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Keywords:

  • congenital thrombotic thrombocytopenic purpura;
  • von Willebrand factor-cleaving protease;
  • microangiopathy;
  • intermediate purity factor VIII concentrate;
  • fresh-frozen plasma

Abstract

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Summary. There is increasing evidence that congenital thrombotic thrombocytopenic purpura (TTP) is caused by an absolute deficiency of von Willebrand factor-cleaving protease. The recent identification of this protease and the development of assays for its detection have enabled its quantification in a number of plasma products, including some commercial intermediate-purity plasma-derived factor VIII preparations. We report the successful, weekly prophylactic use of a commercial intermediate-purity plasma-derived factor VIII concentrate in the treatment of a 14-year-old girl with severe congenital TTP who had previously required transfusions of fresh-frozen plasma every 2 weeks from the age of 4 months.

Congenital thrombotic thrombocytopenic purpura (TTP) is caused by a deficiency of von Willebrand factor (VWF)-cleaving protease (Furlan et al, 1997), a metalloprotease first identified in 1996 (Furlan et al, 1996; Tsai, 1996). In order to investigate the protease activity of plasma and plasma products, Allford et al (2000) used a semiquantitative assay of VWF-cleaving protease to screen a number of commercially available plasma-derived factor VIII concentrates for VWF-cleaving protease activity, noting that some intermediate-purity products showed VWF-cleaving protease activity of at least 100% compared with pooled normal plasma. One such product was BPL (Bio Products Laboratory, Elstree, UK) 8Y factor VIII concentrate and we have subsequently investigated its efficacy in the treatment of a patient with severe congenital TTP.

Case Report and Methods

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The patient we describe is an Asian girl, born in 1987 to consanguineous parents (first cousins). She was delivered at 37 weeks gestation by caesarean section for fetal distress and became jaundiced within 12 h of birth. Her haemoglobin (Hb) was 12·9 g/dl and platelets were 39 × 109/l; she received a whole blood exchange transfusion with subsequent clinical improvement and normalization of the platelet count. At 7 weeks of age, she required re-admission to hospital because of pallor and poor feeding. Her Hb had fallen to 5·2 g/dl, the platelet count was 32 × 109/l and the blood film showed features of a microangiopathic haemolytic anaemia. Following red cell transfusion, her haemoglobin and platelet count normalized. At the age of 11 weeks, the patient was referred to Birmingham Children's Hospital for further investigations. At that time, her Hb was 5·8 g/dl, platelets 49 × 109/l, reticulocytes 7·6% with microangiopathic features on blood film examination. Renal function was normal, as was a routine coagulation screen. However, VWF antigen was increased at 300% and VWF multimer gel electrophoresis revealed the presence of an ultra-high molecular-weight component. A diagnosis of congenital TTP was made and a treatment regime consisting of regular infusions of fresh-frozen plasma (FFP) at a dose of 15 ml/kg was initiated. It was noted that if treatment was delayed beyond a 14 d interval, microangiopathic features recurred and the patient became thrombocytopenic, requiring hospitalization on one occasion because of progressive somnolence and fever, which responded to repeated infusions of FFP. This treatment regimen was continued for 10 years, at which time conventional FFP was substituted with solvent detergent-treated pooled plasma (Octaplas; Octapharma, Coventry, UK) with no change in efficacy. At the age of 13 years, the patient and her parents consented to a trial of therapy with the intermediate-purity plasma-derived factor VIII concentrate BPL 8Y, including an initial dose finding study.

VWF-cleaving protease activity was measured using the modified method described by Allford et al (2000). This is a semiquantitative assay in which VWF-cleaving protease activity is expressed as a ratio, comparing the extent of degradation of VWF in the patient's plasma with that of a positive and a negative control: negative values indicate the presence of ultra-large (UL) VWF multimers.

Results

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The patient was found to have undetectable levels of VWF-cleaving protease without evidence of an inhibitor being present. Figure 1 shows the effects of the first dose of 8Y on the platelet count and the serum lactate dehydrogenase (LDH) level with an improvement in the platelet count occurring within 24 h and a maximum increase detected at 7–9 d. The ratio of protease activity increased from −0·06 pre-8Y infusion to +0·06 at 1 h and +0·03 at 6 h post infusion. At 24 h, the ratio had returned to −0·06. The maximum protease activity achieved, as measured at 1 h post infusion, was equivalent to 3% that of pooled normal plasma.

image

Figure 1. Platelet count and LDH levels following a single dose of 8Y factor VIII concentrate.

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Sodium dodecyl sulphate-agarose electrophoresis of the patient's VWF multimers demonstrated a paradoxical increase in UL VWF multimers as the platelet count recovered following 8Y therapy.

