The Role of VWF:Ag II in Patients with Acquired von Willebrand Disease

von Willebrand Disease

M. Krause, C. Caron, I. Stier-Brück, T. Vigh and I. Scharrer

Introduction

Acquired von Willebrand syndrome (AVWS) is a rare disease, characterized by a late onset of bleeding diathesis, and absence of personal and family history of bleeding.

The clinical symptoms are similar to those of congenital von Willebrand syndrome (VWS). The majority of cases of acquired von Willebrand disease are associated with myeloproliferative, lymphoproliferative or autoimmune disorders, neoplasia, drugs and agents, and other disorders. Laboratory features are associated with nor- mal or prolonged bleeding time and/or activated partial thromboplastin time (APTT), a decreased ristocetin cofactor activity (VWF:RCo) or collagen binding activity (VWF:CBA), a reduced VWF:RCo/Ag or VWF:CBA/Ag ratio, and a reduced concentration of all the multimeric forms or selective loss of the largest multimeric form of von Willebrand factor simulating a type 1 or 2 von Willebrand disease. An additional test to characterize of acquired von Willebrand disease is the measure- ment of VWF:AgII.

Von Willebrand factor is a multimeric glycoprotein, that originates from the VWF precursor pro VWF and results in mature VWF and in the VWF propeptide, called VWF:AgII. Von Willebrand factor is synthesized by vascular endothelial cells and megakaryocytes. The biological functions of VWF and VWF:AgII are differing, VWF:AgII is contributed to intracellular, posttranslational multimerisation and tar- geting of VWF to storage vesicles. Systemic VWF propeptide levels can reflect more endothelial secretion processes than VWF levels do.

In patients with acquired von Willebrand disease, VWF is normally synthesized but rapidly removed from the circulation. Normal or increased levels of VWF:AgII compared to decreased levels of VWF:Ag show an accelerated removal of VWF:Ag from plasma. Decreased levels of VWF compared to low VWF:AgII levels might be a predictor of a decrease in VWF synthesis.

The aim of the present study was to assess the role of VWF:AgII in the diagno- sis of acquired von Willebrand disease in our patients.

I. Scharrer/W. Schramm (Ed.)

34

Hemophilia Symposium Hamburg 2003

” Springer Medizin Verlag Heidelberg 2005

Methods Blood Sampling

With informed consent, blood samples were collected by peripheral vein puncture into 3.8% trisodium citrate (Sarstedt tubes, Germany). Platelet poor plasma was prepared by centrifugation at 3000 g for 40 minutes at 4°C, aliquoted in polystyrene tubes, stored at -70°C and thawed immediately before the assay.

Assays of Coagulations Factors

APTT (normal range: 28–38 sec): conventional method; FVIII:C (normal range: 60- 150%): one-stage clotting assay, ACL; VWF:RCo (normal range: 62-131%): turbidi- metric method, BCT; VWF:Ag (normal range: 58-135%): in-house ELISA;

VWF:AgII (normal values: 81+/-17%): in-home ELISA; autoantibodies to VWF (AbVWF): in-house ELISA [de Romeuf C, Mazurier C. Comparison between plasma von Willebrand factor (VWF) and VWF antigen II levels in normal individuals and patients with von Willebrand disease. Thromb Haemost 1998; 80:37-41]

Other Methods

Platelet count: conventional methods; Bleeding time (normal range: <9.30 minutes):

template method, Simplate; Platelet function analyzer PFA-100, DADE AG (normal range collagen/ADP: 71-118 seconds; collagen/epinephrine: 85-165 seconds); Multi- meric analysis of von Willebrand factor: sodium dodecyl sulphate agarose gel elec- trophoresis

Statistical Analysis

The Chi-squared test was used for group comparison and P-values. P-values less than 0.05 were considered significant. All statistical analyses were performed using the BIAS program (BIAS. Windows 7.04, Germany).

Subjects

11 patients (female: 5/male: 6) with acquired von Willebrand syndrome, associated with Valproat replacement (n=3), essential thrombocythemia (ET; n=2), chronic lymphatic leukemia (CLL; n=1), chronic myeloid leukemia (CML; n=1), thrombotic thrombocytopenic purpura (TTP; n=1), Paget von Schroetter syndrome (n=1) and idiopathic form (n=2), were studied. At first onset, patients were aged 8-71 years.

None of the patients enrolled had bleeding and family histories for the von

Willebrand disease.

Laboratory characteristics in our patients shows Table 1.

22 healthy subjects and 19 patients with inherited VWS type 1 and 13 patients with VWS type 2 constituted the control group.

