Background Thrombotic thrombocytopenic purpura is a potentiallyfatal disease characterized by widespread platelet thrombi inthe microcirculation. In the normal circulation, von Willebrandfactor is cleaved by a plasma protease. We explored the hypothesisthat a deficiency of this protease predisposes patients withthrombotic thrombocytopenic purpura to platelet thrombosis.
Methods We studied the activity of von Willebrand factor–cleavingprotease and sought inhibitors of this protease in plasma frompatients with acute thrombotic thrombocytopenic purpura, patientswith other diseases, and normal control subjects. We also investigatedthe effect of shear stress on the ristocetin cofactor activityof purified von Willebrand factor in the cryosupernatant fractionof the plasma samples.
Results Thirty-nine samples of plasma from 37 patients withacute thrombotic thrombocytopenic purpura had severe deficiencyof von Willebrand factor–cleaving protease. No deficiencywas detected in 16 samples of plasma from patients with thromboticthrombocytopenic purpura in remission or in 74 plasma samplesfrom normal subjects, randomly selected hospitalized patientsor outpatients, or patients with hemolysis, thrombocytopenia,or thrombosis from other causes. Inhibitory activity againstthe protease was detected in 26 of the 39 plasma samples (67percent) obtained during the acute phase of the disease. Theinhibitors were IgG antibodies. Shear stress increased the ristocetincofactor activity of von Willebrand factor in the cryosupernatantof plasma samples obtained during the acute phase, but decreasedthe activity in cryosupernatant of plasma from normal subjects.
Conclusions Inhibitory antibodies against von Willebrand factor–cleavingprotease occur in patients with acute thrombotic thrombocytopenicpurpura. A deficiency of this protease is likely to have a criticalrole in the pathogenesis of platelet thrombosis in this disease.
Thrombotic thrombocytopenic purpura is characterized by widespreadplatelet thrombi in arterioles and capillaries.1,2 Despite therapeuticadvances,3 the age-adjusted mortality associated with the diseasenearly tripled from 1971 to 1991.4 Among those who survive theacute phase, relapse is not uncommon.5 Infection with the humanimmunodeficiency virus (HIV) and other retroviruses may contributeto the increased frequency of the disease.6
Both endothelial-cell injury7,8 and intravascular platelet aggregation9have been implicated in the pathogenesis of thrombotic thrombocytopenicpurpura. Immunohistologic studies have demonstrated abundantvon Willebrand factor in the thrombotic lesions.10 Abnormalmultimers of von Willebrand factor, initially described in patientswith chronic relapsing thrombotic thrombocytopenic purpura,11are also common in the acute phase.12 However, the type of abnormalityvaries; many patients have fewer large multimers than normal,whereas others have normal levels of large multimers or evenunusually large forms.
Von Willebrand factor is secreted from endothelial cells asan extra large polymer of a polypeptide joined by disulfidebonds13 and cleaved in the circulation at the peptide bond betweentyrosine at position 842 and methionine at position 84314 bya 200-kd plasma metalloproteinase.15,16 Cleavage by the enzymedecreases the size of von Willebrand factor to dimers of 176-kdand 140-kd fragments.15,17 The enzyme, which is present in thecryosupernatant fraction of the plasma, requires a calcium orzinc cation for its activity.18 It is inhibited by tetracyclinesbut resistant to batimastat, a synthetic matrix metalloproteinase–specificinhibitor.18
In plasma the protease has little effect on von Willebrand factorunless the factor is unfolded by high levels of shear stressor other means.17 This suggests that in patients with thromboticthrombocytopenic purpura, the multimers of von Willebrand factorought to be relatively small, because the abnormal shear stresscaused by platelet thrombi in the microcirculation should enhanceproteolysis of von Willebrand factor. However, in some patientswith acute thrombotic thrombocytopenic purpura, the size ofthe multimers is normal or very large. These findings pointto a defect in the proteolysis of von Willebrand factor. Sucha defect was suspected11 and recently described19 in patientswith the chronic relapsing form of thrombotic thrombocytopenicpurpura. In this study, we investigated the activity of vonWillebrand factor–cleaving protease in patients with acuteepisodes of thrombotic thrombocytopenic purpura and the mechanismsby which a deficiency of the protease might lead to plateletthrombosis.
Methods
Subjects
The diagnosis of acute thrombotic thrombocytopenic purpura wasbased on the standard criteria3 of thrombocytopenia (a plateletcount of less than 100x103 per cubic millimeter), microangiopathichemolytic anemia (as indicated by erythrocyte fragmentationon peripheral-blood smears) with a negative Coombs' test, andthe absence of identifiable causes of these abnormalities, suchas disseminated intravascular coagulation, cancer, or preeclampsia.None of the patients had a serum creatinine concentration ofmore than 4 mg per deciliter (354 µmol per liter) or requiredrenal dialysis. Thirty patients were treated at our institutions,and seven were referred from other hospitals. Six patients wereknown to have had prior episodes of the disease or additionalepisodes after the collection of plasma samples. Two of thepatients had HIV infection, one had a history of systemic lupuserythematosus, and one had rheumatoid factor and antibodiesto DNA.
Blood samples were collected in tubes containing citrate anticoagulantbefore or after the institution of plasma-exchange therapy orduring remission when platelet counts were normal and stable.We also examined plasma samples from 35 normal subjects or outpatientsor hospitalized patients without thrombotic thrombocytopenicpurpura; 21 patients with miscellaneous autoimmune or blooddisorders, including disseminated intravascular coagulopathy(1 patient), autoimmune thrombocytopenia (2 patients), drug-inducedthrombocytopenia (2 patients), the HELLP syndrome (hemolysis,elevated liver enzymes, and low platelet count in associationwith preeclampsia, 1 patient), deep-vein thrombosis (1 patient),lupus anticoagulant with thrombosis (1 patient), myeloproliferativedisorders (4 patients), sickle cell anemia (5 patients), hemophiliaA (1 patient), and von Willebrand's disease (3 patients); and18 patients with heparin-induced thrombocytopenia. Sixteen ofthe 18 plasma samples from patients with heparin-induced thrombocytopeniawere kindly provided by Dr. G.P. Visentin of the Blood Centerof Southeastern Wisconsin in Milwaukee. The heparin-inducedserotonin-release assay was positive in all 17 of the plasmasamples that were tested. Eight of the patients with heparin-inducedthrombocytopenia had thrombotic complications. The investigationprotocol was approved by the institutional review boards ofthe participating centers.
