- Email: [email protected]
ADAMTS-13 activity in von Willebrand disease
Thrombosis Research (2006) 117, 685 — 688
intl.elsevierhealth.com/journals/thre
BRIEF COMMUNICATION
ADAMTS-13 activity in von Willebrand disease Paolo Perutelli *, Stefano Amato, Angelo C. Molinari Hemostasis and Thrombosis Unit, Department of Hematology and Oncology, IRCCS G. Gaslini, Largo G. Gaslini, 5, I-16147 Genova, Italy Received 6 April 2005; received in revised form 11 May 2005; accepted 18 May 2005 Available online 5 July 2005
KEYWORDS ADAMTS-13; von Willebrand disease; von Willebrand factor
Introduction Human von Willebrand factor (VWF) is a plasma glycoprotein that mediates shear-dependent platelet adhesion to subendothelium and platelet— platelet interactions [1]. VWF is synthesized by endothelial cells as an ultralarge molecule (ULVWF) which may contain more than 40 subunits (up to 20,000 kDa). Upon secretion, the size of the ULVWF is reduced by a specific metalloprotease (ADAMTS-13) [2]. ADAMTS-13 cleaves the Tyr842—Met843 peptide bond in the VWF A2 domain, generating smaller multimers and proteolysis fragments [3].
* Corresponding author. Tel.: +39 10 5636331; fax: +39 10 386204. E-mail address: [email protected] (P. Perutelli).
Uncleaved ULVWF is hyperreactive in its capacity to bind to the platelet glycoprotein Ib-IX complex [4]. Thus, downregulation of VWF multimeric size helps avoid unwanted platelet agglutination [5]. Indeed, severely deficient ADAMTS-13 activity has been demonstrated in most patients with thrombotic thrombocytopenic purpura (TTP) [6], a disorder that is characterized by the occurrence of microvascular thrombi made up of platelet aggregates containing ULVWF [7]. Little is known about the biological mechanisms that regulate ADAMTS-13 activity. An inverse relationship between ADAMTS-13 activity and VWF plasma levels was found by Mannucci et al. [8,9]. They investigated ADAMTS-13 in patients with type 3 von Willebrand disease (VWD) with undetectable VWF plasma levels and showed that activity was approximately one third higher than it was in healthy individuals with normal VWF. Infusion of desmopressin (DDAVP) in healthy volunteers resulted in a two- to threefold increase in VWF, and a 10—20% decrease in ADAMTS-13 activity, whereas following experimental administration of DDAVP to type 3 VWD patients, VWF was still undetectable, and ADAMTS-13 was unmodified. On the other hand, therapy with plasma concentrates in such patients resulted in a transient increase in VWF, along with a decrease in ADAMTS-13. Based on
0049-3848/$ - see front matter D 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.05.018
686
P. Perutelli et al.
these findings, it was hypothesized that VWF levels regulate ADAMTS-13 [9]. ADAMTS-13 has never been measured in a large series of patients with type 1 VWD. The VWF susceptibility to ADAMTS-13 proteolysis has been recently investigated in individuals with type 1 VWD, but only qualitative results were presented [10]. We examined 62 type 1 patients whose ADAMTS13 activity we assumed would range between that of normal individuals and that of type 3 VWD. Moreover, we intended to broaden our knowledge on ADAMTS-13 in VWD by investigating some patients with type 2 VWD, since ADAMTS-13 was reported to be within the normal range in a few patients with qualitative variants (6 patients with type 2M Vicenza and 4 patients with type 2A), although results have not been shown [8].
Materials and methods Plasma samples After obtaining informed consent, blood samples were taken from 54 healthy, volunteer donors (25 males and 29 females; median age 35 years) and 80 patients with VWD (38 males and 42 females; median age 32.5 years). VWD types included: 62 type 1, 8 type 2A, 2 type 2B, 1 homozygous type 2N and 7 type 3. Diagnosis of VWD was made according to the published criteria [11]. Venous blood was anticoagulated with 129 mM sodium citrate and centrifuged at 1500g for 20 min. Platelet-poor plasma was collected, then snap frozen and stored at 80 8C until assay.
