413: 488-494.
Dong JF, Moake JL, Nolasco L, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood. 2002;100: 4033-4039.
Padilla A, Moake JL, Bernardo A, et al. P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface. Blood. 2004;103: 2150-2156.
Zheng X, Nishio K, Majerus eM, Sadler Je. Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS-13. J Biol Chem. 2003;278: 30136-30141.
Soejima K, Matsumoto M, Kokame K, et al. ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage. Blood. 2003;102: 3232-3237.
Dong JF, Bernardo A, Nolasco L et al. ADAMTS-13 proteolysis of ultra-large von Willebrand factor multimers on the endothelial surface requires protease docking and is inhibited by thrombospondin-1 . Blood. 2002;100: 123a.
Zhang W, Motto D, Ginsburg D. ADAMTS-13 binds VWF via its C-terminal CUB2 domain . Blood. 2004;104: 39a.
Majerus eM, Anderson PJ, Sadler Je. Characterization of the binding interaction between ADAMTS-13 and von Willebrand factor . 2005;104: 150a.
Gao G, Westling J, Thompson VP, Howell TD, Gottschall Pe, Sandy JD. Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation. J Biol Chem. 2002;277: 11034-11041.
Kashiwagi M, enghild JJ, Gendron C, et al. Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J Biol Chem. 2003;279: 10109-10119.
Dong JF, Whitelock J, Bernardo A, Ball C, Cruz MA. Variations among normal individuals in the cleavage of endothelial-derived ultra-large von Willebrand factor under flow. J Thromb Haemost. 2004;2: 1460-1466.
Nishio K, Anderson PJ, Zheng XL, Sadler Je. Binding of platelet glycoprotein Ibalpha to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS-13. Proc Natl Acad Sci U S A. 2004;101: 10578-10583.(Zhenyin Tao, Yongtao Wang)
the Hematology Division, Department of Medicine, Washington University, St Louis, MO.
Abstract
A disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13) cleaves the A2 domain of von Willebrand factor (VWF), converting the ultralarge (UL) and hyperactive VWF multimers freshly released from endothelial cells to smaller and less active forms found in plasma. Recombinant ADAMTS-13 lacking the C-terminal region is active under static conditions, but its functions under flow conditions have not been determined. Here, we show that VWF-cleaving activity measured under flow was preserved in an ADAMTS-13 mutant lacking the second to eighth thrombospondin-1 motifs and the complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, but was severely deficient in a mutant that was further truncated to remove the spacer domain. We also show that the mutant lacking the TSP-1 and CUB domains was hyperactive under flow, suggesting that the C-terminal region may negatively regulate ADAMTS-13 activity. The wild type and the mutant without the spacer were more active in the presence of plasma, raising the possibility of ADAMTS-13 cofactors in plasma.
Introduction
Thrombotic thrombocytopenic purpura (TTP) is caused by widespread microvascular thrombi rich in platelets and von Willebrand factor (VWF).1,2 The disorder is associated with the deficiency of the VWF-cleaving metalloprotease, a disintegrin-like and metalloprotease with thrombospondin type 1-motif 13 (ADAMTS-13), which cleaves the Y842/M843 peptide bond in the VWF A2 domain,3-6 converting the ultralarge (UL) and hyperactive VWF multimers newly released from endothelial cells to smaller and less active forms.7 ADAMTS-13 deficiency, caused either by congenital defects of the ADAMTS-13 gene or the presence of autoantibodies against the metalloprotease,8 results in the accumulation of ULVWF multimers in plasma and systemic thrombosis in the microvasculature.1 ADAMTS-13 activity is mostly measured under static conditions that require denaturing the VWF substrate. We recently showed that freshly released ULVWF multimers are anchored to the endothelial surface and form long platelet-decorated strings under venous and arterial shear stresses.9,10 These ULVWF strings are cleaved within seconds to minutes by normal plasma, but not plasma from patients with TTP,9 suggesting that ULVWF proteolysis in vivo may occur on the surfaces of endothelial cells and is regulated by fluid shear stress.
ADAMTS-13 contains multiple domains characteristic of the ADAMTS family of metalloproteases. experiments performed under static conditions suggest that many of these domains are dispensable for protease activity, as recombinant ADAMTS-13 mutants lacking much of the C-terminal region, including 7 thrombospondin-1 (TSP-1) motifs and 2 complement components C1r/C1s, Uegf sea urchin fibropellins, and bone morphogenic protein 1 (CUB) domains, can still cleave plasma VWF.11,12 However, more than 50% of mutations identified in patients with congenital TTP were located in the deleted regions. One possibility for such discrepancy is that the C-terminal regions are required for ADAMTS-13 to function in vivo under flow conditions. To examine this possibility and further investigate the relationship between ADAMTS-13 structure and function, we examined the activity of 2 ADAMTS-13 truncation mutants under flow.