Control plasma consisted of pooled normal plasma used in theclinical coagulation laboratory. Plasma was separated from wholeblood by a two-stage centrifugation15 and stored at –70°C.Cryosupernatant fractions were obtained by centrifugation at4°C from plasma samples thawed on ice.15 Aliquots of plasmafrom two patients were also studied before being frozen, afterhigh-speed centrifugation at 30,000xg for 60 minutes and afterpassage through 0.2-µm filters.
Preparation of von Willebrand Factor
Von Willebrand factor was purified from pooled normal plasmaby precipitation with glycine salts and gel filtration.17 Thefractions eluted from the chromatographic column that containedthe largest multimers at a concentration of 150 to 250 µgper milliliter were exposed to guanidine (1.5 mol per liter)and used as the substrate for the protease assay.
Multimers of von Willebrand factor were analyzed by sodium dodecylsulfate–agarose-gel electrophoresis as previously described.18The size of the multimers was represented by the distance fromthe starting point of electrophoresis to the peak of multimerdistribution, as determined by densitometry, divided by thepeak distance of control von Willebrand factor (the normalizedpeak distance).18 An increase in the normalized peak distanceindicated a decrease in the size of the multimers. Concentrationsof von Willebrand factor were determined by an enzyme-linkedimmunosorbent assay as described previously.20
Assay of Protease Activity
The assay of von Willebrand factor–cleaving protease activityin plasma was based on the generation, from purified von Willebrandfactor, of dimers of 176-kd and 140-kd fragments, which appearedas 350-kd and 200-kd bands, respectively, on sodium dodecylsulfate–polyacrylamide-gel electrophoresis.15 Plasma sampleswere diluted 1:3 with a TRIS saline buffer containing 50 mmolof TRIS per liter and 50 mmol of sodium chloride per liter (pH8.0) in the presence or absence of 5 mmol of EDTA per liter.Von Willebrand factor was added to the test samples at a dilutionof 1:10. After a 60-minute incubation at 37°C, the reactionwas terminated by the addition of a 1:5 dilution of a buffercontaining 0.125 mmol of TRIS per liter (pH 6.8), 20 g of sodiumdodecyl sulfate per liter, 5 percent glycerol (vol/vol), and5 mmol of EDTA per liter.18 The optical intensity of von Willebrandfactor fragments, after immunoblotting and autoradiography,was determined by scanning densitometry.18 The difference betweenthe intensity of the 350-kd band in the reaction mixture withoutEDTA and that in the mixture with EDTA represented the amountgenerated by the protease in the test sample. The 350-kd bandgenerated by serially diluted samples of control plasma, expressedas a percentage of that generated in an undiluted sample ofcontrol plasma, showed a linear correlation with the plasmaconcentration. Therefore, the optical intensity of the 350-kdband generated in the assay was used to represent the proteaseactivity in the test sample.
Sixty-seven of the plasma samples from control subjects andpatients with thrombotic thrombocytopenic purpura were obtainedat the University of Miami and were randomly distributed ina blinded fashion to the laboratory where the assay was performed.Many of the plasma samples had been frozen at –70°C.Analysis of stored samples of control plasma showed that freezingfor at least 20 years did not adversely affect the proteaseactivity.
Preparation of IgG
IgG was isolated from plasma with staphylococcal protein A–agarosecolumns equilibrated in a buffer of 50 mmol of TRIS per liter(pH 8.0) and 0.5 mol of sodium chloride per liter. IgG was elutedwith a buffer of 0.1 mol of glycine per liter (pH 2.8) and 0.5mol of sodium chloride per liter (pH 2.8). The eluates, whichwere immediately neutralized by a 1:10 dilution of TRIS (1 molper liter, pH 8.0), were dialyzed extensively against the TRISsaline buffer before being concentrated on polyethylene glycol20,000. Protein concentrations were determined by a Coomassiebrilliant blue dye–binding assay.15 Bovine immune globulinwas used as the reference.
Detection of Inhibitor by Mixing Studies
For the mixing studies, plasma samples were incubated at 56°Cfor 60 minutes to inactivate the protease. Control plasma wasthen incubated at 37°C for 30 minutes with an equal volumeof the test plasma or IgG. The protease activity in the mixturewas expressed as a percentage of that in control plasma incubatedwith heated control plasma or control IgG.
In IgG-neutralization studies, IgG antibody was incubated withvarious concentrations of rabbit IgG antibodies against humanIgG Fab region or Fc region or IgM mu chain (Sigma Chemical,St. Louis) before its inhibitory activity was determined bymixing studies.
Mixing studies were also performed with various dilutions ofsome plasma samples from patients with acute thrombotic thrombocytopenicpurpura. Ten microliters of control plasma was mixed with 30µl of undiluted or serially diluted plasma samples fromthe patients. The von Willebrand factor–cleaving proteaseactivity in 10 µl of control plasma incubated with 30µl of undiluted or serially diluted heated control plasmawas used as the reference.
Effect of Shear Stress on von Willebrand Factor
Purified von Willebrand factor that contained large multimerswas dissolved to a final concentration of 5 to 10 µg permilliliter in the cryosupernatant fraction of control plasmaor plasma samples from patients with thrombotic thrombocytopenicpurpura. Aliquots of von Willebrand factor were forced at controlledflow rates through stainless-steel tubing (length, 610 cm; internalradius, 0.0254 cm) at room temperature with a syringe pump.17In this device, the aliquots were exposed to the highest rateof shear stress of 6404 per second for 15 seconds. The ristocetincofactor activity in the aliquots was determined in triplicatewithin 60 minutes after the procedure as previously described.18The values were compared with those of control aliquots thathad not been exposed to shear stress with the use of Student'st-test.