VWF and ADAMTS-13 assays Plasma VWF (VWF:Ag) was assessed using a commercial ELISA kit (Imubind, American Diagnostica, Stamford, CT, USA). A pool of citrated plasma obtained from 10 healthy women (not pregnant and not on oral contraceptives) and 10 healthy men
Table 1 Controls VWD type VWD type VWD type VWD type VWD type VWD type
was used as the reference plasma. Undiluted reference plasma (arbitrarily defined as containing 100% VWF:Ag) and dilutions between 1:2 and 1:128 were tested in the same assay. ADAMTS-13 assay was carried out as described by Gerritsen et al. [12]. Dilutions of the reference plasma pool from 1:5 to 1:320 were used to obtain a calibration curve of the protease activity. The 1:20 dilution was defined as 100% activity. The nonparametric Mann—Whitney U-test was used for comparison between groups. All P values were based upon two-tailed tests, and values less than 0.05 were considered statistically significant. Statistical analysis was performed using SPSS for Windows (SPSS Inc, Chicago, IL, USA).
Results Healthy donors and patients with type 3 VWD were chosen as being representative of normal and undetectable VWF levels. They exhibited normal and increased ADAMTS-13 activity, respectively (Table 1). Type 1, 2A and 2B VWD patients with low, but detectable VWF levels showed significantly increased ADAMTS-13 activity. Their median values ranged between those of healthy controls and patients with type 3 VWD (Table 1 and Fig. 1). The highest ADAMTS-13 value we observed in controls was 123%. All type 3 VWD patients showed higher ADAMTS-13, as did 22/62 type 1 patients, 3/8 type 2A and 2/2 type 2B. Thus, 27 out of 72 (37.5%) VWD patients with low, but measurable VWF had higher than normal ADAMTS13 activity. The patient with homozygous type 2N VWD had normal VWF:Ag and ADAMTS-13. On average, statistically significant differences were observed between controls and VWD patients, and among the various VWD types, but not between type 1 and type 2. By evaluating ADAMTS-13 activity as a function of plasma VWF levels (normal in healthy controls
VWF: Ag and ADAMTS-13 activity in controls and VWD patients
1 2A/B 2A 2B 2N 3
n
VWF:Ag (%)
54 62 10 8 2 1 7
95; 84—117 28; 15—38 32; 25—47 30; 24—50 37 105 b1
ADAMTS-13 (%) (62—156) (5—60) (16—55) (16—55) (34—40)
100; 90—109 (58—123) 118; 109—125 (70—134) 122; 110—129 (90—133) 116; 106—129 (90—130) 130 (127—133) 101 135; 130—138 (126—143)
Stat vs. controls: p b 0.001; vs. VWD3: p b 0.001 vs. controls: p b 0.005; vs. VWD3: p b 0.05
vs. controls: p b 0.001
Median value, interquartile range, observed range (in parentheses) and statistical comparison of ADAMTS-13 activity are shown.
ADAMTS-13 activity in von Willebrand disease
687
160 Controls type 1 VWD type 2A VWD type 2B VWD type 2N VWD type 3 VWD
140
ADAMTS-13 (%)
120
100
80
60
40 0
50
100
150
200
VWF (%)
Figure 1 ADAMTS-13 activity plotted as a function of VWF levels.
and in type 2N VWD, low in type 1 VWD, and undetectable in type 3 VWD), an inverse relationship between VWF and ADAMTS-13 was shown (r = 0.53, p b 0.001). On the other hand, a positive correlation between VWF and ADAMTS-13 was found in type 2A and 2B VWD (r = 0.63, p b 0.06).
