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Keywords:

  • clearance;
  • desmopressin;
  • hemophilia A;
  • Type 1 von Willebrand disease;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Summary.  The mechanism of von Willebrand factor (VWF) clearance is not fully understood. The factors that affect VWF clearance, and the normal in vivo mechanism of clearance, may be relevant to the pathogenesis of Type 1 von Willebrand disease (VWD), in which there is a partial deficiency of VWF. In order to investigate the clearance of VWF in Type 1 VWD, the current study assessed the half-life of VWF antigen (t½ VWF:Ag) in Type 1 VWD patients and individuals with mild hemophilia A following the administration of 1-deamino-8-d-arginine vasopressin (DDAVP; desmopressin). To date 20 individuals have been assessed, 13 with Type 1 VWD and seven with mild hemophilia A. The median t½ VWF:Ag in the Type 1 VWD and mild hemophilia A groups were 4.6 h and 9.5 h, respectively. The difference between the t½ VWF:Ag for the two groups was significant, P < 0.02. Analysis of the data showed a correlation between the t½ VWF:Ag and the baseline VWF:Ag level prior to administration of DDAVP: lower baseline VWF:Ag levels were associated with a shorter t½ VWF:Ag. These data suggest that increased clearance of VWF may be the pathogenic mechanism in some cases of Type 1 VWD.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

The commonest inherited bleeding disorder is von Willebrand disease (VWD), which results from either a quantitative or qualitative deficiency of the glycoprotein, von Willebrand factor (VWF). VWD can be divided into three broad subtypes [1] on the basis of phenotypic data: Type 1 (partial quantitative deficiency), Type 2 (qualitative variants) and Type 3 (severe quantitative deficiency). The commonest subtype is Type 1, in which there is a proportionate decrease in both the VWF antigen and the functional assays of VWF. Since the cloning of the VWF gene [2], there has been an increased understanding of the molecular basis of VWD, especially Type 2 VWD. This is not the case for Type 1 VWD, in which variable expression and penetrance [3], and the size of the VWF gene have hampered molecular investigation. The molecular basis of autosomal Type 1 VWD has only been determined in a limited number of individuals [4,5], while other studies have found inconsistent linkage between the VWF gene and the phenotype and of a partial quantitative deficiency of VWF [6,7]. These observations are consistent with a partial quantitative deficiency of VWF, representing a complex disorder in which a molecular diagnosis is currently not attainable in all cases. Recently, work on a murine model of Type 1 VWD has drawn attention to the possibility that genes outside the VWF locus may be responsible for this phenotype [8]. In this murine model the partial quantitative deficiency of VWF results from an increased clearance of VWF. This raises the question: does a similar pathogenic mechanism operate in humans?

The synthetic analog of vasopressin, 1-deamino-8-d-arginine vasopressin (DDAVP), has been used in the management of VWD and mild hemophilia A for a considerable time. Early studies demonstrated an increase in factor (F) VIII [9,10] and VWF levels [11] after the infusion of DDAVP. Following these initial studies, DDAVP was shown to be effective in the prevention and treatment of bleeding episodes in individuals with mild hemophilia A and VWD [12]. In the present study, the half-life (t½) of VWF antigen was measured following administration of DDAVP to individuals with Type 1 VWD and mild hemophilia A, to assess the relative clearance in these two groups.

Patients and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

Subjects

Patients were recruited from the haemophilia centers at the University Hospital of Wales, Cardiff and the Royal Free Hospital, London. Patients were enrolled after giving informed consent. The study received local ethics committee approval at both centres. Patients with Type 1 VWD and mild hemophilia A between the ages of 19 and 58 years were recruited. Type 1 VWD was defined as a partial quantitative deficiency of VWF according to the International Society on Thrombosis and Haemostasis recommendations [1]. Individuals with a partial quantitative deficiency of VWF had a VWF ristocetin cofactor activity : VWF antigen ratio (VWF:Rco : VWF:Ag) > 0.7, a normal VWF multimeric pattern, and VWF:Ag and VWF:ΡCo levels < 50 IU dL−1. Patients on any regular medication, who had known arterial or thrombotic disease, or a confirmed inhibitor (present or historical), were excluded from the study. Patients were excluded if they had abnormal urea, creatinine or liver function tests.