Results
Protease Activity
We studied 39 plasma samples obtained from 37 patients duringacute episodes of thrombotic thrombocytopenic purpura (Figure 1A).Two of the patients were studied during two acute episodes,at least two years apart. Thirty-eight samples had no detectablevon Willebrand factor–cleaving protease activity, whereasone sample had a level that was 3 percent of the control value.The protease activity of 23 samples obtained after plasma-exchangetherapy was instituted ranged from 0 to 70 percent (mean [±SD],16±20 percent). As compared with the protease activityof plasma samples from subjects without thrombotic thrombocytopenicpurpura (Figure 1B), 22 of these samples had lower-than-normalactivity. Samples with protease activity that was more than57 percent of the value measured in control plasma samples wereconsidered normal.
Figure 1. Von Willebrand Factor–Cleaving Protease Activity in Plasma Samples from 37 Patients with Thrombotic Thrombocytopenic Purpura and 74 Control Subjects.
The protease activity in each sample was reflected by the intensity of the 350-kd band generated from purified von Willebrand factor and expressed as a percentage of the activity in control plasma. Panel A shows the protease activity of 39 plasma samples obtained during acute episodes of thrombotic thrombocytopenic purpura (TTP) before plasma-exchange therapy, 23 samples obtained after therapy, and 16 samples obtained during remission. Diamonds represent five samples, and circles one sample. Panel B shows the protease activity of plasma samples obtained from 35 randomly selected subjects without thrombotic thrombocytopenic purpura, 21 patients with miscellaneous autoimmune or blood disorders, and 18 patients with heparin-induced thrombocytopenia. Panel C shows the protease activity of plasma samples obtained from seven patients with thrombotic thrombocytopenic purpura during both an acute episode and remission. Panel D shows a composite immunoblot of 200-kd, 350-kd, and larger fragments generated from purified von Willebrand factor in control plasma (lanes 1 and 2) but not in plasma samples from two patients with acute thrombotic thrombocytopenic purpura (lanes 3, 4, 5, and 6). Each sample was assayed in duplicate. EDTA was absent from the buffer in lanes 1, 3, and 5 and present in lanes 2, 4, and 6.
Plasma samples from subjects without thrombotic thrombocytopenicpurpura were also studied (Figure 1B). Protease activity rangedfrom 68 to 126 percent of the value in control samples (mean,102±15 percent) in 35 samples from normal subjects oroutpatients or hospitalized patients without thrombotic thrombocytopenicpurpura, from 45 to 145 percent (mean, 101±25 percent)in 21 samples from patients with miscellaneous autoimmune orblood disorders, and from 48 to 160 percent (mean, 106±33percent) in 18 samples from patients with heparin-induced thrombocytopenia.These values did not differ significantly from those measuredin 18 plasma samples obtained from patients with thromboticthrombocytopenic purpura in remission (range, 48 to 150 percent;mean, 91±28 percent) (Figure 1A).
Seven patients with thrombotic thrombocytopenic purpura werestudied during both an acute episode and remission (Figure 1C).In each case, the protease activity rose to a normal or a nearlynormal level at remission. Figure 1D shows the absence of proteaseactivity in plasma samples from two patients. Twenty-six of39 plasma samples (67 percent) from patients with acute thromboticthrombocytopenic purpura had poorly defined bands at 300 kd(as shown in Figure 1D for Patient 2) that were not seen insubjects without thrombotic thrombocytopenic purpura. Thesebands were not derived from the added von Willebrand factor,since they were present in the unadulterated plasma.
Multimer analysis showed that after incubation with the cryosupernatantof control plasma, von Willebrand factor was cleaved to smallerforms, whereas it remained unchanged after incubation with plasmasamples from patients with thrombotic thrombocytopenic purpura(data not shown).
In one patient, plasma samples were obtained from the time ofan acute episode until remission and analyzed. Plasma samplesobtained on days 1, 2, 5, and 22 had protease activity thatwas 0, 30, 65, and 95 percent of the control value, respectively,with corresponding platelet counts of 2x106, 2x106, 45x106,and 212x106 per cubic millimeter. The extraneous 300-kd bandswere present on day 1 but disappeared in subsequent samples.High-speed centrifugation or filtration to remove platelet microparticlesfrom the plasma samples did not affect the protease activity,nor did storage at –70°C.
Inhibitors of the Protease
Mixing studies were performed to detect inhibitors of von Willebrandfactor–cleaving protease (Figure 2A). The protease activityin mixtures of control plasma and plasma samples from 21 subjectswith miscellaneous autoimmune or blood disorders ranged from70 to 125 percent of the value in control samples (mean, 98±14percent). Of the 39 samples obtained from patients during acuteepisodes of thrombotic thrombocytopenic purpura, 26 (67 percent)reduced the level of protease activity in control plasma bymore than 3 SD. No inhibitory activity was detected in 16 samplesobtained during a remission of the disease; the protease activityranged from 71 to 152 percent (mean, 103±19 percent).Proteolysis in control plasma was inhibited by incubation witha plasma sample from a patient with thrombotic thrombocytopenicpurpura (lanes 1 and 2 in Figure 2B) but not by incubation withheated control plasma (lanes 3 and 4 in Figure 2B).
Figure 2. Detection of an Inhibitor of von Willebrand Factor–Cleaving Protease.