Discussion Apart from TTP, a decreased ADAMTS-13 activity has been reported in physiological and pathological conditions [8]. On the other hand, increased ADAMTS-13 levels were only found in patients with the severe VWF deficit, type 3 VWD. Evidence about the regulation of ADAMTS-13 activity by VWF levels has been provided by Mannucci et al. [9] as well as by our study. In both studies, the collagen-binding assay was used for the measurement of the cleaved VWF. However, other methods for ADAMTS-13 assay in vitro may be less affected by the plasma VWF concentration. We investigated ADAMTS-13 in VWD, for the first time reporting about ADAMTS-13 activity in a numerically representative group of patients with type 1 VWD, and evaluated the protease activity over a continuous spectrum of VWF levels ranging from normal to undetectable. As expected, ADAMTS-13 was within the normal range in the type 2N patient. Actually, the VWF mutation that is responsible for type 2N VWD leads to defective FVIII binding, but does not affect VWF synthesis [13].
As found in type 1 VWD, the patients with type 2A and 2B variants also showed increased ADAMTS13 activity. The median values did not significantly differ from what was observed in type 1 VWD. Interestingly, protease activity was positively correlated to VWF levels in these patients. Type 2A VWD refers to qualitative variants with decreased platelet-dependent function associated with the absence of the high molecular weight VWF multimers [14]. Mutations have been described in type 2A VWF, close to the cleavage site for ADAMTS-13, that induce a conformational change in the molecule, thus increasing its susceptibility to proteolysis by ADAMTS-13 [15,16]. Therefore, in some type 2A patients ADAMTS-13 might also be increased as a result of heightened accessibility to the cleavage site on the VWF A2 domain. On the other hand, the mutations that occur in type 2B VWD do not cause major conformational changes, but increase the affinity of VWF for platelet glycoprotein Iba. As a consequence of this gain of function, the larger VWF multimers are cleared from the circulation [17], resulting in a multimeric pattern similar to what is observed in type 2A variants. ADAMTS-13 downregulates the size of VWF both on the surface of endothelial cells soon after its release [2] and in the circulation under high shear stress conditions, in order to prevent the accumulation of the hemostatically active, unfolded VWF [5]. Increased ADAMTS-13 in type 2B VWD would therefore reflect its enhanced activity in order to limit the spreading of the VWFplatelet interaction. In conclusion, ADAMTS-13 activity in type 1, 2A and 2B VWD is higher than in healthy controls, but lower than in type 3 VWD. As far as types 2A and 2B VWD are concerned, the limited number of patients we investigated makes this paper a preliminary report, offering the basis for larger studies.
Acknowledgments The authors are grateful to Dr. Alessandra Casonato, Padova, who diagnosed the homozygous Arg91Gln mutation in the patient with type 2N VWD.
References [1] Tsai H-M. Shear stress and von Willebrand factor in health and disease. Semin Thromb Hemost 2003;29:479 – 88. [2] Dong J-F, Moake JL, Nolasco L, Bernardo A, Arceneaux W, Shrimpton CN, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the
688
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
P. Perutelli et al. endothelial surface under flowing conditions. Blood 2002;100:4033 – 9. Kokame K, Matsumoto M, Fujimura Y, Miyata T. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood 2004;103:607 – 12. Arya M, Anvari B, Romo GM, Cruz MA, Dong J-F, McIntire LV, et al. Ultralarge multimers of von Willebrand factor forms spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 2002;99:3971 – 7. Furlan M. Proteolytic cleavage of von Willebrand factor by ADAMTS-13 prevents uninvited clumping of blood platelets. J Thromb Haemost 2004;2:1505 – 9. Peyvandi F, Ferrari S, Lavoretano S, Canciani MT, Mannucci PM. von Willebrand factor cleaving protease (ADAMTS-13) and ADAMTS-13 neutralizing autoantibodies in 100 patients with thrombotic thrombocytopenic purpura. Br J Haematol 2004;127:433 – 9. Moake JL. Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura. J Thromb Haemost 2004;2:1515 – 21. Mannucci PM, Canciani MT, Forza I, Lussana F, Lattuada A, Rossi E. Changes in health and disease of the metalloprotease that cleaves von Willebrand factor. Blood 2001;98:2730 – 5. Mannucci PM, Capoferri C, Canciani MT. Plasma levels of von Willebrand factor regulate ADAMTS-13, its major cleaving protease. Br J Haematol 2004;126:213 – 8. Bowen DJ, Collins PW, Lester W, Cumming AM, Keeney S, Grundy P, et al. The prevalence of the cysteine 1584 variant