Study design
ADAMTS-13 activity was measured under flow conditions by a previously described method.9 Briefly, human umbilical vein endothelial cells (HUVeCs) were stimulated with 25 μM histamine and then perfused with washed platelets suspended in either buffer or citrated plasma at 2.5 dyne/cm2 of shear stress. Platelets tethered to newly released ULVWF multimers to form long and platelet-decorated strings on endothelial cells in the absence of ADAMTS-13, whereas perfusion of platelets in normal plasma produced no strings because of their cleavage by plasma ADAMTS-13. Using this system, we examined the ability of 2 truncation mutants to cleave ULVWF strings. The mutants are described previously (Figure 1A)11 and named in this study as MDTCS, which represents the domains remained in the mutant (metalloprotease, disintegrin, the first TSP-1 motif, Cys-rich, and spacer), lacking TSP-1 motifs second through eighth and the C-terminal CUB domains, and MDTC, which also lacks the spacer. Wild-type (WT) ADAMTS-13 and the 2 mutants were transiently expressed in Hela cells and purified through the C-terminal His-tag using a Y-per 6xHis Fusion Protein Purification Kit (Pierce Chemicals, Rockford, IL). We have also tested WT and the mutants expressed in sf9 insect cells.11 expression of the recombinant polypeptides was verified by immunoblotting using an ADAMTS-13 antibody (Bethyl Laboratories, Houston, TX) and a myc-tag antibody (Sigma, St Louis, MO). Protein concentrations were determined using a commercial kit (Pierce Chemicals). WT and ADAMTS-13 mutants suspended in either Tyrode buffer or ADAMTS-13-depleted plasma were perfused over stimulated HUVeCs. ULVWF proteolysis was determined after 2-minute perfusion.
Human blood was used for the studies presented in the manuscript for platelets and ADAMTS-13. The use of human blood has been approved by the institutional review board of the Baylor College of Medicine. All donors signed informed consent in accordance with the Declaration of Helsinki before blood was drawn. ADAMTS-13-depleted plasma was prepared from the plasma of a patient with acquired TTP with a high-titer ADAMTS-13 autoantibody. Briefly, plasma was first incubated overnight with 500 μL protein A/G beads (Pierce Chemicals) at 4°C and then centrifuged at 3000g for 10 minutes at 4°C to remove the beads. The plasma was then incubated with a human ADAMTS-13 antibody (previously purified from the patient) and then with protein A/G beads. ADAMTS-13 activity in plasma thus prepared was undetectable under static and flow conditions, but was restored by the addition of either normal plasma or recombinant ADAMTS-13 (data not shown).
Results and discussion
ADAMTS-13 activity was defined as the number of ULVWF strings present in 20 continuous view fields (x 400). There were 78 ± 24 strings counted when the Tyrode buffer containing platelets was perfused and defined as 0% activity, whereas 0 to 7 strings were detected when recombinant ADAMTS-13 (670 nM) was perfused and defined as 100% activity. At the equal molar concentration, the MDTCS mutant demonstrated 100% cleavage under these conditions, whereas the MDTC mutant cleaved only 26.5 ± 2.5% of strings, significantly less than the percentage cleaved by MDTCS (n = 96, P < .001, Figure 1B). ADAMTS-13 activities at the maximal concentration of 670 nM were comparable between the WT and the MDTCS mutant, but the latter exhibited significantly greater activities at lower concentrations (Figure 1C). Similar results were obtained using recombinant ADAMTS-13 expressed in Hela and sf9 cells (data not shown), suggesting that difference in activity is an intrinsic property of the mutant. This result is consistent with that obtained under static condition,11,12 suggesting that the C-terminal domains are not critical for, but may regulate, the enzymatic activity under flow conditions. This is different from recent studies that the C-terminus CUB domains either directly bind to VWF13,14 or regulate the affinity of VWF-ADAMTS-13 interaction.15 These results suggest that the C-terminal regions (especially CUB domains) may serve as or be a part of the docking site for ULVWF, a critical step for cleaving endothelial cell–anchored ULVWF under flow. The reason for such a discrepancy is unclear, but may suggest that there are either multiple docking sites on ADAMTS-13 or new sites created by the truncations. Additional studies to solve the discrepancy are critical for understanding how ADAMTS-13 cleaves ULVWF under flow conditions.
One potential problem with the studies described in the previous section is that plasma, in which ADAMTS-13 functions in vivo, was not present. We therefore tested the activity of WT and 2 mutants in ADAMTS-13-depleted plasma, which presumably contains any potential ADAMTS-13 modifiers. As shown in Figure 2A, both WT and the MDTCS mutant fully restored the ability of ADAMTS-13-depleted plasma to cleave ULVWF strings at concentrations comparable with those used in buffer. To our surprise, the presence of plasma increased the cleavage of ULVWF strings by the MDTC mutant (800 nM) from 26.5 ± 3.5% in buffer to 54.8 ± 6.8% in plasma (Figure 2A, Student t test, n = 9, P < .05), even though activity was not detected at lower concentrations (400 and 200 nM). To investigate whether plasma also regulates the activity of WT ADAMTS-13, we then measured ULVWF cleavage by various concentrations of WT ADAMTS-13 in the presence and absence of plasma. We found that WT ADAMTS-13 at 84 nM cleaved 19.5 ± 4.3% more strings in the presence of plasma than in its absence (Figure 2B, Student t test, n = 4, P < .05).