In Panel A, von Willebrand factor–cleaving protease activity was measured in a mixture of control plasma and an equal volume of a plasma sample from 21 patients with miscellaneous autoimmune or blood disorders, 37 patients with acute thrombotic thrombocytopenic purpura (39 samples), and 16 patients with thrombotic thrombocytopenic purpura in remission. Panel B shows the results of autoradiography. Von Willebrand factor was cleaved in control plasma incubated with heated control plasma (lanes 3 and 4), whereas there was minimal cleavage in control plasma incubated with a plasma sample from a patient with thrombotic thrombocytopenic purpura (TTP) (lanes 1 and 2). EDTA was absent from the buffer in lanes 1 and 3 and present in lanes 2 and 4. In Panel C, protease activity was measured in 3:1 mixtures of various dilutions of a plasma sample from a patient with thrombotic thrombocytopenic purpura and control plasma.
The plasma samples from patients with thrombotic thrombocytopenicpurpura that reduced the level of protease activity in controlplasma by less than 3 SD but more than 2 SD were further investigated(Figure 2C). When these plasma samples were present in an equalvolume with control plasma, the protease activity was 59 percentof that in the control mixture. When the samples were presentat a ratio of 3 to 1, the activity was 22 percent of that inthe control mixture.
The inhibitory activity in plasma samples from patients withthrombotic thrombocytopenic purpura was not affected by dialysis,was stable at 56°C, and was resistant to serine or cysteineprotease inhibitors such as isoflurophate, phenylmethylsulfonylfluoride, aprotinin, leupeptin, iodoacetamide, and N-ethylmaleimide.However, it was abolished by passage through a protein A–agarosecolumn, suggesting that the inhibition was mediated by IgG.
The IgG antibody isolated from 12 samples of plasma from patientswith acute thrombotic thrombocytopenic purpura and 11 samplesfrom patients after plasma-exchange therapy was instituted wascompared with the inhibitory activity of the patients' plasma.The IgG was isolated in a quantitative manner: 1 ml of plasmawas loaded onto 1 ml of protein–A agarose beads, and IgGantibody was eluted with 4 ml of glycine buffer and concentratedto a final volume of 0.5 ml. The concentrations of the IgG antibodyin the samples ranged from 8.6 to 39.0 mg per milliliter. Theprotease activity in the mixture of control plasma and the IgGfrom the patients was plotted against the protease activityin the mixture of control plasma and the plasma from which theIgG antibody had been isolated (Figure 3). The positive correlation(correlation coefficient, 0.91) indicated that the inhibitoryactivity of plasma samples from the patients with thromboticthrombocytopenic purpura was mediated by the IgG.
Figure 3. Inhibitory Activity of IgG from 12 Patients with Acute Thrombotic Thrombocytopenic Purpura (TTP) and from 11 Patients after Plasma-Exchange Therapy Was Instituted as a Function of the Inhibitory Activity of Plasma Samples from Which IgG Was Isolated.
IgG antibody was isolated from plasma samples from the patients and mixed with control plasma, and protease activity was plotted against the protease activity measured in the mixture of control plasma and the patients' plasma samples.
Figure 4A depicts the concentration-dependent inhibition ofprotease activity by IgG antibody isolated from one of theseplasma samples. At a concentration of 2 mg per milliliter, theIgG antibody inhibited the protease activity in control plasmaby 50 percent. Control IgG exhibited no inhibitory activity.
Figure 4. Inhibition of von Willebrand Factor–Cleaving Protease Activity by an IgG Antibody Isolated from Plasma from a Patient with Thrombotic Thrombocytopenic Purpura (TTP) (Panel A) and Neutralization of the Inhibitory Activity by the Antibody against Human Fab Region (Panel B).
In Panel A, the protease activity of control plasma and IgG antibody isolated from the patient was plotted against the final IgG concentration in the mixture (log scale). The protease activity of the mixture of control plasma and control IgG is also shown. In Panel B, the protease activity of a mixture of control plasma, IgG antibody isolated from the patient, and IgG antibodies to the Fab region (anti-Fab) or the Fc region (anti-Fc) was plotted against the ratio of anti-Fab or anti-Fc to IgG in the mixture (log scale).
To confirm that the inhibition involved an antigen–antibodyinteraction, we treated the patient's IgG with rabbit IgG antibodiesagainst human IgG Fab region or Fc region before the assay (Figure 4B).Only the antibody against the Fab region abolished theinhibitory activity of the patient's IgG antibody, and thiswas true in all six IgG preparations that were tested. In separateexperiments, the inhibitory activity of the IgG antibody fromthe patient's plasma was not affected by the rabbit antibodyagainst human IgM mu chain.
Effects of Shear Stress on von Willebrand Factor
Von Willebrand factor was dissolved in the cryosupernatant ofcontrol plasma or a plasma sample from a patient with thromboticthrombocytopenic purpura, and ristocetin cofactor activity wasstudied before and after shear stress (Figure 5A and Figure 5B).The size and proteolysis of multimers of von Willebrandfactor were also investigated. Cryosupernatants that were depletedof large and intermediate-sized multimers had no ristocetincofactor activity, and exposure to shear stress did not changethe concentration of von Willebrand factor (data not shown).Shear stress at a rate of 6404 per second decreased the sizeof the multimers and increased the number of cleaved fragmentsof von Willebrand factor only in control plasma (Figure 5A andFigure 5B). Only the von Willebrand factor in the cryosupernatantof control plasma had an increase in both the normalized peakdistance (Figure 5C) and von Willebrand factor proteolytic fragments(Figure 5D) that was dependent on the level of shear stress.
Figure 5. Effects of Shear Stress on the Proteolysis of Purified von Willebrand Factor Multimers.
Purified von Willebrand factor was dissolved in the cryosupernatant of control plasma or a plasma sample from a patient with thrombotic thrombocytopenic purpura (TTP). Panel A shows the sizes of the von Willebrand factor multimers on agarose-gel electrophoresis in the cryosupernatants in the presence and absence of shear stress. Panel B shows the frequency of the cleaved 350-kd and 200-kd fragments on polyacrylamide-gel electrophoresis and immunoblotting in the presence and absence of shear stress. Shear stress at a rate of 6404 per second decreased the size of the multimers and increased the numbers of cleaved fragments only in control cryosupernatant. The von Willebrand factor in the cryosupernatant of control plasma had an increase in the normalized peak distance (Panel C) and the amount of 350-kd fragments (Panel D) that was dependent on the rate of shear stress. A normalized peak distance of more than 1 indicates that the size of the multimers was decreased as compared with that measured in the absence of shear stress. The amount of 350-kd fragments was expressed as a percentage of the amount in the absence of shear stress.