VWF:Ag estimation after DDAVP infusion

DDAVP was infused intravenously over 15 min after the patients had rested for a minimum of 20 min. The dose given was 0.3 μg kg−1. Venous blood samples were taken just minutes prior to the start of the DDAVP infusion (time 0, T0) and then hourly for 6 h from the start of the infusion. Whole venous blood was collected into tri-sodium citrate (109 mmol L−1, 10% v/v). Platelet-poor plasma was prepared and stored at −70 °C until assayed. The VWF:Ag was assayed using a standard ELISA assay as previously described [13].

Calculaton of t½ VWF:Ag

The half-life was calculated by calculation of the first-order rate constant for the elimination phase from the slope of the VWF:Ag concentration against time, according to the following formula [14]:

  • image

where C(t) = concentration of VWF:Ag in plasma as a function of time, C0 = VWF:Ag concentration at time 0, e= base for natural logarithms, kE= first-order rate constant for the elimination phase and t = time. Peak VWF:Ag levels are attained by 1 h, and so the 1 h VWF:Ag level was taken as the start of the elimination phase and a best-fit line was calculated by regression analysis from this timepoint onwards. This assumes a distribution phase of up to 1 h for VWF:Ag. The T0 VWF:Ag value was subtracted from post-DDAVP VWF:Ag values before logarithmic transformation and calculation of the best-fit line. This method to calculate VWF half-life data has been used in previous studies [15–17].

Statistics

Results are reported as median values. Spearman's rank correlation and the Mann–Whitney test were used to analyze the data.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

The t½ VWF:Ag was measured in 20 patients in total: 13 patients with Type 1 VWD and seven patients with mild hemophilia A. The median values for the basal levels of FVIII:C, VWF:Ag and VWF:ΡCo for the individuals with Type 1 VWD were 70 IU dL−1, 33 IU dL−1 and 36 IU dL−1, respectively. The median value for the VWF:ΡCo : VWF:Ag ratio was 0.9 (range 0.7–2.3). The corresponding median values for the hemophilia A group were: FVIII:C, 17 IU dL−1; VWF:Ag, 131 IU dL−1; and VWF:ΡCo, 178 IU dL−1.

The median t½ VWF:Ag for the patients with Type 1 VWD was 4.6 h, whereas the median t½ VWF:Ag for the patients with mild hemophilia A was 9.5 h (Table 1). Analysis of the half-life data using the Mann–Whitney test demonstrated a significant difference between the two groups (P < 0.02). Only three of the VWD patients (1V, 10V and 13V; 1 h VWF:Ag levels 48 IU dL−1, 21 IU dL−1 and 48 IU dL−1, respectively) failed to normalize their VWF:Ag level post-DDAVP. Potentially their VWD would be consistent with the Type 1 platelet-low VWD described by Mannucci and colleagues [18]. Further analysis of the data from all 20 individuals showed no correlation between their age and the t½ VWF:Ag (rs = 0.35, P > 0.1). However, there was a strong correlation between the VWF:Ag level at T0 and the t½ VWF:Ag (rs = 0.84, P < 0.002), with low T0 VWF:Ag levels correlating with a short VWF:Ag half-life. Analysis of the two groups revealed that this correlation was solely due to the Type 1 VWD group (rs = 0.89, P < 0.002).

Table 1.  Characteristics of patients, VWF:Ag half-life (t½ VWF:Ag) post-DDAVP, absolute increase of VWF:Ag 1 h post-DDAVP and the ratio of VWF:Ag increase from time 0–1 h
PatientSexABO blood groupAge (years)Baseline FVIII:C (IU dL−1)Baseline VWF:Ag (IU dL−1)Baseline VWF:RCo (IU dL−1)t½ VWF:Ag (h)Ratio of VWF:Ag 1 h post-DDAVP: T0Increase in VWF:Ag (IU dL−1) from T0–1 h post-DDAVP
1 AMaleA571923824026.11.7166
2 AMaleO3133939910.12.3122
3 AMaleB353816830010.11.8135
4 AMaleB54101921789.52192
5 AMaleO56461489.13.3138
6 AMaleO4451311966.92.5200
7 AMaleO261782824.23162
1 VFemaleO332713< 103.63.735
2 VFemaleNT21219< 101.515.9104
3 VFemaleO197845364.53.8127
4 VFemaleA51104404211.42.9101
5 VFemaleNT307043394.63.4102
6 VFemaleA449549414.92.365
7VMaleA521814321.36.171
8VMaleA357633435.22.157
9VMaleNT4513145478.62.465
10VMaleA58451092.6314
11VMaleA364830236.33.3157
12VMaleO4070474655188
13VMaleO335217142.92.529