In summary, we show that the truncation mutant that lacks the second to eighth TSP-1 motifs and the CUB domains (MDTCS) is active under flow conditions, consistent with observations under static conditions.11,12 Furthermore, the mutant appears to be more active than WT ADAMTS-13 (Figure 1C), suggesting that the C-terminal domain may regulate the enzyme activity. This hypothesis is supported by a study showing that the activity and substrate specificity of ADAMTS4 is regulated by the C-terminal domains, including the TSP-1 motif.16,17 In contrast to the hyperactivity of the MDCTS mutant, truncation to further remove the spacer domain (MDTC) rendered the mutant severely deficient in this system.18 More importantly, the metalloprotease is significantly more active in the presence of plasma (Figure 2), suggesting that ADAMTS-13 activity may be regulated by plasma factors. The nature and identity of these cofactors remains to be investigated, but a recent study shows that glycoprotein 1b (GP Ib), the platelet VWF receptor, enhances ADAMTS-13 activity.19 Glycocalicin, the soluble extracellular portion of GP Ib that normally circulates in plasma in various amounts, may therefore be one of such cofactors. Studying how ADAMTS-13 is regulated by plasma factors is important not only for understanding the ADAMTS-13 function in vivo, but also for identifying new factors that affect ULVWF proteolysis and contribute to bleeding or thrombotic phenotypes.
Footnotes
Prepublished online as Blood First edition Paper, March 17, 2005; DOI 10.1182/blood-2004-11-4188.
Supported by National Institutes of Health (NIH) grants HL64796, 1-P50-HL65967, and HL18673. J.-F.D is an established Investigator of the American Heart Association.
Z.T. and Y.W. contributed equally to this work.
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.
References
Moake JL, Rudy CK, Troll JH, et al. Unusually large plasma factor VIII: von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura. N engl J Med. 1982;307: 1432-1435.
Moake JL. Thrombotic thrombocytopenic purpura: the systemic clumping "plague." Annu Rev Med. 2002;53: 75-88.
Gerritsen He, Robles R, Lammle B, Furlan M. Partial amino acid sequence of purified von Willebrand factor-cleaving protease. Blood. 2001;98: 1654-1661.
Furlan M, Robles R, Lammle B. Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood. 1996;87: 4223-4234.
Dent JA, Galbusera M, Ruggeri ZM. Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit. J Clin Invest. 1991;88: 774-782.
Furlan M. Von Willebrand factor: molecular size and functional activity. Ann Hematol. 1996;72: 341-348.
Moake JL. Thrombotic microangiopathies. N engl J Med. 2002;347: 589-600.
Levy GG, Nichols WC, Lian eC, et al. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature. 2001;413: 488-494.
Dong JF, Moake JL, Nolasco L, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood. 2002;100: 4033-4039.
Padilla A, Moake JL, Bernardo A, et al. P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface. Blood. 2004;103: 2150-2156.
Zheng X, Nishio K, Majerus eM, Sadler Je. Cleavage of von Willebrand factor requires the spacer domain of the metalloprotease ADAMTS-13. J Biol Chem. 2003;278: 30136-30141.
Soejima K, Matsumoto M, Kokame K, et al. ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage. Blood. 2003;102: 3232-3237.
Dong JF, Bernardo A, Nolasco L et al. ADAMTS-13 proteolysis of ultra-large von Willebrand factor multimers on the endothelial surface requires protease docking and is inhibited by thrombospondin-1 . Blood. 2002;100: 123a.
Zhang W, Motto D, Ginsburg D. ADAMTS-13 binds VWF via its C-terminal CUB2 domain . Blood. 2004;104: 39a.
Majerus eM, Anderson PJ, Sadler Je. Characterization of the binding interaction between ADAMTS-13 and von Willebrand factor . 2005;104: 150a.
Gao G, Westling J, Thompson VP, Howell TD, Gottschall Pe, Sandy JD. Activation of the proteolytic activity of ADAMTS4 (aggrecanase-1) by C-terminal truncation. J Biol Chem. 2002;277: 11034-11041.
Kashiwagi M, enghild JJ, Gendron C, et al. Altered proteolytic activities of ADAMTS-4 expressed by C-terminal processing. J Biol Chem. 2003;279: 10109-10119.
Dong JF, Whitelock J, Bernardo A, Ball C, Cruz MA. Variations among normal individuals in the cleavage of endothelial-derived ultra-large von Willebrand factor under flow. J Thromb Haemost. 2004;2: 1460-1466.
Nishio K, Anderson PJ, Zheng XL, Sadler Je. Binding of platelet glycoprotein Ibalpha to von Willebrand factor domain A1 stimulates the cleavage of the adjacent domain A2 by ADAMTS-13. Proc Natl Acad Sci U S A. 2004;101: 10578-10583.(Zhenyin Tao, Yongtao Wang)