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PEDIATRICS Vol. 109 No. 2 February 2002, pp. 322-325

EXPERIENCE AND REASON

Thrombotic Thrombocytopenic Purpura Attributable to von Willebrand Factor-Cleaving Protease Inhibitor in an 8-Year-Old Boy

Abbreviations: TTP, thrombotic thrombocytopenic purpura • HUS, hemolytic-uremic syndrome • vWf, von Willebrand factor • SD, standard deviation • IgG, immunoglobulin G • SDS, sodium dodecyl sulphate

	INTRODUCTION

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 
Thrombotic thrombocytopenic purpura (TTP) is a rare problem in children, and until recently, the diagnosis was often difficult to distinguish from atypical forms of hemolytic-uremic syndrome (HUS). The recent demonstration that either congenital deficiency in von Willebrand factor (vWf)-cleaving protease or the presence of inhibitors of this protease are central to the pathophysiology of TTP allows a more objective method to differentiate the condition from HUS and also clarifies the rationale for the successful results of treatment with plasma exchange.^1–6 We report an 8-year-old boy with acute TTP attributable to the presence of inhibitors of vWf-cleaving protease who responded to plasma exchange and corticosteroid therapy.

	METHODS

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 
The assay of vWf-cleaving metalloprotease activity was based on the generation, from exogenous vWf substrate, of dimers of the 176 kDa and the 140 kDa fragment.³ Normal control plasma obtained from the clinical coagulation laboratory was used as a reference and assumed to contain 100% of the metalloprotease activity. The mean (± standard deviation [SD]) metalloprotease activity observed in 61 normal adults without TTP is 102% ± 12%, and the level is not different in children <10 years of age.⁷ Procedures for determining the inhibitor titer of a sample by mixing studies have been described previously⁸ and were modified from the Bethesda method that was originally designed for the determination of inhibitor titers of coagulation factor VIII. All test samples were treated at 56°C for 60 minutes to inactivate endogenous vWf-cleaving protease activity. An aliquot of a test sample or its serial dilutions was added to an equal volume of pooled normal plasma. After incubation at 37°C for 60 minutes, the protease activity in the mixture was determined. A sample causing an inhibition of normal protease activity by 50% was assumed to contain 1 U/mL inhibitor. The mean (± SD) titer in normal plasma samples is 0.0 ± 0.1 U/mL.⁸

Immunoglobulin G (IgG) molecules were isolated from plasma samples with a staphylococcal protein A column, and their inhibitory activities were determined by mixing studies.³

vWf multimers were analyzed by using 1% sodium dodecyl sulphate (SDS) agarose gel electrophoresis.³

	CASE REPORT

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 
An 8-year-old boy was well until 8 days before admission when he developed fever, nausea, vomiting, and headache. He voided dark brownish-red urine without discomfort. He was assessed at a regional hospital where blood tests showed a hemoglobin of 6.0 g/dL, platelet count of 7 x 10⁹/L, white blood count of 6.5 x 10⁹/L with 56% neutrophils, and a creatinine of 0.9 mg/dL. There was no history of contact with infectious illness, exposure to sick animals, pets, chemicals or toxins, or travel. There was a family history of systemic lupus erythematosus in the maternal grandmother, maternal great aunt, and maternal uncle.

Physical examination revealed pallor, scleral icterus, and a scattered petechial rash over his trunk, and hemorrhages in the buccal mucosa. His blood pressure was 100/70 mm Hg. He had a grade 2/6 systolic ejection murmur. His neurologic examination was normal and symmetrical. No retinal hemorrhages were noted. His examination was otherwise unremarkable.

The serum creatinine was 0.9 mg/dL on admission and declined during therapy to 0.5 mg/dL. Similarly, the serum lactate dehydrogenase declined from 1083 U/L to 213 U/L. A blood smear showed a microangiopathic hemolytic anemia with characteristic fragmented cells, helmet cells, and schistocytes. The haptoglobin level was 0, prothrombin time was abnormal at 15.2 seconds (normal: 11.2–13.2 seconds), partial thromboplastin time was 25 seconds, and fibrinogen was 202 mg/dL. A direct Coombs’ test was positive. The serum electrolytes, amylase, and lipase were normal. A urinalysis on the day of admission revealed a specific gravity of 1.024, pH 6, 1+ protein, was positive for urobilinogen, and had greater than 100 red blood cells per high-powered field and many hyaline and coarse granular casts. The antistreptolysin O titre was 200 U/L, C₃ was 70 mg/dL (normal: 91–161 mg/dL), erythrocyte sedimentation rate was 50 mm/hour, C-reactive protein was less than 0.4, antinuclear antibody was positive with a titer of 1:80 and a homogeneous pattern but anticentromere antibody, anti-native-DNA antibody, and antiphospholipid antibody-IgG and -IgM were negative, and antineutrophil cytoplasmic antibody was less than 6 U/mL. A throat swab, blood culture, rectal swab for Escherichia coli 0157:H7, and mycoplasma, human immunodeficiency virus, and hepatitis B and C antibody screening tests were negative. Chest and sinus radiographs were normal.

