Fatal congenital thrombotic thrombocytopenic purpura with apparent ADAMTS13 inhibitor: in vitro inhibition of ADAMTS13 activity by hemoglobi
http://www.100md.com
《血液学杂志》
the Department of Hematology and Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland
Baxter BioScience, Orth, Austria
the Department of Pediatrics, University Hospital, Tübingen, Germany.
Abstract
Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an ADAMTS13 activity of approximately 50%. Sequencing of the ADAMTS13 gene revealed an intronic 687-2A>G substitution affecting exon 7, homozygous in the propositus and heterozygous in both parents, confirming constitutional ADAMTS13 deficiency. ADAMTS13 activity of normal plasma was inhibited by incubation with the propositus' serum, suggesting alloantibody formation to ADAMTS13. However, immunoglobulin purified from serum had no ADAMTS13 inhibitory effect, whereas the immunoglobulin-depleted hemolytic serum inhibited ADAMTS13 activity of normal plasma, suggesting an inhibitory effect of hemolysis products. Incubation of hemoglobin, recombinant and from lysed erythrocytes, with normal plasma revealed an ADAMTS13 inhibitory effect at hemoglobin concentrations of 2 g/L or higher.
Introduction
Severe deficiency of ADAMTS13 activity (< 5% of normal plasma) is a strong risk factor for thrombotic thrombocytopenic purpura (TTP).1-3 There are 2 pathogenetic mechanisms: homozygous or compound heterozygous mutations of the ADAMTS13 gene in congenital TTP,4-10 and inhibition of ADAMTS13 by autoantibodies in acquired TTP.2,3,11 Nonneutralizing autoantibodies,12 possibly leading to increased clearance of ADAMTS13 from the circulation, or interfering with its interaction with endothelial cells13 or the von Willebrand factor (VWF) A1/A3 domains,14 may be additional mechanisms.
Investigation of premortem serum of a pediatric TTP patient revealed a severe ADAMTS13 deficiency. Sequencing of the ADAMTS13 gene identified a homozygous mutation confirming constitutional ADAMTS13 deficiency. Surprisingly, the patient's severely hemolytic serum had a pronounced ADAMTS13 inhibitory effect that was not attributable to purified immunoglobulin (Ig) but caused by hemolysis products present in the sample. Subsequent experiments addressed an ADAMTS13 inhibitory effect of hemoglobin.
Study design
Patient
The propositus, a Turkish boy,15 had suffered from episodes of severe Coombs-negative hemolytic anemia and thrombocytopenia since birth. He died at the age of 7 years from a severe attack of this disease, until then classified as atypical Evans syndrome. Autopsy revealed microvascular occlusions by fibrin-poor platelet thrombi, suggesting the diagnosis of TTP. Strongly hemolytic serum from whole blood obtained before death and stored at 4°C was shipped frozen to our laboratory. The serum had a hemoglobin concentration of 17 g/L (1.7 g/dL), probably due to in vitro hemolysis.
Informed consent for publication was obtained from the parents; the study was conducted according to the guidelines on research on human subjects of the responsible Ethics Committee (Kantonale Ethikkommission, Bern).
ADAMTS13 activity and routine inhibitor screening
ADAMTS13 activity was determined by immunoblotting of purified VWF substrate degraded by BaCl2-activated ADAMTS13 in serum or plasma. For inhibitor screening, the residual ADAMTS13 activity of pooled normal human plasma (NHP) was measured after 1:1 (vol/vol) incubation with patient serum/plasma. Assays were performed as described.2,16
Inhibition of ADAMTS13 activity by hemoglobin
There were 2 solutions of concentrated hemoglobin prepared. Solution 1 consisted of recombinant human hemoglobin (Baxter, Boulder, CO) 50 g/L in 0.15 M NaCl, 5 mM sodium phosphate, 0.03% TWEEN 80. Solution 2 was obtained from lysed washed erythrocytes. Citrated blood from a healthy donor was centrifuged for 10 minutes at 4000g, and the supernatant plasma and buffy coat were discarded. The sediment was washed 3 times with physiologic saline, and incubated 1:3 (vol/vol) with distilled water for 10 minutes at 37°C. After centrifugation for 10 minutes at 4000g, the supernatant solution contained 84 g/L hemoglobin.