[11]
[12]
[13]
[14] [15]
[16]
[17]
von Willebrand factor is increased in type 1 von Willebrand disease: co-segregation with increased susceptibility to ADAMTS-13 proteolysis but not clinical phenotype. Br J Haematol 2005;128:830 – 6. Federici AB, Castaman G, Mannucci PM. Guidelines for the diagnosis and management of von Willebrand disease in Italy. Haemophilia 2002;8:607 – 21. Gerritsen H, Turecek P, Schwarz HP, La ¨mmle B, Furlan M. Assay of von Willebrand factor (VWF)-cleaving protease based on decreased collagen binding affinity of degraded VWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999;82:1386 – 9. Nishino M, Girma J-P, Rothschild C, Fressinaud E, Meyer D. New variant of von Willebrand disease with defective binding to factor VIII. Blood 1989;74:1591 – 9. Sadler JE. A revised classification of von Willebrand disease. Thromb Haemost 1994;71:520 – 5. Tsai H-M, Sussman II, Ginsburg D, Lankhof H, Sixma JJ, Nagel RL. Proteolytic cleavage of recombinant von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1. Blood 1997;89:1954 – 62. O’Brien LA, Sutherland JJ, Hegadorn C, Benford K, Racz H, Rapson D, et al. A novel type 2A (group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers. J Thromb Haemost 2004;2:1135 – 42. Ruggeri ZM. Type IIB von Willebrand disease: a paradox explains how von Willebrand factor works. J Thromb Haemost 2004;2:2 – 6.
intl.elsevierhealth.com/journals/thre
BRIEF COMMUNICATION
ADAMTS-13 activity in von Willebrand disease Paolo Perutelli *, Stefano Amato, Angelo C. Molinari Hemostasis and Thrombosis Unit, Department of Hematology and Oncology, IRCCS G. Gaslini, Largo G. Gaslini, 5, I-16147 Genova, Italy Received 6 April 2005; received in revised form 11 May 2005; accepted 18 May 2005 Available online 5 July 2005
KEYWORDS ADAMTS-13; von Willebrand disease; von Willebrand factor
Introduction Human von Willebrand factor (VWF) is a plasma glycoprotein that mediates shear-dependent platelet adhesion to subendothelium and platelet— platelet interactions [1]. VWF is synthesized by endothelial cells as an ultralarge molecule (ULVWF) which may contain more than 40 subunits (up to 20,000 kDa). Upon secretion, the size of the ULVWF is reduced by a specific metalloprotease (ADAMTS-13) [2]. ADAMTS-13 cleaves the Tyr842—Met843 peptide bond in the VWF A2 domain, generating smaller multimers and proteolysis fragments [3].
* Corresponding author. Tel.: +39 10 5636331; fax: +39 10 386204. E-mail address: [email protected] (P. Perutelli).
Uncleaved ULVWF is hyperreactive in its capacity to bind to the platelet glycoprotein Ib-IX complex [4]. Thus, downregulation of VWF multimeric size helps avoid unwanted platelet agglutination [5]. Indeed, severely deficient ADAMTS-13 activity has been demonstrated in most patients with thrombotic thrombocytopenic purpura (TTP) [6], a disorder that is characterized by the occurrence of microvascular thrombi made up of platelet aggregates containing ULVWF [7]. Little is known about the biological mechanisms that regulate ADAMTS-13 activity. An inverse relationship between ADAMTS-13 activity and VWF plasma levels was found by Mannucci et al. [8,9]. They investigated ADAMTS-13 in patients with type 3 von Willebrand disease (VWD) with undetectable VWF plasma levels and showed that activity was approximately one third higher than it was in healthy individuals with normal VWF. Infusion of desmopressin (DDAVP) in healthy volunteers resulted in a two- to threefold increase in VWF, and a 10—20% decrease in ADAMTS-13 activity, whereas following experimental administration of DDAVP to type 3 VWD patients, VWF was still undetectable, and ADAMTS-13 was unmodified. On the other hand, therapy with plasma concentrates in such patients resulted in a transient increase in VWF, along with a decrease in ADAMTS-13. Based on
0049-3848/$ - see front matter D 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2005.05.018