He was treated with plasma exchange for 9 consecutive days from the second to the eleventh day after admission. He was also treated with a single daily dose of intravenous Solumedrol, 10 mg/kg for 2 days from the second day of admission and then 2 mg/kg for the next 8 days. Thereafter, he was treated with prednisone 30 mg 2 times daily (2.0 mg/kg/day) for 1 month. The prednisone was slowly tapered and eventually discontinued after 7 months total therapy.

He responded to this treatment with a progressive increase in his platelet count and hemoglobin concentration, a decrease in lactate dehydrogenase, and a disappearance of schistocytes. His platelet counts and vWf-cleaving protease levels are shown in Fig 1, A and B.

View larger version (17K): [in this window] [in a new window]   Fig 1. A, Changes in the platelet count with treatment and time. Days of plasma exchange therapy are shown by the bar. B, Changes in the vWf-cleaving protease level with treatment and time. The mean level (± SD) in healthy participants is 102% ± 12%. C, The course of the inhibitor titer of the vWf-cleaving protease in plasma obtained from the patient. The mean titer (± SD) in healthy participants is 0.0 ± 0.1 U/mL. The days of plasma exchange therapy are indicated by . Days after admission: day 1 was the day of admission. vWf protease (%) represents the level vWf-cleaving metalloprotease activity in the plasma sample. Inhibitor titer (U/mL) represents the titer of inhibitor of the protease in the plasma sample. During the days when the patient was being treated with plasma exchange, blood samples were obtained before each session.

 
Mixing studies detected the presence of inhibitors of the vWf-cleaving protease. As depicted in Fig 1C, the inhibitor titers were 2.8 U/mL and 2.9 U/mL before the initiation of plasma exchange, declined to 0.2 U/mL on day 4 after 1 session of plasma exchange therapy, and were undetectable on day 6, after 3 sessions of therapy.

The inhibitory activity in the plasma was removed by passage through a staphylococcal protein A column. The IgG molecules eluted from the column inhibited the protease activity in normal control plasma. On day 2, a concentration of 1.1 mg/mL of IgG inhibited protease activity by 50% in mixtures containing 12% plasma from a healthy subject.

Analysis of vWf multimeric size distribution by SDS agarose gel electrophoresis (Fig 2) shows a decrease of large multimers at presentation and the appearance of ultra-large multimers after 3 sessions of plasma exchange on day 6, when the vWf protease level was raised to 34% and the platelet count to 53x10⁹/L. By day 28, the multimer pattern was normal, whereas the protease level was 76%.

View larger version (32K): [in this window] [in a new window]   Fig 2. Analysis of vWf multimers with 1% SDS agarose gel electrophoresis. The number above the middle 3 electrophoresis lanes refers to the days subsequent to admission. The electrophoresis lanes on each side represent normal control (C) plasma. As a reference, the expected position of IgM in a similar gel is indicated.

 
Both his mother and maternal grandmother had normal vWf-cleaving protease levels of 111% and 93%, respectively.

	DISCUSSION

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 
TTP is characterized by widespread platelet thrombi in arterioles and capillaries but without perivascular inflammation, endothelial cell loss, or subendothelial exposure.⁹

Acquired TTP is rare in childhood. Sartori et al¹⁰ reviewed TTP and identified 15 children reported by 1993. By comparison, HUS is common. Based on the overlap in clinical manifestations, some investigators have proposed that TTP and HUS represent variable expressions of a single entity.¹¹ However, immunohistologic studies demonstrate abundant vWf but little fibrin in the thrombotic lesions of TTP, the opposite of that seen with HUS.^7,9 In addition, TTP but not HUS is associated with a deficiency in vWf-cleaving protease.^2,7 These observations support the view that TTP and HUS are distinct entities.

vWf is secreted from endothelial cells as an ultra-large polymer that maximizes adhesion of platelets to the subendothelium under high shear stress conditions.^12–15 When exposed to high levels of shear stress as encountered in the arterioles and capillaries, large vWf multimers are unfolded from a globular to an elongated chain configuration.¹⁶ This configurational transformation by shear stress may be essential for vWf to maintain contact with the subendothelial matrix and explains why vWf is uniquely capable of supporting platelet adhesion and aggregation in high shear environments. Unfolding by shear stress also makes vWf susceptible to the protease,¹² presumably by exposing the cleavage sites. Conversely, vWf in a globular configuration is not susceptible to the protease. This scheme explains why the vWf of normal plasma coexists with the protease without undergoing proteolysis in a test tube. In the circulation, whenever a cleavage site is exposed during passage through the arterioles and capillaries, the vWf molecule is immediately proteolyzed by the protease. The brief transit time in the arterioles and capillaries suggests that each microcirculatory passage will not cause a complete unfolding of vWf; rather, it is more likely that only a few cleavage sites will be exposed during each passage. As a result, large multimers exist in normal circulation. According to this scheme, the vWf-cleaving protease is essential to prevent the highly adhesive unfolded forms of vWf from accumulation in the circulation. In support of this model, exposure of vWf to shear stress in the environment of normal plasma increases proteolysis of vWf and decreases the adhesive activity. In contrast, exposure to shear stress in the environment of TTP plasma causes no additional proteolysis of vWf but increases the adhesive activity.³