Inhibition of ADAMTS13 activity by hemoglobin was assessed by routine ADAMTS13 inhibitor assay.16 NHP was incubated 1:1 (vol/vol) for 2 hours at 37°C with hemoglobin solution 1 or 2 prediluted in 0.01 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 7.4 (TBS). Final hemoglobin concentrations after addition to NHP ranged from 0.5 to 40 g/L.
Preliminary experiments addressed a complexation of divalent metal ions required for ADAMTS13 activity, such as calcium or zinc,17,18 as a hypothetical mechanism by which hemoglobin might inhibit ADAMTS13. NHP was incubated 1:1 (vol/vol) with recombinant hemoglobin, 20 g/L, for 1 hour at 37°C. This mixture was diluted 1:10 with TBS and activated with increasing final concentrations of BaCl2 between 10 and 75 mM.
Purification of total Ig
Total Ig was purified from the propositus' serum by adsorption to Ig-Therasorb (Miltenyi Biotech, Teterow, Germany; Sepharose CL-4B coupled with polyclonal sheep antibodies to human Ig). The purified Ig and the Ig-depleted serum were concentrated to original serum levels by centrifugation through filter membranes (Biomax; Millipore, Bedford, MA; molecular weight cut-off, 30 kDa).
Investigation of the ADAMTS13 gene
Amplification and sequencing of polymerase chain reaction (PCR) products were performed as described.6 SpliceView program (Institute of Biomedical Technologies, Segrate, Italy) was used for predicting alternative splice sites resulting from the detected mutation.
Results and discussion
ADAMTS13 activity was less than 3% in premortem serum of the propositus, and approximately 50% in plasma of both parents (Figure 1A). Sequencing of all 29 exons of the ADAMTS13 gene, including their intron/exon boundaries, revealed a point mutation (687-2A>G) at the 3' splice acceptor site of intron 6 affecting exon 7, between amino acids 228 to 229, homozygous in the propositus and heterozygous in both parents. We hypothesize that this mutation could result in 2 alternative splice sites, causing either a frameshift and premature termination of the polypeptide, or an in-frame insertion of 8 amino acids. These findings confirmed constitutional ADAMTS13 deficiency with hereditary TTP in the propositus, misdiagnosed as atypical Evans syndrome until autopsy. His fatal course illustrates the importance of rapid and comprehensive diagnostics in patients suffering from congenital thrombotic microangiopathies including determination of ADAMTS13 activity and ADAMTS13 inhibitors because plasma infusion is a highly effective treatment.19-21
Pronounced inhibition of the ADAMTS13 activity of NHP was observed after incubation with an equal volume of the propositus' hemolytic serum, corresponding to a titer of 1 to 2 Bethesda units (BU)/mL and suggested alloantibody formation to ADAMTS13 triggered by repeated transfusions of blood products containing ADAMTS13. However, total Ig purified from the propositus' serum had no ADAMTS13 inhibitory effect (Figure 1B). In contrast, the Ig-depleted hemolytic serum inhibited ADAMTS13 activity of NHP to a similar extent as the original serum. This indicated that the observed inhibition was not caused by antibodies to ADAMTS13 but was related to other factors present in the sample, possibly originating from lysed erythrocytes.
Subsequent experiments addressed a possible ADAMTS13 inhibitory capacity of hemoglobin. NHP was incubated with hemoglobin, either recombinant or purified from lysed erythrocytes at various concentrations. Slight inhibition of the ADAMTS13 activity of NHP ( 1 BU/mL) was observed after incubation with hemoglobin at a final concentration of 2 g/L (Figure 1C). Moderate inhibition (1-2 BU/mL) occurred at 5 g/L, and strong inhibition ( 2 BU/mL) occurred at 10 g/L or higher. The ADAMTS13 inhibitory effect was similar for recombinant and erythrocyte-derived hemoglobin, indicating that it can be linked specifically to the hemoglobin molecule.
To exclude an artifact related to our assay, NHP was incubated 1:1 (vol/vol) with hemoglobin solutions 1 and 2 (final hemoglobin concentrations, 10 g/L and 25 g/L each) for 2 hours at 37°C. Identical aliquots were investigated for their ADAMTS13 activity by 3 other laboratories using different assays.22-24 All laboratories found a severe or borderline severe ADAMTS13 deficiency (< 5% or 5%-9% of the normal), instead of an activity of 50% corresponding to the proportion of NHP in the mixture.