686
P. Perutelli et al.
these findings, it was hypothesized that VWF levels regulate ADAMTS-13 [9]. ADAMTS-13 has never been measured in a large series of patients with type 1 VWD. The VWF susceptibility to ADAMTS-13 proteolysis has been recently investigated in individuals with type 1 VWD, but only qualitative results were presented [10]. We examined 62 type 1 patients whose ADAMTS13 activity we assumed would range between that of normal individuals and that of type 3 VWD. Moreover, we intended to broaden our knowledge on ADAMTS-13 in VWD by investigating some patients with type 2 VWD, since ADAMTS-13 was reported to be within the normal range in a few patients with qualitative variants (6 patients with type 2M Vicenza and 4 patients with type 2A), although results have not been shown [8].
Materials and methods Plasma samples After obtaining informed consent, blood samples were taken from 54 healthy, volunteer donors (25 males and 29 females; median age 35 years) and 80 patients with VWD (38 males and 42 females; median age 32.5 years). VWD types included: 62 type 1, 8 type 2A, 2 type 2B, 1 homozygous type 2N and 7 type 3. Diagnosis of VWD was made according to the published criteria [11]. Venous blood was anticoagulated with 129 mM sodium citrate and centrifuged at 1500g for 20 min. Platelet-poor plasma was collected, then snap frozen and stored at 80 8C until assay.
VWF and ADAMTS-13 assays Plasma VWF (VWF:Ag) was assessed using a commercial ELISA kit (Imubind, American Diagnostica, Stamford, CT, USA). A pool of citrated plasma obtained from 10 healthy women (not pregnant and not on oral contraceptives) and 10 healthy men
Table 1 Controls VWD type VWD type VWD type VWD type VWD type VWD type
was used as the reference plasma. Undiluted reference plasma (arbitrarily defined as containing 100% VWF:Ag) and dilutions between 1:2 and 1:128 were tested in the same assay. ADAMTS-13 assay was carried out as described by Gerritsen et al. [12]. Dilutions of the reference plasma pool from 1:5 to 1:320 were used to obtain a calibration curve of the protease activity. The 1:20 dilution was defined as 100% activity. The nonparametric Mann—Whitney U-test was used for comparison between groups. All P values were based upon two-tailed tests, and values less than 0.05 were considered statistically significant. Statistical analysis was performed using SPSS for Windows (SPSS Inc, Chicago, IL, USA).
Results Healthy donors and patients with type 3 VWD were chosen as being representative of normal and undetectable VWF levels. They exhibited normal and increased ADAMTS-13 activity, respectively (Table 1). Type 1, 2A and 2B VWD patients with low, but detectable VWF levels showed significantly increased ADAMTS-13 activity. Their median values ranged between those of healthy controls and patients with type 3 VWD (Table 1 and Fig. 1). The highest ADAMTS-13 value we observed in controls was 123%. All type 3 VWD patients showed higher ADAMTS-13, as did 22/62 type 1 patients, 3/8 type 2A and 2/2 type 2B. Thus, 27 out of 72 (37.5%) VWD patients with low, but measurable VWF had higher than normal ADAMTS13 activity. The patient with homozygous type 2N VWD had normal VWF:Ag and ADAMTS-13. On average, statistically significant differences were observed between controls and VWD patients, and among the various VWD types, but not between type 1 and type 2. By evaluating ADAMTS-13 activity as a function of plasma VWF levels (normal in healthy controls
VWF: Ag and ADAMTS-13 activity in controls and VWD patients
1 2A/B 2A 2B 2N 3
n
VWF:Ag (%)
54 62 10 8 2 1 7
95; 84—117 28; 15—38 32; 25—47 30; 24—50 37 105 b1
ADAMTS-13 (%) (62—156) (5—60) (16—55) (16—55) (34—40)
100; 90—109 (58—123) 118; 109—125 (70—134) 122; 110—129 (90—133) 116; 106—129 (90—130) 130 (127—133) 101 135; 130—138 (126—143)
Stat vs. controls: p b 0.001; vs. VWD3: p b 0.001 vs. controls: p b 0.005; vs. VWD3: p b 0.05
vs. controls: p b 0.001
Median value, interquartile range, observed range (in parentheses) and statistical comparison of ADAMTS-13 activity are shown.
