First report of inhibitory von Willebrand factor alloantibodies in type 2B von Willebrand disease

Antibodies to von Willebrand factor (VWF) in patients with congenital von Willebrand disease (VWD) are rare; the prevalence in type 3 VWD patients is 6–10%. These antibodies are associated with loss of haemostatic response to VWF concentrates and allergic reactions, even anaphylactic, in rare cases. The majority of these alloantibodies occur in patients with large VWF gene deletions, although VWF antibodies have also been described in patients with nonsense or frameshift mutations (James et al, 2013). There are no reported cases of VWF alloantibodies in non-type 3 VWD. We report the first case of VWF antibodies with inhibitor activity in a 35-year-old woman with type 2B VWD. Anti-VWF antibodies are mostly characterized as polyclonal IgG class alloantibodies (Berntorp et al, 2013). Target specificity has been demonstrated towards the GP1b-binding

A1 and the collagen binding A3 domain of VWF in selected cases (Van Genderen et al, 1994; Tout et al, 2000). In addition, some antibodies have been reported to partially inhibit factor VIII (FVIII) coagulation activity (FVIII:C). To date, no single common epitope has been shown to be involved (L opez-Fernandez et al, 1988; Batlle et al, 1997; Tout et al, 2000). We further analysed the properties of the VWF alloantibody in the presented type 2B VWD patient. A 35-year-old woman was previously diagnosed with type 2B VWD; mutation analysis revealed a 3922C>T nucleotide substitution in VWF, resulting in a R1308C amino acid substitution in exon 28 coding for the VWF-A1 domain (Verweij et al, 1988; Ahmad et al, 2013). Incidental treatment consisted of desmopressin and tranexamic acid, she had received cryoprecipitate once in childhood. The current



Fig 1. In vivo recovery of von Willebrand factor antigen (VWF:Ag), ristocetin cofactor activity (VWF:RCo) and factor VIII (FVIII) coagulant activity and inhibition of VWF:RCo in mixing studies. (A) The left y-axis represents the observed (coagulation) factor level or activity as a percentage in response to the various treatment modalities. Platelet count is plotted on the right y-axis. The symbols on the horizontal axis represent; I: Pregnancy first trimester, II: Pregnancy third trimester, three consecutive Haemate P administrations during the caesarean section (CS, t = 0 at the start of delivery, t = 4 h, t = 18 h). PPH: primary postpartum haemorrhage, *Day 1–4 postpartum, LPPH: late postpartum haemorrhage, 1 and 24 h after Wilfactin, Test: desmopressin (DDAVP) test dose, 03 lg/kg i.v.; ⊕1 and 4 h after DDAVP test dose. The platelet counts during CS are imprecise due to clumping and transfusion of one unit of a 5-donor pool platelet concentrate after CS. (B) The y-axis represents VWF activity (VWF:RCo), the x-axis represents the percentage of patient plasma. VWF:RCo is progressively inhibited by increasing amounts of patient plasma (black bars) and loses part of its inhibiting activity when incubated at 37°C (grey).

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 424–439

Correspondence episode started with a grade 1 allergic reaction marked by an erythematous skin rash and generalized oedema in response to the administration of plasma VWF concentrate (Haemate P; CSL Behring, Breda, the Netherlands) during a haemorrhagic caesarean section due to uterine atonia and placental abruption. The allergic reaction did not recur after simultaneous administration of intravenous (IV) antihistamines with the consecutive Haemate P administration and could also have been provoked by a simultaneous blood transfusion. Five weeks later she presented with secondary post-partum haemorrhage and a different plasma-derived VWF product (Wilfactin; Sanquin, Amsterdam, the Netherlands) was administered combined with IV antihistamines. No allergic reaction occurred, however, hardly any recovery of FVIII:C and VWF ristocetin cofactor activity (VWF:RCo) was noted at 1 and 24 h after this treatment. The bleeding ceased within 3 weeks with the additional use of desmopressin and tranexamic acid in combination with curettage for placental retention. We measured the recovery of FVIII:C, VWF antigen and VWF:RCo on three separate occasions: each time after the IV administration of Haemate P and Wilfactin and once after IV desmopressin (Fig 1A). The lack of VWF:RCo recovery after the administration of VWF concentrate 5 weeks after exposure to Haemate P, in contrast to the recovery of autologous VWF:RCo after desmopressin, strongly suggests the

presence of inhibitory alloantibodies against exogenouslyadministered VWF. The fall in platelet count after desmopressin administration phenotypically supports the Type 2B genotype. To determine the inhibiting activity of the anti-VWF antibodies ex vivo, VWF:RCo mixing studies were performed in which patient plasma was mixed with normal pool plasma. Anti-VWF antibody titrations were performed via enzymelinked immunosorbent assay (ELISA). The VWF:RCo only partially corrected upon mixing with an equal volume of normal plasma, which is indicative for the presence of a VWF inhibitor. VWF:RCo was further corrected with an increasing concentration of normal plasma pool (NPP) from 335% (50% NPP) to 375% (75% NPP). The inhibitory function declined after 2 h incubation at 37°C, with VWF:RCo correcting from 50% (50% NPP) to 100% (75% NPP) (Fig 1B). This loss of inhibitory activity after incubation is different from the patterns observed in FVIII inhibitors. Also, the VWF:RCo inhibitor activity remained stable for at least 16 months, despite no additional exposures to VWF concentrates. We speculated that the anti-VWF antibody might be directed against the VWF-A1 domain because of the patients’ specific gene mutation. Further assessment of antibody domain specificity was therefore attempted by direct coating of the VWF-A1 domain and VWF-delta-A1 (VWF lacking the A1 domain). By direct immobilization of VWF on a surface, the