The ADAMTS13 inhibitory capacity of hemoglobin depended upon incubation temperature and time. ADAMTS13 activity of NHP was completely inhibited after incubation with 30 g/L hemoglobin for 3 hours at 37°C but only reduced to 25% at 4°C. Incubation for 5 minutes to 2 hours revealed mild inhibition ( 1 BU/mL) after 5 minutes, moderate inhibition (1-2 BU/mL) after 15 to 30 minutes, and strong inhibition ( 2 BU/mL) after 1 to 2 hours (not shown).
ADAMTS13 inhibition by hemoglobin was attenuated, but not completely reverted by the addition of increasing BaCl2 concentrations up to 40 mM.
Inhibition of ADAMTS13 activity occurred at hemoglobin concentrations potentially found in strong intravascular hemolysis, as observed in incompatible erythrocyte transfusion, immunemediated hemolytic anemias, or others.25 These conditions are not usually associated with TTP, and it is unclear whether short-lived elevation of free hemoglobin in plasma will lead to a clinically relevant ADAMTS13 inhibition. In our case, the serum hemoglobin concentration of 17 g/L probably resulted from in vitro hemolysis in stored native whole blood. Nevertheless, some practical conclusions can be drawn from our observations. Marked hemolysis and the presence of hemoglobin apparently influence most of the current ADAMTS13 assays.2,22-24 Lowering of ADAMTS13 activity values should be expected under these circumstances and inhibitor screening may lead to the erroneous assumption of ADAMTS13 inhibitory antibodies. Whether the ADAMTS13 inhibitory capacity of hemoglobin is of any physiologic or pathophysiologic relevance (eg, in severe intravascular hemolysis) will need further study.
Acknowledgements
We thank Dr W. Bhm (Miltenyi Biotech, Teterow, Germany) for providing Ig-Therasorb columns, and Drs U. Budde (Laboratory Keeser and Arndt, Hamburg, Germany), M. Bhm (University Hospital Frankfurt, Germany), and J.-P. Girma (INSERM U.143, Le Kremlin-Bicêtre, France) for performing confirmatory determinations of ADAMTS13 activity.
Footnotes
Prepublished online as Blood First Edition Paper, September 14, 2004; DOI 10.1182/blood-2004-06-2096.
Supported by grants from the Swiss National Foundation for Scientific Research (32-66756.01; B.L.) and from the Fondation pour la Recherche sur l'Arteriosclerose et la Thrombose (J.A.K.H.).
J.-D.S. and J.A.K.H. 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
Furlan M, Robles R, Solenthaler M, et al. Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood. 1997;89: 3097-3103.
Furlan M, Robles R, Galbusera M, et al. Von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolyticuremic syndrome. N Engl J Med. 1998;339: 1578-1584.
Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339: 1585-1594.
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.
Kokame K, Matsumoto M, Soejima K, et al. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity. Proc Natl Acad Sci U S A. 2002;99: 11902-11907.
Antoine G, Zimmermann K, Plaimauer B, et al. ADAMTS13 gene defects in 2 brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13. Br J Haematol. 2003;120: 821-824.
Schneppenheim R, Budde U, Oyen F, et al. Von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP. Blood. 2003;101: 1845-1850.
Assink K, Schiphorst R, Allford S, et al. Mutation analysis and clinical implications of von Willebrand factor-cleaving protease deficiency. Kidney Int. 2003;63: 1995-1999.
Matsumoto M, Kokame K, Soejima K, et al. Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome. Blood. 2004;103: 1305-1310.
Veyradier A, Lavergne JM, Ribba AS, et al. Ten candidate ADAMTS13 mutations in 6 French families with congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome). J Thromb Haemost. 2004;2: 424-429.
Furlan M, Robles R, Solenthaler M, et al. Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood. 1998;91: 2839-2846.
Scheiflinger F, Knbl P, Trattner B, et al. Non-neutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS13) in a patient with thrombotic thrombocytopenic purpura. Blood. 2003;102: 3241-3243.
Dong JF, Moake JL, Nolasco L, et al. ADAMTS13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood. 2002;100: 4033-4039.
Dong JF, Moake JL, Bernardo A, et al. ADAMTS13 metalloprotease interacts with the endothelial cellderived ultra-large von Willebrand factor. J Biol Chem. 2003;278: 29633-29639.
Studt JD, Kremer Hovinga JA, Alberio L, et al. Von Willebrand factor-cleaving protease (ADAMTS13) activity in thrombotic microangiopathies: diagnostic experience 2001/2002 of a single research laboratory. Swiss Med Wkly. 2003;133: 325-332.