ADAMTS-13 activity in von Willebrand disease
687
160 Controls type 1 VWD type 2A VWD type 2B VWD type 2N VWD type 3 VWD
140
ADAMTS-13 (%)
120
100
80
60
40 0
50
100
150
200
VWF (%)
Figure 1 ADAMTS-13 activity plotted as a function of VWF levels.
and in type 2N VWD, low in type 1 VWD, and undetectable in type 3 VWD), an inverse relationship between VWF and ADAMTS-13 was shown (r = 0.53, p b 0.001). On the other hand, a positive correlation between VWF and ADAMTS-13 was found in type 2A and 2B VWD (r = 0.63, p b 0.06).
Discussion Apart from TTP, a decreased ADAMTS-13 activity has been reported in physiological and pathological conditions [8]. On the other hand, increased ADAMTS-13 levels were only found in patients with the severe VWF deficit, type 3 VWD. Evidence about the regulation of ADAMTS-13 activity by VWF levels has been provided by Mannucci et al. [9] as well as by our study. In both studies, the collagen-binding assay was used for the measurement of the cleaved VWF. However, other methods for ADAMTS-13 assay in vitro may be less affected by the plasma VWF concentration. We investigated ADAMTS-13 in VWD, for the first time reporting about ADAMTS-13 activity in a numerically representative group of patients with type 1 VWD, and evaluated the protease activity over a continuous spectrum of VWF levels ranging from normal to undetectable. As expected, ADAMTS-13 was within the normal range in the type 2N patient. Actually, the VWF mutation that is responsible for type 2N VWD leads to defective FVIII binding, but does not affect VWF synthesis [13].
As found in type 1 VWD, the patients with type 2A and 2B variants also showed increased ADAMTS13 activity. The median values did not significantly differ from what was observed in type 1 VWD. Interestingly, protease activity was positively correlated to VWF levels in these patients. Type 2A VWD refers to qualitative variants with decreased platelet-dependent function associated with the absence of the high molecular weight VWF multimers [14]. Mutations have been described in type 2A VWF, close to the cleavage site for ADAMTS-13, that induce a conformational change in the molecule, thus increasing its susceptibility to proteolysis by ADAMTS-13 [15,16]. Therefore, in some type 2A patients ADAMTS-13 might also be increased as a result of heightened accessibility to the cleavage site on the VWF A2 domain. On the other hand, the mutations that occur in type 2B VWD do not cause major conformational changes, but increase the affinity of VWF for platelet glycoprotein Iba. As a consequence of this gain of function, the larger VWF multimers are cleared from the circulation [17], resulting in a multimeric pattern similar to what is observed in type 2A variants. ADAMTS-13 downregulates the size of VWF both on the surface of endothelial cells soon after its release [2] and in the circulation under high shear stress conditions, in order to prevent the accumulation of the hemostatically active, unfolded VWF [5]. Increased ADAMTS-13 in type 2B VWD would therefore reflect its enhanced activity in order to limit the spreading of the VWFplatelet interaction. In conclusion, ADAMTS-13 activity in type 1, 2A and 2B VWD is higher than in healthy controls, but lower than in type 3 VWD. As far as types 2A and 2B VWD are concerned, the limited number of patients we investigated makes this paper a preliminary report, offering the basis for larger studies.