In acquired TTP, antibodies to the vWf-cleaving protease or to a cofactor presumably develop in response to an infectious or exogenous triggering event.^2,3,17 The antibodies suppress the protease level and allow large, unfolded forms of vWf multimers to accumulate, which leads to vWf-platelet binding and microvascular thrombosis. In support of this model, increased levels of vWf have been detected on the surface of platelets during the active phase of the disease.¹⁸

Our patient presented with undetectable vWf-cleaving protease activity and had inhibitors to the protease. Neutralization by the protease infused during plasma exchange presumably accounted for the rapid decline of the inhibitor titer. However, the slight decrease of protease activity that persisted during remission suggested that inhibitor production continued at a low level.

The changes in the vWf multimers shown in Fig 2 demonstrate the typical biphasic pattern observed in adults with idiopathic and ticlopidine-associated TTP: a decrease of large multimers at presentation and an appearance of ultra-large multimers when the vWf protease was raised by plasma exchange.^17,19 We interpret these results as evidence supporting the scheme that the absence of protease activity allows vWf to bind to platelets with a resultant decrease of the large multimers at presentation (day 2). Inadequate vWf proteolysis would result in accumulation of partially or completely unfolded forms of vWf in the circulation. This led to platelet-platelet binding and platelet thrombi in the arterioles and capillaries. Additional increase in shear stress caused by microvascular thrombi resulted in additional unfolding of vWf. This set off a cycle of systemic arteriolar thrombosis characteristic of the disorder and accounts for the decrease of large multimers detected during the stage of severe thrombocytopenia. Thereafter, when the vWf-cleaving protease level rose in response to plasma exchange, vWf-platelet binding decreased and ultra large multimers reappeared (day 6). The multimeric size returned to normal when the protease level reached greater than 50% (day 28).

The effectiveness of plasma therapy in adult patients with TTP has been investigated in a randomized, control study.²⁰ A survival rate of 60% is achieved with simple plasma infusion, whereas 80% survival is expected in patients treated with plasma exchange. Presumably plasma infusion is effective by replenishing the missing protease. Plasma exchange might achieve higher efficacy by allowing patients to receive a larger volume of plasma and by removal of the inhibitors. Experience with plasma exchange in children with TTP is anecdotal. Uncertainty in distinguishing the diagnosis from HUS probably contributes to the paucity of experience. In the report by Sartori et al, 6 children were treated with plasma infusion, but none with plasma exchange.¹⁰ More recent case reports, and our case, suggest plasma exchange is beneficial in children with TTP.^21,22 The low titers of inhibitors in our case and in most adult TTP cases probably contribute to the effectiveness of plasma therapy.⁸

	CONCLUSION

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 
An 8-year-old boy with TTP presented with a deficiency of vWf-cleaving protease attributable to the presence of an inhibitor. The deficiency led to a pattern of vWf abnormalities comparable with that observed in adult patients with idiopathic and ticlopidine-associated TTP. Similar to these adult cases, the patient responded promptly to plasma exchange and corticosteroid therapy. Measurement of vWf-cleaving protease and its inhibitor is useful to clarify the diagnosis of TTP and monitor the results of treatment.

William Lane M. Robson, MD, FAAP, FRCP(C), FRCP(Glasg) and Han-Mou Tsai, MD

University of Oklahoma
Health Sciences Center
Oklahoma City, OK 73104
Unified Division of Hematology
Albert Einstein College of Medicine and Montefiore
Medical Center
Bronx, NY 10467

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	ACKNOWLEDGMENTS

 
This study was supported, in part, by a grant (R01 HL62131 to H-M. T.) from the National Heart, Lung, and Blood Institute of the National Institutes of Health.

We thank Loretta Westcott and Debbie Porter of the Greenville Hospital System library for their assistance with document research; Larry Gluck, MD, and Ernie Moore, RN, for plasma exchange support; James Hay, MD, and Cary Stroud, MD, for pediatric hematology support; and Anping Li for technical assistance.

	FOOTNOTES

 
Received for publication Mar 22, 2001; Accepted Jul 16, 2001.

Address correspondence to William Lane M. Robson, MD, University of Oklahoma, Health Sciences Center, 940 NE 13th St, Rm 2B2307, Oklahoma City, OK 73104. E-mail: lanerobson{at}msn.com

	REFERENCES

TOP INTRODUCTION METHODS CASE REPORT DISCUSSION CONCLUSION REFERENCES

 

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PEDIATRICS (ISSN 1098-4275). ©2002 by the American Academy of Pediatrics

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This Article Extract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services E-mail this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My File Cabinet Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Robson, W. L. M. Articles by Tsai, H.-M. Search for Related Content PubMed PubMed Citation Articles by Robson, W. L. M. Articles by Tsai, H.-M. Related Collections Blood Related AAP Red Book topics: Escherichia coli Diarrhea... Social Bookmarking                         What's this?