Studt JD, Bhm M, Budde U, et al. Measurement of von Willebrand factor-cleaving protease (ADAMTS13) activity in plasma: a multicenter comparison of different assay methods. J Thromb Haemost. 2003;1: 1882-1887.
Furlan M, Robles R, Lmmle 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.
Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood. 1996;87: 4235-4244.
Kinoshita S, Yoshioka A, Park YD, et al. Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura. Int J Hematol. 2001;74: 101-108.
Barbot J, Costa E, Guerra M, et al. Ten years of prophylactic treatment with fresh-frozen plasma in a child with chronic relapsing thrombotic thrombocytopenic purpura as a result of a congenital deficiency of von Willebrand factor-cleaving protease. Br J Haematol. 2001;113: 649-651.
Furlan M, Lmmle B. Aetiology and pathogenesis of thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome: the role of von Willebrand factor-cleaving protease. Best Pract Res Clin Haematol. 2001;14: 437-454.
Gerritsen HE, Turecek PL, Schwarz HP, et al. 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: 1386-1389.
Bhm M, Vigh T, Scharrer I. Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (ADAMTS13). Ann Hematol. 2002;81: 430-435.
Obert B, Tout H, Veyradier A, et al. Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to VWF. Thromb Haemost. 1999;82: 1382-1385.
Pimstone NR. Renal degradation of hemoglobin. Semin Hematol. 1972;9: 31-42.(Jan-Dirk Studt, Johanna A)
Baxter BioScience, Orth, Austria
the Department of Pediatrics, University Hospital, Tübingen, Germany.
Abstract
Severe ADAMTS13 deficiency in thrombotic thrombocytopenic purpura (TTP) is either constitutional and caused by ADAMTS13 mutations, or acquired and most often due to ADAMTS13 inhibitory autoantibodies. In strongly hemolytic serum of a pediatric patient, diagnosed with TTP postmortem, ADAMTS13 activity was less than 3%. Both parents had an ADAMTS13 activity of approximately 50%. Sequencing of the ADAMTS13 gene revealed an intronic 687-2A>G substitution affecting exon 7, homozygous in the propositus and heterozygous in both parents, confirming constitutional ADAMTS13 deficiency. ADAMTS13 activity of normal plasma was inhibited by incubation with the propositus' serum, suggesting alloantibody formation to ADAMTS13. However, immunoglobulin purified from serum had no ADAMTS13 inhibitory effect, whereas the immunoglobulin-depleted hemolytic serum inhibited ADAMTS13 activity of normal plasma, suggesting an inhibitory effect of hemolysis products. Incubation of hemoglobin, recombinant and from lysed erythrocytes, with normal plasma revealed an ADAMTS13 inhibitory effect at hemoglobin concentrations of 2 g/L or higher.
Introduction
Severe deficiency of ADAMTS13 activity (< 5% of normal plasma) is a strong risk factor for thrombotic thrombocytopenic purpura (TTP).1-3 There are 2 pathogenetic mechanisms: homozygous or compound heterozygous mutations of the ADAMTS13 gene in congenital TTP,4-10 and inhibition of ADAMTS13 by autoantibodies in acquired TTP.2,3,11 Nonneutralizing autoantibodies,12 possibly leading to increased clearance of ADAMTS13 from the circulation, or interfering with its interaction with endothelial cells13 or the von Willebrand factor (VWF) A1/A3 domains,14 may be additional mechanisms.
Investigation of premortem serum of a pediatric TTP patient revealed a severe ADAMTS13 deficiency. Sequencing of the ADAMTS13 gene identified a homozygous mutation confirming constitutional ADAMTS13 deficiency. Surprisingly, the patient's severely hemolytic serum had a pronounced ADAMTS13 inhibitory effect that was not attributable to purified immunoglobulin (Ig) but caused by hemolysis products present in the sample. Subsequent experiments addressed an ADAMTS13 inhibitory effect of hemoglobin.
Study design
Patient
The propositus, a Turkish boy,15 had suffered from episodes of severe Coombs-negative hemolytic anemia and thrombocytopenia since birth. He died at the age of 7 years from a severe attack of this disease, until then classified as atypical Evans syndrome. Autopsy revealed microvascular occlusions by fibrin-poor platelet thrombi, suggesting the diagnosis of TTP. Strongly hemolytic serum from whole blood obtained before death and stored at 4°C was shipped frozen to our laboratory. The serum had a hemoglobin concentration of 17 g/L (1.7 g/dL), probably due to in vitro hemolysis.