The current prophylactic dosing schedule was established after gradually reducing the amount of 8Y administered in order to determine the minimum dose necessary to maintain the platelet count and LDH within the normal range. Figure 2 shows the effect of the first dose of 8Y, equivalent to 30 units/kg, on the patient's plasma VWF antigen levels, VWF Ristocetin Cofactor (RiCof) and factor VIII coagulant (FVIII:C) activities. For ease of administration and to minimize this iatragenic increase in clotting factors, a once weekly dosage regime was introduced, the initial dose requirement being 10 units/kg. Using this dosing regime led to an approximate doubling in VWF antigen and RiCof measurements at 1 h post dose but only an approximate 20% increase in FVII:C. The patient remained in remission for 6 months using this treatment regimen but then developed symptomatic thrombocytopenia and an elevated LDH, following an episode of fever associated with an upper respiratory tract infection: this relapse, however, responded to an additional three doses of 8Y given on alternate days. Following this, the dosage was increased to 750 units (15 units/kg) once weekly and the patient has continued in remission on this dose for the last 8 months.

image

Figure 2. Plasma VWF antigen (VWF Ag) levels, Ristocetin Co-factor activity (RiCof) and FVIII:C following the first dose of 8Y factor VIII concentrate.

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Despite a more frequent dosing regime than was previously necessary with FFP, infusion time has been reduced considerably and the patient reports an overall improvement in the quality of her life: school attendance and performance has improved, and she is currently learning to self-administer 8Y concentrate as part of our haemophilia centre home treatment programme.

Our patient has been vaccinated against Hepatitis A and B and has shown no evidence of exposure to Hepatitis C or human immunodeficiency virus (HIV) throughout the 14 years of treatment with plasma and plasma products.

Discussion

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Historically, individuals with congenital TTP have received regular infusions of FFP, the frequency of infusions being dependent on the severity of the condition. However, there has been a small number of published case reports describing a total of six patients with chronic relapsing TTP and TTP-like syndromes whose response to intermediate-purity plasma-derived factor VIII concentrate has been variable (Miura et al, 1984; Hara et al, 1986; Karpman et al, 1996; Allford et al, 2000). The mechanism of action of 8Y factor VIII concentrate and other similar plasma concentrates in the treatment of congenital TTP is unclear, although there is some evidence to suggest that factor VIII concentrates with high intrinsic VWF-cleaving protease activity may be more efficacious than those with a lower protease activity in the treatment of congenital TTP (Allford et al, 2000). These authors demonstrated at least 100% VWF-cleaving protease in 8Y compared with pooled normal plasma; using the same methodology, protease activity was assayed in the two batches of 8Y concentrate used in the treatment of our patient and were found to have levels of activity similar to pooled normal plasma on a volume for volume basis. We were only able to detect low levels of protease activity in the patient's plasma within the first 24 h following each dose of 8Y. This contrasts with the report by Barbot et al (2001) who demonstrated detectable levels of VWF-cleaving protease activity in a congenital TTP patient for at least 4 d following 3·6 ml/kg of FFP; these varying results may, however, simply reflect differing sensitivities of the assays currently used. Furlan et al (1999) studied VWF-cleaving protease activity in the plasma of two patients following FFP infusion, predicting an exceptionally long half-life of approximately 2 to 3 d. It does, therefore, appear likely that even low levels of VWF-cleaving protease may be sufficient to degrade large adhesive VWF multimers under steady state conditions, with suggested effective levels varying from 5% (Barbot et al, 2001) to 1% (Allford et al, 2000). However, current assay techniques do not allow such an accurate determination of serum protease activity.

The low level of protease activity measured in our patient after 8Y concentrate suggests a possible alternative explanation of its efficacy. Miura et al (1984) suggested that the normal factor VIII–VWF complex present in intermediate-purity FVIII concentrate may compete with the abnormal VWF complexes present in TTP plasma and thereby prevent these abnormal complexes binding to and agglutinating newly formed platelets. Such a mechanism could contribute to the prolonged effect of 8Y concentrate in our patient.

Whether the effectiveness of 8Y concentrate in the treatment of congenital TTP reflects its intrinsic VWF-cleaving protease activity remains unclear at present and may remain so until such time that a purified protease concentrate becomes available for human trials; the possibility of such a concentrate has been greatly advanced by the recent identification of the gene mutations responsible for this disease (Levy et al, 2001) and by the purification of VWF-cleaving protease (Fujikawa et al, 2001; Gerritsen et al, 2001).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

The authors are grateful to S. O'Brien, D. Marshall and G. Taylor, Haemophilia nurses at Birmingham Children's Hospital, for their help in this study.

References

  1. Top of page
  2. Abstract
  3. Case Report and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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