Acknowledgments The authors are grateful to Dr. Alessandra Casonato, Padova, who diagnosed the homozygous Arg91Gln mutation in the patient with type 2N VWD.
References [1] Tsai H-M. Shear stress and von Willebrand factor in health and disease. Semin Thromb Hemost 2003;29:479 – 88. [2] Dong J-F, Moake JL, Nolasco L, Bernardo A, Arceneaux W, Shrimpton CN, et al. ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the
688
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
P. Perutelli et al. endothelial surface under flowing conditions. Blood 2002;100:4033 – 9. Kokame K, Matsumoto M, Fujimura Y, Miyata T. VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood 2004;103:607 – 12. Arya M, Anvari B, Romo GM, Cruz MA, Dong J-F, McIntire LV, et al. Ultralarge multimers of von Willebrand factor forms spontaneous high-strength bonds with the platelet glycoprotein Ib-IX complex: studies using optical tweezers. Blood 2002;99:3971 – 7. Furlan M. Proteolytic cleavage of von Willebrand factor by ADAMTS-13 prevents uninvited clumping of blood platelets. J Thromb Haemost 2004;2:1505 – 9. Peyvandi F, Ferrari S, Lavoretano S, Canciani MT, Mannucci PM. von Willebrand factor cleaving protease (ADAMTS-13) and ADAMTS-13 neutralizing autoantibodies in 100 patients with thrombotic thrombocytopenic purpura. Br J Haematol 2004;127:433 – 9. Moake JL. Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura. J Thromb Haemost 2004;2:1515 – 21. Mannucci PM, Canciani MT, Forza I, Lussana F, Lattuada A, Rossi E. Changes in health and disease of the metalloprotease that cleaves von Willebrand factor. Blood 2001;98:2730 – 5. Mannucci PM, Capoferri C, Canciani MT. Plasma levels of von Willebrand factor regulate ADAMTS-13, its major cleaving protease. Br J Haematol 2004;126:213 – 8. Bowen DJ, Collins PW, Lester W, Cumming AM, Keeney S, Grundy P, et al. The prevalence of the cysteine 1584 variant
[11]
[12]
[13]
[14] [15]
[16]
[17]
von Willebrand factor is increased in type 1 von Willebrand disease: co-segregation with increased susceptibility to ADAMTS-13 proteolysis but not clinical phenotype. Br J Haematol 2005;128:830 – 6. Federici AB, Castaman G, Mannucci PM. Guidelines for the diagnosis and management of von Willebrand disease in Italy. Haemophilia 2002;8:607 – 21. Gerritsen H, Turecek P, Schwarz HP, La ¨mmle B, Furlan M. Assay of von Willebrand factor (VWF)-cleaving protease based on decreased collagen binding affinity of degraded VWF: a tool for the diagnosis of thrombotic thrombocytopenic purpura (TTP). Thromb Haemost 1999;82:1386 – 9. Nishino M, Girma J-P, Rothschild C, Fressinaud E, Meyer D. New variant of von Willebrand disease with defective binding to factor VIII. Blood 1989;74:1591 – 9. Sadler JE. A revised classification of von Willebrand disease. Thromb Haemost 1994;71:520 – 5. Tsai H-M, Sussman II, Ginsburg D, Lankhof H, Sixma JJ, Nagel RL. Proteolytic cleavage of recombinant von Willebrand factor mutants R834W and R834Q: inhibition by doxycycline and by monoclonal antibody VP-1. Blood 1997;89:1954 – 62. O’Brien LA, Sutherland JJ, Hegadorn C, Benford K, Racz H, Rapson D, et al. A novel type 2A (group II) von Willebrand disease mutation (L1503Q) associated with loss of the highest molecular weight von Willebrand factor multimers. J Thromb Haemost 2004;2:1135 – 42. Ruggeri ZM. Type IIB von Willebrand disease: a paradox explains how von Willebrand factor works. J Thromb Haemost 2004;2:2 – 6.