Informed consent for publication was obtained from the parents; the study was conducted according to the guidelines on research on human subjects of the responsible Ethics Committee (Kantonale Ethikkommission, Bern).
ADAMTS13 activity and routine inhibitor screening
ADAMTS13 activity was determined by immunoblotting of purified VWF substrate degraded by BaCl2-activated ADAMTS13 in serum or plasma. For inhibitor screening, the residual ADAMTS13 activity of pooled normal human plasma (NHP) was measured after 1:1 (vol/vol) incubation with patient serum/plasma. Assays were performed as described.2,16
Inhibition of ADAMTS13 activity by hemoglobin
There were 2 solutions of concentrated hemoglobin prepared. Solution 1 consisted of recombinant human hemoglobin (Baxter, Boulder, CO) 50 g/L in 0.15 M NaCl, 5 mM sodium phosphate, 0.03% TWEEN 80. Solution 2 was obtained from lysed washed erythrocytes. Citrated blood from a healthy donor was centrifuged for 10 minutes at 4000g, and the supernatant plasma and buffy coat were discarded. The sediment was washed 3 times with physiologic saline, and incubated 1:3 (vol/vol) with distilled water for 10 minutes at 37°C. After centrifugation for 10 minutes at 4000g, the supernatant solution contained 84 g/L hemoglobin.
Inhibition of ADAMTS13 activity by hemoglobin was assessed by routine ADAMTS13 inhibitor assay.16 NHP was incubated 1:1 (vol/vol) for 2 hours at 37°C with hemoglobin solution 1 or 2 prediluted in 0.01 M tris(hydroxymethyl)aminomethane, 0.15 M NaCl, pH 7.4 (TBS). Final hemoglobin concentrations after addition to NHP ranged from 0.5 to 40 g/L.
Preliminary experiments addressed a complexation of divalent metal ions required for ADAMTS13 activity, such as calcium or zinc,17,18 as a hypothetical mechanism by which hemoglobin might inhibit ADAMTS13. NHP was incubated 1:1 (vol/vol) with recombinant hemoglobin, 20 g/L, for 1 hour at 37°C. This mixture was diluted 1:10 with TBS and activated with increasing final concentrations of BaCl2 between 10 and 75 mM.
Purification of total Ig
Total Ig was purified from the propositus' serum by adsorption to Ig-Therasorb (Miltenyi Biotech, Teterow, Germany; Sepharose CL-4B coupled with polyclonal sheep antibodies to human Ig). The purified Ig and the Ig-depleted serum were concentrated to original serum levels by centrifugation through filter membranes (Biomax; Millipore, Bedford, MA; molecular weight cut-off, 30 kDa).
Investigation of the ADAMTS13 gene
Amplification and sequencing of polymerase chain reaction (PCR) products were performed as described.6 SpliceView program (Institute of Biomedical Technologies, Segrate, Italy) was used for predicting alternative splice sites resulting from the detected mutation.
Results and discussion
ADAMTS13 activity was less than 3% in premortem serum of the propositus, and approximately 50% in plasma of both parents (Figure 1A). Sequencing of all 29 exons of the ADAMTS13 gene, including their intron/exon boundaries, revealed a point mutation (687-2A>G) at the 3' splice acceptor site of intron 6 affecting exon 7, between amino acids 228 to 229, homozygous in the propositus and heterozygous in both parents. We hypothesize that this mutation could result in 2 alternative splice sites, causing either a frameshift and premature termination of the polypeptide, or an in-frame insertion of 8 amino acids. These findings confirmed constitutional ADAMTS13 deficiency with hereditary TTP in the propositus, misdiagnosed as atypical Evans syndrome until autopsy. His fatal course illustrates the importance of rapid and comprehensive diagnostics in patients suffering from congenital thrombotic microangiopathies including determination of ADAMTS13 activity and ADAMTS13 inhibitors because plasma infusion is a highly effective treatment.19-21
Pronounced inhibition of the ADAMTS13 activity of NHP was observed after incubation with an equal volume of the propositus' hemolytic serum, corresponding to a titer of 1 to 2 Bethesda units (BU)/mL and suggested alloantibody formation to ADAMTS13 triggered by repeated transfusions of blood products containing ADAMTS13. However, total Ig purified from the propositus' serum had no ADAMTS13 inhibitory effect (Figure 1B). In contrast, the Ig-depleted hemolytic serum inhibited ADAMTS13 activity of NHP to a similar extent as the original serum. This indicated that the observed inhibition was not caused by antibodies to ADAMTS13 but was related to other factors present in the sample, possibly originating from lysed erythrocytes.