Figure 6 demonstrates that high levels of shear stress increasedthe ristocetin cofactor activity of the von Willebrand factorin the cryosupernatant of plasma from a patient with thromboticthrombocytopenic purpura and decreased the activity in the cryosupernatantof control plasma. For example, a shear rate of 4307 per seconddecreased the ristocetin cofactor activity of the von Willebrandfactor in the control cryosupernatant by 15 percentage points(P<0.03) and increased the activity in the cryosupernatantfrom the patient by 22 percentage points (P<0.05).
Figure 6. Effect of Shear Stress on Ristocetin Cofactor Activity of Purified von Willebrand Factor.
Shear stress decreased the ristocetin cofactor activity of von Willebrand factor in the cryosupernatant of control plasma, but increased it in the cryosupernatant of plasma from a patient with thrombotic thrombocytopenic purpura (TTP). The ristocetin cofactor activity is expressed as a percentage of the activity in the absence of shear stress.
Discussion
We found a severe deficiency of von Willebrand factor–cleavingprotease activity in plasma samples from 37 patients during39 episodes of acute thrombotic thrombocytopenic purpura. Bycontrast, samples obtained during remission had normal proteaseactivity. In one patient who was studied serially from the timeof an acute episode to remission, protease activity graduallybecame normal.
The protease deficiency was specific for thrombotic thrombocytopenicpurpura. It was not detected in patients with thrombocytopenia,thrombosis, or hemolysis from other causes, suggesting thatthe deficiency did not result from hemolysis, thrombocytopenia,platelet activation, or thrombosis. Nonetheless, we have notruled out the possibility that other diseases may be associatedwith a deficiency of this protease.
Plasma from normal subjects contained trace amounts of 350-kdand 200-kd fragments of von Willebrand factor, whereas plasmafrom patients with thrombotic thrombocytopenic purpura did notalways have these fragments. When present, such fragments mayhave been produced before the protease activity was suppressedby inhibitors. Many plasma samples from patients with acutethrombotic thrombocytopenic purpura also had 300-kd bands. Thesebands differed from the 350-kd band produced by the proteaseor that produced by plasmin.17 Whether cysteine proteases orplatelet microparticles, which have been detected in plasmafrom patients with thrombotic thrombocytopenic purpura,21,22generated these bands in vivo and what role, if any, they havein the disease remain to be determined.
We found that plasma from most of our patients contained inhibitorsof von Willebrand factor–cleaving protease. In each ofthe samples that we studied, the inhibition was mediated byIgG antibodies. These antibodies were probably directed againstthe protease, but they may also have been directed against unknownprotease cofactors. Since the incubation of plasma from patientswith thrombotic thrombocytopenic purpura with purified von Willebrandfactor before the addition of control plasma resulted in lessinhibition than did incubation of the plasma with control plasmafollowed by the addition of von Willebrand factor (data notshown), it is unlikely that the inhibitors were directed againstthe cleavage sites of the von Willebrand factor substrate.
Four patients with the chronic relapsing form of thromboticthrombocytopenic purpura were reported to have reduced levelsof von Willebrand factor–cleaving protease activity.19In none of these patients was an inhibitor of the protease detected.However, as reported by Furlan et al.23 elsewhere in this issueof the Journal, inhibitors of this protease activity have beendetected in patients with acute thrombotic thrombocytopenicpurpura.
A link between thrombotic thrombocytopenic purpura and abnormalimmune reactions has been suggested.24,25,26 Treatment withglucocorticoids or immunoadsorption with staphylococcal proteinA may induce a remission of the disease,27 but even in the absenceof immunosuppression thrombotic thrombocytopenic purpura doesnot recur in most patients who are successfully treated withplasmapheresis. The transient nature of acute thrombotic thrombocytopenicpurpura raises the possibility that the antibodies to the proteaserepresent a deranged response to certain triggering events.
How does a deficiency of von Willebrand factor–cleavingprotease lead to platelet thrombosis in thrombotic thrombocytopenicpurpura? Shear stress unfolds von Willebrand factor28 and enhancesits proteolysis by protease in normal plasma.15,17 Shear stressmay also expose the platelet-binding sites of von Willebrandfactor. Our finding that the increase in ristocetin cofactoractivity was dependent on the level of shear stress supportsthis possibility. Thus, in the absence of the protease, large,unfolded multimers may be more likely to bind platelets. Thismay explain why in patients with thrombotic thrombocytopenicpurpura the numbers of large multimers are frequently decreased12and the binding of von Willebrand factor to platelets is increased.29In our capillary-tube device, von Willebrand factor was shearedat a rate of 4307 per second for 22 seconds. This brief exposure,and perhaps differences in temperature and other unknown factors,may explain why in a recent study the shear stress requiredto increase ristocetin cofactor activity in vitro (77 dyn persquare centimeter) was higher than that encountered in normalcirculation (60 dyn per square centimeter).30
The presence of inhibitory antibodies to von Willebrand factor–cleavingprotease may explain why in our study thrombotic thrombocytopenicpurpura responded to plasma infusion or plasma exchange. Presumably,patients with low titers of inhibitor would have a responseto simple plasma infusion, whereas patients with high titerswould require plasmapheresis to remove the inhibitory antibodiesand supply normal protease. Plasmapheresis without plasma infusionis relatively ineffective in patients with thrombotic thrombocytopenicpurpura,31 perhaps because it does not increase the proteaseactivity quickly.