Fig 2. ELISA antibody measurement. The red lines represent patient plasma and the black lines represent control plasma [normal plasma pool (NPP)]. (A) The two upper panels depict IgM/IgG antibodies against directly coated purified human von Willebrand factor (VWF) (left) and against Haemate P (right). (B) The lower panels represent IgM/IgG antibodies against captured VWF (left) and directly coated VWF delta A1 (right). Low plasma dilutions resulted in aspecific binding (above dotted line). ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 424–439


Correspondence structure of the molecule changes to its active platelet binding conformation (Savage et al, 1996; Groot et al, 2009). IgM and IgG class antibodies were detected against purified human VWF and Haemate P that was directly immobilized on a microtitre plate (Fig 2A). However, no reactivity towards VWF could be observed when VWF was in resting conformation (i.e., captured via coating with an anti-VWF antibody), indicating possible antibody specificity towards the active conformation of VWF. Antibodies directed against VWF-delta-A1 could not be detected, further supporting this possible antibody specificity (Fig 2B). No anti-FVIII antibodies were detected with either the Nijmegen Bethesda assay or ELISA with immobilized recombinant FVIII. The inhibitory activity of the patients’ VWF antibodies in the VWF:RCo assay (Fig 1B) in combination with the results from the coating experiments (Fig 2) suggest the presence of antibodies against the active platelet-binding conformation of VWF. VWF interacts with platelet receptor GP1b via its A1domain, therefore antibody reactivity towards VWF lacking the A1-domain was assessed. No antibody binding could be observed, suggesting that the antibodies in this patient bind to a region comprising part of the A1-domain. This finding is in contrast to suggestions in earlier studies that a single epitope on VWF will not be found in VWD inhibitor patients (L opez-Fernandez et al, 1988; Batlle et al, 1997; Tout et al, 2000). However, we could not demonstrate antibody reactivity towards the A1-domain itself. The heterozygous nature of the R1308C mutation implies that the patients’ VWF also contains normal A1-domains. However, the patients’ VWF will be composed of both normal VWF and VWF exhibiting the R1308C mutation. We hypothesize that the nature of the mutation will lead to preferred exposition of the mutated A1 domain. This could explain the immune response against the administered large amounts of normal VWF and the observed relative good response to desmopressin with significant drop in platelet count. Although the pathophysiological mechanism is not fully understood, this case clearly demonstrates that antibody formation should be considered in non-type 3 VWD when insufficient recovery of VWF:RCo is observed after administration of VWF concentrate.

References Ahmad, F., Jan, R., Kannan, M., Obser, T., Hassan, M.I., Oyen, F., Budde, U., Saxena, R. & Schneppenheim, R. (2013) Characterisation of mutations and molecular studies of type 2 von Willebrand disease. Thrombosis and Haemostasis, 109, 39–46. Batlle, J., Loures, E., Vila, P., Hernandez, M.C., Mendez, J.A., Torea, J., Rendal, E., Couselo, M.J., Filgueira, A. & L opez Fernandez, M.F. (1997) Alloantibody from a patient with severe von Willebrand disease inhibits von Willebrand factor-FVIII interaction. Annals of Hematology, 75, 111–115.


Acknowledgements We thank the patient for cooperation and additional blood sampling.

Authorship M. Baaij co-wrote the manuscript, performed laboratory research, analysed and interpreted data. K.P.M. van Galen initiated case publication, co-wrote the manuscript, analysed and interpreted data. R.T. Urbanus supervised laboratory assessments, analysed and interpreted data, commented on intellectual content of the manuscript. J. Nigten initiated publication, commented on intellectual content of manuscript and laboratory assessments. H.C.J. Eikenboom commented on the laboratory assessments and intellectual content of the manuscript, expert contributions to the manuscript on the genetics of VWD. R.E.G. Schutgens supervised and commented on the intellectual content of the manuscript and accountability of the final version. There were no potential conflicts of interest. Marije Baaij1 Karin P. M. van Galen2 Rolf T. Urbanus1 Jeannet Nigten3 Jeroen H. C. Eikenboom4 Roger E. G. Schutgens2 1

Department of Clinical Chemistry and Haematology, University Medical

Centre Utrecht, 2Department of Haematology/Van Creveldkliniek, University Medical Centre Utrecht, 3Laboratory of Clinical Chemistry, Haematology and Immunology, Diakonessenhuis Utrecht, and 4Department of Thrombosis and Haemostasis and Einthoven Laboratory for Experimental Vascular Medicine, Leiden University Medical Centre, the Netherlands. E-mail: [email protected]