Subsequent experiments addressed a possible ADAMTS13 inhibitory capacity of hemoglobin. NHP was incubated with hemoglobin, either recombinant or purified from lysed erythrocytes at various concentrations. Slight inhibition of the ADAMTS13 activity of NHP ( 1 BU/mL) was observed after incubation with hemoglobin at a final concentration of 2 g/L (Figure 1C). Moderate inhibition (1-2 BU/mL) occurred at 5 g/L, and strong inhibition ( 2 BU/mL) occurred at 10 g/L or higher. The ADAMTS13 inhibitory effect was similar for recombinant and erythrocyte-derived hemoglobin, indicating that it can be linked specifically to the hemoglobin molecule.
To exclude an artifact related to our assay, NHP was incubated 1:1 (vol/vol) with hemoglobin solutions 1 and 2 (final hemoglobin concentrations, 10 g/L and 25 g/L each) for 2 hours at 37°C. Identical aliquots were investigated for their ADAMTS13 activity by 3 other laboratories using different assays.22-24 All laboratories found a severe or borderline severe ADAMTS13 deficiency (< 5% or 5%-9% of the normal), instead of an activity of 50% corresponding to the proportion of NHP in the mixture.
The ADAMTS13 inhibitory capacity of hemoglobin depended upon incubation temperature and time. ADAMTS13 activity of NHP was completely inhibited after incubation with 30 g/L hemoglobin for 3 hours at 37°C but only reduced to 25% at 4°C. Incubation for 5 minutes to 2 hours revealed mild inhibition ( 1 BU/mL) after 5 minutes, moderate inhibition (1-2 BU/mL) after 15 to 30 minutes, and strong inhibition ( 2 BU/mL) after 1 to 2 hours (not shown).
ADAMTS13 inhibition by hemoglobin was attenuated, but not completely reverted by the addition of increasing BaCl2 concentrations up to 40 mM.
Inhibition of ADAMTS13 activity occurred at hemoglobin concentrations potentially found in strong intravascular hemolysis, as observed in incompatible erythrocyte transfusion, immunemediated hemolytic anemias, or others.25 These conditions are not usually associated with TTP, and it is unclear whether short-lived elevation of free hemoglobin in plasma will lead to a clinically relevant ADAMTS13 inhibition. In our case, the serum hemoglobin concentration of 17 g/L probably resulted from in vitro hemolysis in stored native whole blood. Nevertheless, some practical conclusions can be drawn from our observations. Marked hemolysis and the presence of hemoglobin apparently influence most of the current ADAMTS13 assays.2,22-24 Lowering of ADAMTS13 activity values should be expected under these circumstances and inhibitor screening may lead to the erroneous assumption of ADAMTS13 inhibitory antibodies. Whether the ADAMTS13 inhibitory capacity of hemoglobin is of any physiologic or pathophysiologic relevance (eg, in severe intravascular hemolysis) will need further study.
Acknowledgements
We thank Dr W. Bhm (Miltenyi Biotech, Teterow, Germany) for providing Ig-Therasorb columns, and Drs U. Budde (Laboratory Keeser and Arndt, Hamburg, Germany), M. Bhm (University Hospital Frankfurt, Germany), and J.-P. Girma (INSERM U.143, Le Kremlin-Bicêtre, France) for performing confirmatory determinations of ADAMTS13 activity.
Footnotes
Prepublished online as Blood First Edition Paper, September 14, 2004; DOI 10.1182/blood-2004-06-2096.
Supported by grants from the Swiss National Foundation for Scientific Research (32-66756.01; B.L.) and from the Fondation pour la Recherche sur l'Arteriosclerose et la Thrombose (J.A.K.H.).
J.-D.S. and J.A.K.H. 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
Furlan M, Robles R, Solenthaler M, et al. Deficient activity of von Willebrand factor-cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura. Blood. 1997;89: 3097-3103.
Furlan M, Robles R, Galbusera M, et al. Von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolyticuremic syndrome. N Engl J Med. 1998;339: 1578-1584.