Results

The mean level of VWF:AgII (69%; range:131-47%) was increased as compared to the mean level of VWF:Ag (51%; range:74-6%) in the patients with AVWS. In con- trast to the patients with inherited VWS (VWF:AgII – mean level: 64% in type 1;

mean level: 81% in type 2) and the healthy subjects (VWF:AgII-level: 81%), the mean level of VWF:AgII in our study group was not different. Furthermore, in 10/11 patients with AVWS the levels of VWF:AgII were normal or increased. In only two of the patients VWF:AgII levels were decreased. The mean value of VWF:AgII/Ag ratio in the patients was 1.35 as compared to the ratio 1.63 in type 1 VWS, 2.53 in type 2 VWS and 0.76 in healthy controls. In 8/11 patients VWF:AgII/Ag ratio was about 1, and in 3 of these patients about 5 (range: >22-6.55).

We found in 3/11 patients VWF:AgII/Ag ratio lower than 1, associated with VWF:RCo levels <35% (range: 35-19%), compatible with an inherited VWS, but lack of bleeding history inspite of surgery and deliveries. None of the patients with Valproat treatment had ratio about 5 or positive autoantibodies to VWF.

In our study group we found positive autoantibodies to VWF in 3/11 patients (27%), only one of them had VWF:AgII/Ag ratio higher than 5, but in all of these patients the ratio was about 1. These patients were associated with CLL, CML or TTP.

The laboratory findings in our patients with AVWS are documented in Table 2.

Table 1. Laboratory characteristics

Pts. FVIII:C vWF:Ag vWF:RCo vWF:RCo/Ag multimeric

(%) (%) (%) form

1# 74 74 35 0.47 type 1

2# 47 13 26 2.00 type 1

3# 61 48 35 0.73 type 1

4# 21 20 <5 0.25 n.a.

5# 5 <6.25 <5 0.80 type 1

6# 61 51 64 1.25 type 1

7# 84 63 49 0.77 type 1

8# 46 69 19 0.27 type 1

9# 74 36 61 1.69 n.a.

10# 95 61 43 0.70 type 1

11# 128 56 56 1.00 type 1

Discussion and Conclusions

The acquired von Willebrand disease was diagnosed in all of our patients based upon the data from an international registry of acquired von Willebrand syndrome, and the guidelines for the diagnosis of the acquired von Willebrand syndrome the Scientific and Standardization Committee Communication of the International Society on Thrombosis and Hemostasis (SSC-ISTH).

Von Willebrand factor is synthesized normally in the majority of patients with AVWS, however it is rapidly removed by pathogenic mechanisms, associated with presence of autoantibodies to VWF, adsorption of VWF and proteolytic degradation of VWF. Patients with inherited VWS shows low VWF:AgII levels, as an indicator of a decreased VWF synthesis, compared to normal or increased VWF:AgII levels in patients with AVWS. A number of studies have shown that elevated VWF:AgII levels may occur in acute phases of vascular disease, which more reflect the endothelial cell activation.

In our study group of the patients with AVWS we found normal or increased levels of VWF:AgII compared to decreased levels of VWF:Ag, which reflects an accel- erated removal of VWF from the circulation. The ratio VWF:AgII/Ag of all patients was higher, but not significantly higher, as compared to the healthy controls (1.35 vs. 0.76; p=0.87). In 8/11 patients (73%) we found VWF:AgII/Ag ratio >1.0. These results are similar to the observations made by other investigators.

In the international registry of acquired von Willebrand syndrome, the prevalence of autoantibodies to VWF was 16% (27/170), that is similar to that reported in other studies. We found in our patients a strikingly high prevalence of autoantibodies (27%, 3/11). The presence of autoantibodies may be influence both the risk of bleed- ing and the response to treatment. In only one of our patients with antibodies bleeding episodes in case of surgery was documented. The bleeding tendency was not different in patients with autoantibodies compared to patients without auto- Table 2. Laboratory results

Pts. vWF:AgII (%) Ratio vWF:AgII/Ag Anti vWF IgG/IgM

1# 69 0.93 neg/neg

2# 113 8.69 neg/pos+++

3# 47 0.98 neg/neg

4# 131 6.55 neg/neg

5# 110 >22 neg/neg

6# 77 1.51 neg/neg

7# 85 1.35 neg/neg

8# 68 0.99 neg/neg

9# 66 1.83 neg/neg

10# 64 1.05 pos+/neg

11# 61 1.09 weak pos/neg

antibodies, but the patients with VWF:AgII/Ag ratio upper 5 were characterized by severe bleeding events.

– In our present study of patients with acquired von Willebrand syndrome we could demonstrate, that VWF:Ag II levels, the VWF:AgII/VWF:Ag ratio and the presence of antibodies to VWF appears to be helpful in the characterization be- tween inherited von Willebrand syndrome and acquired von Willebrand syn- drome and should be performed in suspected acquired von Willebrand syn- drome.

– This suggests an important role of VWF:AgII in pathogenesis, in the diagnosis and management of acquired von Willebrand syndrome.

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