We are indebted to Drs. G.P. Visentin, H. Billett, and V. Chandrasyekaranfor supplying plasma samples and to Drs. Ronald L. Nagel andIra I. Sussman for their generous support of the study and criticalcomments.
Source Information
From the Division of Hematology, Montefiore Medical Center and Albert Einstein College of Medicine, Bronx, N.Y. (H.-M.T.); and the Hemophilia and Thrombosis Center and Sylvester Cancer Center, University of Miami and Veterans Affairs Medical Center, Miami (E.C.-Y.L.).
Address reprint requests to Dr. Tsai at Montefiore Medical Center, Division of Hematology, 111 E. 210th St., Bronx, NY 10467.
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(2009). A First-in-Man Phase I and Pharmacokinetic Study on CHR-2797 (Tosedostat), an Inhibitor of M1 Aminopeptidases, in Patients with Advanced Solid Tumors. Clin. Cancer Res.
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(2009). Autoimmune hemophilia at rescue. haematol
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(2009). A comparison of thrombotic thrombocytopenic purpura in an inception cohort of patients with and without systemic lupus erythematosus. Rheumatology (Oxford)
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Laje, P., Shang, D., Cao, W., Niiya, M., Endo, M., Radu, A., DeRogatis, N., Scheiflinger, F., Zoltick, P. W., Flake, A. W., Zheng, X. L.
(2009). Correction of murine ADAMTS13 deficiency by hematopoietic progenitor cell-mediated gene therapy. Blood
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Peyvandi, F., Lavoretano, S., Palla, R., Feys, H. B., Vanhoorelbeke, K., Battaglioli, T., Valsecchi, C., Canciani, M. T., Fabris, F., Zver, S., Reti, M., Mikovic, D., Karimi, M., Giuffrida, G., Laurenti, L., Mannucci, P. M.
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(2007). Sunitinib induced hypertension, thrombotic microangiopathy and reversible posterior leukencephalopathy syndrome. Ann Oncol
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Bennett, C. L., Kim, B., Zakarija, A., Bandarenko, N., Pandey, D. K., Buffie, C. G., McKoy, J. M., Tevar, A. D., Cursio, J. F., Yarnold, P. R., Kwaan, H. C., De Masi, D., Sarode, R., Raife, T. J., Kiss, J. E., Raisch, D. W., Davidson, C., Sadler, J. E., Ortel, T. L., Zheng, X. L., Kato, S., Matsumoto, M., Uemura, M., Fujimura, Y.
(2007). Two Mechanistic Pathways for Thienopyridine-Associated Thrombotic Thrombocytopenic Purpura: A Report From the SERF-TTP Research Group and the RADAR Project. J Am Coll Cardiol
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(2007). Thrombotic Thrombocytopenic Purpura in Humans and Mice. Arterioscler. Thromb. Vasc. Bio.
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Hunt, B.J., Tueger, S., Pattison, J., Cavenagh, J., D'Cruz, D.P.
(2007). Microangiopathic haemolytic anaemia secondary to lupus nephritis: an important differential diagnosis of thrombotic thrombocytopenic purpura. Lupus
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Ferrari, S., Scheiflinger, F., Rieger, M., Mudde, G., Wolf, M., Coppo, P., Girma, J.-P., Azoulay, E., Brun-Buisson, C., Fakhouri, F., Mira, J.-P., Oksenhendler, E., Poullin, P., Rondeau, E., Schleinitz, N., Schlemmer, B., Teboul, J.-L., Vanhille, P., Vernant, J.-P., Meyer, D., Veyradier, A., for the French and Clinical Biological Network on,
(2007). Prognostic value of anti-ADAMTS13 antibody features (Ig isotype, titer, and inhibitory effect) in a cohort of 35 adult French patients undergoing a first episode of thrombotic microangiopathy with undetectable ADAMTS13 activity. Blood
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(2007). Evaluation and Management of Patients With Thrombotic Thrombocytopenic Purpura. J Intensive Care Med
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Mannucci, P. M., Peyvandi, F.
(2007). TTP and ADAMTS13: When Is Testing Appropriate?. ASH Education Book
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Bongers, T. N., de Maat, M. P.M., van Goor, M.-L. P.J., Bhagwanbali, V., van Vliet, H. H.D.M., Gomez Garcia, E. B., Dippel, D. W.J., Leebeek, F. W.G.
(2006). High von Willebrand Factor Levels Increase the Risk of First Ischemic Stroke: Influence of ADAMTS13, Inflammation, and Genetic Variability. Stroke
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Hirata, S., Okamoto, H., Ohta, S., Kobashigawa, T., Uesato, M., Kawaguchi, Y., Tateishi, M., Hara, M., Kamatani, N., Tsai, H.-M.
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(2006). Clinical Outcome of Thrombotic Microangiopathy after Living-Donor Liver Transplantation Treated with Plasma Exchange Therapy. CJASN
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(2006). Novel compound heterozygote mutations (H234Q/R1206X) of the ADAMTS13 gene in an adult patient with Upshaw-Schulman syndrome showing predominant episodes of repeated acute renal failure. Nephrol Dial Transplant
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Chauhan, A. K., Motto, D. G., Lamb, C. B., Bergmeier, W., Dockal, M., Plaimauer, B., Scheiflinger, F., Ginsburg, D., Wagner, D. D.
(2006). Systemic antithrombotic effects of ADAMTS13. JEM
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(2006). Severe secondary deficiency of von Willebrand factor-cleaving protease (ADAMTS13) in patients with sepsis-induced disseminated intravascular coagulation: its correlation with development of renal failure. Blood
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Anderson, P. J., Kokame, K., Sadler, J. E.
(2006). Zinc and Calcium Ions Cooperatively Modulate ADAMTS13 Activity. J. Biol. Chem.
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Sadler, J. E.
(2006). Thrombotic Thrombocytopenic Purpura: A Moving Target. ASH Education Book
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Plaimauer, B., Fuhrmann, J., Mohr, G., Wernhart, W., Bruno, K., Ferrari, S., Konetschny, C., Antoine, G., Rieger, M., Scheiflinger, F.