Tsai HM, Lian EC. Antibodies to von Willebrand factor-cleaving protease in acute thrombotic thrombocytopenic purpura. N Engl J Med. 1998; 339: 1585-1594.
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.
Kokame K, Matsumoto M, Soejima K, et al. Mutations and common polymorphisms in ADAMTS13 gene responsible for von Willebrand factor-cleaving protease activity. Proc Natl Acad Sci U S A. 2002;99: 11902-11907.
Antoine G, Zimmermann K, Plaimauer B, et al. ADAMTS13 gene defects in 2 brothers with constitutional thrombotic thrombocytopenic purpura and normalization of von Willebrand factor-cleaving protease activity by recombinant human ADAMTS13. Br J Haematol. 2003;120: 821-824.
Schneppenheim R, Budde U, Oyen F, et al. Von Willebrand factor cleaving protease and ADAMTS13 mutations in childhood TTP. Blood. 2003;101: 1845-1850.
Assink K, Schiphorst R, Allford S, et al. Mutation analysis and clinical implications of von Willebrand factor-cleaving protease deficiency. Kidney Int. 2003;63: 1995-1999.
Matsumoto M, Kokame K, Soejima K, et al. Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome. Blood. 2004;103: 1305-1310.
Veyradier A, Lavergne JM, Ribba AS, et al. Ten candidate ADAMTS13 mutations in 6 French families with congenital thrombotic thrombocytopenic purpura (Upshaw-Schulman syndrome). J Thromb Haemost. 2004;2: 424-429.
Furlan M, Robles R, Solenthaler M, et al. Acquired deficiency of von Willebrand factor-cleaving protease in a patient with thrombotic thrombocytopenic purpura. Blood. 1998;91: 2839-2846.
Scheiflinger F, Knbl P, Trattner B, et al. Non-neutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS13) in a patient with thrombotic thrombocytopenic purpura. Blood. 2003;102: 3241-3243.
Dong JF, Moake JL, Nolasco L, et al. ADAMTS13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood. 2002;100: 4033-4039.
Dong JF, Moake JL, Bernardo A, et al. ADAMTS13 metalloprotease interacts with the endothelial cellderived ultra-large von Willebrand factor. J Biol Chem. 2003;278: 29633-29639.
Studt JD, Kremer Hovinga JA, Alberio L, et al. Von Willebrand factor-cleaving protease (ADAMTS13) activity in thrombotic microangiopathies: diagnostic experience 2001/2002 of a single research laboratory. Swiss Med Wkly. 2003;133: 325-332.
Studt JD, Bhm M, Budde U, et al. Measurement of von Willebrand factor-cleaving protease (ADAMTS13) activity in plasma: a multicenter comparison of different assay methods. J Thromb Haemost. 2003;1: 1882-1887.
Furlan M, Robles R, Lmmle 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.
Tsai HM. Physiologic cleavage of von Willebrand factor by a plasma protease is dependent on its conformation and requires calcium ion. Blood. 1996;87: 4235-4244.
Kinoshita S, Yoshioka A, Park YD, et al. Upshaw-Schulman syndrome revisited: a concept of congenital thrombotic thrombocytopenic purpura. Int J Hematol. 2001;74: 101-108.
Barbot J, Costa E, Guerra M, et al. Ten years of prophylactic treatment with fresh-frozen plasma in a child with chronic relapsing thrombotic thrombocytopenic purpura as a result of a congenital deficiency of von Willebrand factor-cleaving protease. Br J Haematol. 2001;113: 649-651.
Furlan M, Lmmle B. Aetiology and pathogenesis of thrombotic thrombocytopenic purpura and haemolytic uraemic syndrome: the role of von Willebrand factor-cleaving protease. Best Pract Res Clin Haematol. 2001;14: 437-454.
Gerritsen HE, Turecek PL, Schwarz HP, et al. 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: 1386-1389.
Bhm M, Vigh T, Scharrer I. Evaluation and clinical application of a new method for measuring activity of von Willebrand factor-cleaving metalloprotease (ADAMTS13). Ann Hematol. 2002;81: 430-435.
Obert B, Tout H, Veyradier A, et al. Estimation of the von Willebrand factor-cleaving protease in plasma using monoclonal antibodies to VWF. Thromb Haemost. 1999;82: 1382-1385.
Pimstone NR. Renal degradation of hemoglobin. Semin Hematol. 1972;9: 31-42.(Jan-Dirk Studt, Johanna A)