Keywords: von Willebrand disease, von Willebrand factor, antibodies, platelets First published online 7 April 2015 doi: 10.1111/bjh.13395

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remission. Journal of Thrombosis and Haemostasis: JTH, 7, 962–969. James, P.D., Lillicrap, D. & Mannucci, P.M. (2013) Alloantibodies in von Willebrand Disease. Blood, 122, 636–640. L opez-Fernandez, M.F., Martin, R., L opez-Berges, C., Ramos, F., Bosch, N. & Batlle, J. (1988) Further specificity characterization of von Willebrand factor inhibitors developed in two patients with severe von Willebrand disease. Blood, 72, 116–120. Savage, B., Saldıvar, E. & Ruggeri, Z.M. (1996) Initiation of platelet adhesion by arrest onto fibrin-

ª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 424–439

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Frontline therapy of severe aplastic anaemia with fludarabine, cyclophosphamide and ciclosporin

Acquired aplastic anaemia (AA) represents the failure of haematopoiesis resulting in a hypocellular bone marrow and pancytopenia. Severe aplastic anaemia (SAA) has a high rate of mortality without treatment. Antithymocyte globulin (ATG) and ciclosporin (CsA)-based immunosuppressive therapy is the standard treatment for SAA patients who are not eligible for haematopoietic stem cell transplantation (Scheinberg & Young, 2012), but the response is often incomplete and the cost of a course of ATG is too high for the patients in less developed countries. Moderate-dose cyclophosphamide is effective and cheap for treating SAA, but its toxicity and safety are debatable (Brodsky et al, 2010; Zhang et al, 2013; Scheinberg et al, 2014). Fludarabine is highly selective on lymphocytes and in vitro study on lymphocytes in chronic lymphocytic leukaemia showed that fludarabine inhibits repair of cyclophosphamide-induced DNA interstrand cross links, thus combination of cyclophosphamide with fludarabine produced more additive apoptotic cell death than the sum of each alone (Yamauchi et al, 2001). Based on past experience, we conducted a small scale study with eight SAA patients between September 2012 and October 2013 who received fludarabine, cyclophosphamide and CsA (FCC) as frontline therapy. The study was approved by Institutional Review Board of Blood Disease Hospital and was conducted in accordance with the Declaration of Helsinki. All of the patients or their legal guardians provided signed informed consent. Fludarabine was used at 50 mg/d intravenously for 5 d and cyclophosphamide was used at 10 mg/kg/d intravenously for 4 d. Additional cyclophosphamide of 10 mg/kg/week (range, 2–7 times) was used until the patient achieved transfusionindependence or emerging toxicity. Oral CsA was administered every 8 h and adjusted to maintain a serum level of 200–400 ng/ml or based on renal toxicity or tolerance. The protocol is demonstrated in Figure S1. Red cell and platelet transfusions were performed to maintain a haemoglobin level >70 g/l and platelet count >20 9 109/l. Broad-spectrum antibiotics were administered at first fever and adjusted according to microbial cultures and imaging studies. Criteria for haemaª 2015 John Wiley & Sons Ltd British Journal of Haematology, 2015, 171, 424–439

tological response was in accordance with the current definition (Marsh et al, 2009) and the toxicity of the protocol was evaluated according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0 (http:// Five males and three females with a median age of 355 (14–65) years were enrolled in this study. Patient characteristics are shown in Table I. Patient 2 also had mixed connective tissue disease, hepatic cirrhosis and esophageal varix. Among the three patients (Patients 3, 4 and 6) with Grade 3 infections at the time of hospitalization, Patient 4 was diagnosed with pulmonary fungal infection according to the clinical symptoms and image result. At admission, two patients (Patients 3 and 5) had received CsA alone or with granulocyte-colony stimulating factor for 1 and 25 months, respectively. The median follow-up time was 12 (range, 5–18) months with all patients achieving haematological response within 6 months (Table I and Fig 1). The median time to reach neutrophil count ≥05 9 109/l and platelet count ≥20 9 109/l was 205 (2–57) d and 35 (20–90) d, respectively. Total medical cost was US $12 919 (US $8115–36 646). Patient 2 ceased oral CsA after 4 months due to haemorrhage from esophageal varices. Her last follow-up was at 5 months and by that time she had became transfusion-dependent again. The median haemoglobin concentration, neutrophil count and platelet count of the other seven patients at last followup were 124 (85–143) g/l, 179 (153–355) 9 109/l and 140 (77–319) 9 109/l, respectively, and two patients achieved complete haematological response. The lymphocyte percentage and absolute count decreased significantly at 1 week after treatment. Lymphocyte subtypes analysis showed that CD3+ CD4+ T lymphocytes reduced significantly and remained at a low level for a long time (Figure S2). The bone marrow was ameliorated significantly at 6 months. Five patients showed an increased bone marrow cellularity and four patients were tested for bone marrow stem/progenitor cell colony-forming assay and showed great 427

First report of inhibitory von Willebrand factor alloantibodies in type 2B von Willebrand disease.

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