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Nolasco, L. H., Turner, N. A., Bernardo, A., Tao, Z., Cleary, T. G., Dong, J.-f., Moake, J. L.
(2005). Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers. Blood
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Zhou, W., Dong, L., Ginsburg, D., Bouhassira, E. E., Tsai, H.-M.
(2005). Enzymatically Active ADAMTS13 Variants Are Not Inhibited by Anti-ADAMTS13 Autoantibodies: A NOVEL THERAPEUTIC STRATEGY?. J. Biol. Chem.
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Fakhouri, F., Vernant, J.-P., Veyradier, A., Wolf, M., Kaplanski, G., Binaut, R., Rieger, M., Scheiflinger, F., Poullin, P., Deroure, B., Delarue, R., Lesavre, P., Vanhille, P., Hermine, O., Remuzzi, G., Grunfeld, J.-P.
(2005). Efficiency of curative and prophylactic treatment with rituximab in ADAMTS13-deficient thrombotic thrombocytopenic purpura: a study of 11 cases. Blood
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Rieger, M., Mannucci, P. M., Hovinga, J. A. K., Herzog, A., Gerstenbauer, G., Konetschny, C., Zimmermann, K., Scharrer, I., Peyvandi, F., Galbusera, M., Remuzzi, G., Bohm, M., Plaimauer, B., Lammle, B., Scheiflinger, F.
(2005). ADAMTS13 autoantibodies in patients with thrombotic microangiopathies and other immunomediated diseases. Blood
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Uemura, M., Tatsumi, K., Matsumoto, M., Fujimoto, M., Matsuyama, T., Ishikawa, M., Iwamoto, T.-a., Mori, T., Wanaka, A., Fukui, H., Fujimura, Y.
(2005). Localization of ADAMTS13 to the stellate cells of human liver. Blood
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Galbusera, M., Bresin, E., Noris, M., Gastoldi, S., Belotti, D., Capoferri, C., Daina, E., Perseghin, P., Scheiflinger, F., Fakhouri, F., Grunfeld, J.-P., Pogliani, E., Remuzzi, G.
(2005). Rituximab prevents recurrence of thrombotic thrombocytopenic purpura: a case report. Blood
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Levy, G. G., Motto, D. G., Ginsburg, D.
(2005). ADAMTS13 turns 3. Blood
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De Cristofaro, R., Peyvandi, F., Palla, R., Lavoretano, S., Lombardi, R., Merati, G., Romitelli, F., Di Stasio, E., Mannucci, P. M.
(2005). Role of Chloride Ions in Modulation of the Interaction between von Willebrand Factor and ADAMTS-13. J. Biol. Chem.
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Majerus, E. M., Anderson, P. J., Sadler, J. E.
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Noris, M., Bucchioni, S., Galbusera, M., Donadelli, R., Bresin, E., Castelletti, F., Caprioli, J., Brioschi, S., Scheiflinger, F., Remuzzi, G., for the International Registry of Recurrent and Fa,
(2005). Complement Factor H Mutation in Familial Thrombotic Thrombocytopenic Purpura with ADAMTS13 Deficiency and Renal Involvement. J. Am. Soc. Nephrol.
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Dragon-Durey, M.-A., Loirat, C., Cloarec, S., Macher, M.-A., Blouin, J., Nivet, H., Weiss, L., Fridman, W. H., Fremeaux-Bacchi, V.
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Motto, D.
(2005). Hemoglobin versus ADAMTS13. Blood
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Studt, J.-D., Hovinga, J. A. K., Antoine, G., Hermann, M., Rieger, M., Scheiflinger, F., Lammle, B.
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Kawahara, M., Kanno, M., Matsumoto, M., Nakamura, S., Fujimura, Y., Ueno, S.
(2004). Diffuse neurodeficits in intravascular lymphomatosis with ADAMTS13 inhibitor. Neurology
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Uchida, T., Wada, H., Mizutani, M., Iwashita, M., Ishihara, H., Shibano, T., Suzuki, M., Matsubara, Y., Soejima, K., Matsumoto, M., Fujimura, Y., Ikeda, Y., Murata, M.
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Lee, J.-L., Lee, J.-H., Kim, M.-K., Cho, H. S., Bae, Y. K., Cho, K. H., Bae, S. H., Ryoo, H. M., Lee, K. H., Hyun, M. S.
(2004). A Case of Bone Marrow Necrosis with Thrombotic Thrombocytopenic Purpura as a Manifestation of Occult Colon Cancer. Jpn J Clin Oncol
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Nishio, K., Anderson, P. J., Zheng, X. L., Sadler, J. E.
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Klaus, C., Plaimauer, B., Studt, J.-D., Dorner, F., Lammle, B., Mannucci, P. M., Scheiflinger, F.
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Espinosa, G, Bucciarelli, S, Cervera, R, Lozano, M, Reverter, J-C, de la Red, G, Gil, V, Ingelmo, M, Font, J, Asherson, R A
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Zheng, X. L., Kaufman, R. M., Goodnough, L. T., Sadler, J. E.
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Studt, J.-D., Hovinga, J. A. K., Radonic, R., Gasparovic, V., Ivanovic, D., Merkler, M., Wirthmueller, U., Dahinden, C., Furlan, M., Lammle, B.
(2004). Familial acquired thrombotic thrombocytopenic purpura: ADAMTS13 inhibitory autoantibodies in identical twins. Blood
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Pimanda, J. E., Ganderton, T., Maekawa, A., Yap, C. L., Lawler, J., Kershaw, G., Chesterman, C. N., Hogg, P. J.
(2004). Role of Thrombospondin-1 in Control of von Willebrand Factor Multimer Size in Mice. J. Biol. Chem.
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Matsumoto, M., Kokame, K., Soejima, K., Miura, M., Hayashi, S., Fujii, Y., Iwai, A., Ito, E., Tsuji, Y., Takeda-Shitaka, M., Iwadate, M., Umeyama, H., Yagi, H., Ishizashi, H., Banno, F., Nakagaki, T., Miyata, T., Fujimura, Y.
(2004). Molecular characterization of ADAMTS13 gene mutations in Japanese patients with Upshaw-Schulman syndrome. Blood
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Zakarija, A., Bandarenko, N., Pandey, D. K., Auerbach, A., Raisch, D. W., Kim, B., Kwaan, H. C., McKoy, J. M., Schmitt, B. P., Davidson, C. J., Yarnold, P. R., Gorelick, P. B., Bennett, C. L.
(2004). Clopidogrel-Associated TTP: An Update of Pharmacovigilance Efforts Conducted by Independent Researchers, Pharmaceutical Suppliers, and the Food and Drug Administration. Stroke
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Bowen, D. J., Collins, P. W.
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Kokame, K., Matsumoto, M., Fujimura, Y., Miyata, T.
(2004). VWF73, a region from D1596 to R1668 of von Willebrand factor, provides a minimal substrate for ADAMTS-13. Blood
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Sadler, J. E., Moake, J. L., Miyata, T., George, J. N.
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Majerus, E. M., Zheng, X., Tuley, E. A., Sadler, J. E.
(2003). Cleavage of the ADAMTS13 Propeptide Is Not Required for Protease Activity. J. Biol. Chem.
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Soejima, K., Matsumoto, M., Kokame, K., Yagi, H., Ishizashi, H., Maeda, H., Nozaki, C., Miyata, T., Fujimura, Y., Nakagaki, T.
(2003). ADAMTS-13 cysteine-rich/spacer domains are functionally essential for von Willebrand factor cleavage. Blood
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Scheiflinger, F., Knobl, P., Trattner, B., Plaimauer, B., Mohr, G., Dockal, M., Dorner, F., Rieger, M.
(2003). Nonneutralizing IgM and IgG antibodies to von Willebrand factor-cleaving protease (ADAMTS-13) in a patient with thrombotic thrombocytopenic purpura. Blood
102: 3241-3243
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Varadi, K., Schreiner, J., Plaimauer, B., Rieger, M., Scheiflinger, F., Knobl, P., Turecek, P. L., Schwarz, H. P.
(2003). ADAMTS13 autoantibody detection by quantitative immunoblotting. Blood
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Zheng, X., Nishio, K., Majerus, E. M., Sadler, J. E.
(2003). Cleavage of von Willebrand Factor Requires the Spacer Domain of the Metalloprotease ADAMTS13. J. Biol. Chem.
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Chang, J. C., Naqvi, T.
(2003). Thrombotic Thrombocytopenic Purpura Associated with Bone Marrow Metastasis and Secondary Myelofibrosis in Cancer. The Oncologist
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Studt, J.-D., Hovinga, J. A. K., Furlan, M., Lammle, B.
(2003). Discrepant activity levels of von Willebrand factor-cleaving protease (ADAMTS-13) in congenital thrombotic thrombocytopenic purpura. Blood
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Vesely, S. K., George, J. N., Lammle, B., Studt, J.-D., Alberio, L., El-Harake, M. A., Raskob, G. E.
(2003). ADAMTS13 activity in thrombotic thrombocytopenic purpura-hemolytic uremic syndrome: relation to presenting features and clinical outcomes in a prospective cohort of 142 patients. Blood
102: 60-68
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Savasan, S., Lee, S.-K., Ginsburg, D., Tsai, H.-M.
(2003). ADAMTS13 gene mutation in congenital thrombotic thrombocytopenic purpura with previously reported normal VWF cleaving protease activity. Blood
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Tsai, H.-M.
(2003). Advances in the Pathogenesis, Diagnosis, and Treatment of Thrombotic Thrombocytopenic Purpura. J. Am. Soc. Nephrol.
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Elliott, M. A., Nichols, W. L. Jr, Plumhoff, E. A., Ansell, S. M., Dispenzieri, A., Gastineau, D. A., Gertz, M. A., Inwards, D. J., Lacy, M. Q., Micallef, I. N. M., Tefferi, A., Litzow, M. R.
(2003). Posttransplantation Thrombotic Thrombocytopenic Purpura: A Single-Center Experience and a Contemporary Review. Mayo Clin Proc.
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Tsai, H.-M.
(2003). Platelet Activation and the Formation of the Platelet Plug: Deficiency of ADAMTS13 Causes Thrombotic Thrombocytopenic Purpura. Arterioscler. Thromb. Vasc. Bio.
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Zheng, X., Pallera, A. M., Goodnough, L. T., Sadler, J. E., Blinder, M. A.
(2003). Remission of Chronic Thrombotic Thrombocytopenic Purpura after Treatment with Cyclophosphamide and Rituximab. ANN INTERN MED
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George, J. N., Vesely, S. K.
(2003). Thrombotic Thrombocytopenic Purpura: From the Bench to the Bedside, but Not Yet to the Community. ANN INTERN MED
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Dong, J.-f., Moake, J. L., Nolasco, L., Bernardo, A., Arceneaux, W., Shrimpton, C. N., Schade, A. J., McIntire, L. V., Fujikawa, K., Lopez, J. A.
(2002). ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions. Blood
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Tsai, H.-M., Lammle, B., Bianchi, V., Alberio, L., Furlan, M., Remuzzi, G., Galbusera, M., Mannucci, P. M.
(2002). Deficiency of ADAMTS13 and thrombotic thrombocytopenic purpura. Blood
100: 3839-3842
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Plaimauer, B., Zimmermann, K., Volkel, D., Antoine, G., Kerschbaumer, R., Jenab, P., Furlan, M., Gerritsen, H., Lammle, B., Schwarz, H. P., Scheiflinger, F.
(2002). Cloning, expression, and functional characterization of the von Willebrand factor-cleaving protease (ADAMTS13). Blood
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