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References

The method employed in this study to calculate the clearance of VWF:Ag has been previously employed to estimate the t½ VWF:Ag in normal individuals [15, 16]. In the latter study, the 95% confidence interval (CI) for the t½ VWF:Ag for normal control groups was 10.5–14.3 h (n = 9). The data for the t½ VWF:Ag in the first report were given as the mean clearance in minutes ± standard error. The t½ VWF:Ag values for the normal controls (n = 7) and mild hemophilia group (n = 11) were 563 ± 35 min and 593 ± 41 min, respectively. From these data, the respective 95% CI for the t½ VWF:Ag of each group can be calculated as 8.2–10.5 h and 8.5–11.2 h [16], and show agreement for estimates of VWF clearance in these two groups. The data presented in the current study for seven mild hemophilia A patients compare well with the published data, with a median t½ VWF:Ag of 9.5 h. In contrast, the individuals with Type 1 VWD in the present study have a median t½ VWF:Ag of 4.6 h. The difference between the two groups in the current study is significant, P < 0.02. This contrasts with the finding of Mannucci et al.[16], who did not find a significant difference in the t½ VWF:Ag between ‘classical’ VWD (mean t½ VWF:Ag of 451 ± 98 min, n = 11) and either a normal control group or individuals with mild hemophilia A. However, their data did show a wider range of the 95% CI in the ‘classical’ VWD group compared with the other two groups, with a calculated 95% CI of 4.3–10.7 h for the t½ VWF:Ag in the 11 VWD patients investigated. Together these data suggest that a shorter t½ VWF:Ag may be prevalent among individuals with Type 1 VWD compared to normal individuals or individuals with mild hemophilia A.

Studies have demonstrated discordant values of clearance between functional assays of VWF and VWF:Ag post-DDAVP. This difference reflects the effects of rapid proteolysis of VWF, and loss of the highest molecular weight multimers of VWF on functional assays, as well as the clearance of the glycoprotein [15,19,20]. As the aim of this study was to assess the clearance of VWF in vivo, the clearance of VWF:Ag was measured, in order to avoid this potential confounding effect.

Data from a murine model of autosomal dominant Type 1 VWD has demonstrated that an increased clearance of murine VWF accounts for the phenotype [8]. Similarly, the data from the present study suggest that increased clearance may be the mechanism underlying low VWF levels in some patients with Type 1 VWD. Increased clearance may reflect either a change in the VWF molecule per se (such as an amino-acid substitution or altered glycosylation) that leads to its increased clearance, or it may reflect increased functionality of the VWF clearance mechanism in some individuals. The multimer profile for each of the Type 1 patients included in this study appeared normal, therefore if an amino-acid substitution or change in glycosylation were present, it was not evident using this analytical approach.

Further analysis of the data demonstrated a correlation between the baseline VWF:Ag level, which is higher in individuals with mild hemophilia A, and the t½ VWF:Ag value. One possible explanation for this result is that the clearance of VWF becomes saturated at higher VWF concentrations. Information on the in vivo pathways involved in VWF clearance and their kinetics is limited. However, pharmacokinetic (PK) studies with cryoprecipitate and high-purity VWF concentrates in Type 3 VWD have obtained values for t½ VWF:Ag that are comparable to the values obtained in mild hemophilia A and normal individuals post-DDAVP [21–23]. This suggests that saturation of the clearance pathway is an unlikely explanation for the correlation of t½ VWF:Ag and baseline VWF:Ag level. A PK study with VWF concentrate in individuals with Type 3 VWD demonstrated that the volume of distribution is equivalent to the plasma volume [24]. This is consistent with the lack of an extravascular compartment for VWF, which if present, would be associated with a rapid fall-off due to equilibration of VWF between these compartments and may have influenced the t½ VWF:Ag calculated in this study. Data from animal studies suggest that the composition of the oligosaccharide side-chains on VWF may play an important role in VWF clearance [8, 25]. This limited data suggest that factors intrinsic to VWF may be an important determinant of VWF clearance.

The data from the present study have potential therapeutic relevance. The standard assessment for the suitability of DDAVP therapy in VWD relies on the measurement of FVIII and VWF levels 30–60 min after an intravenous infusion. The responsiveness of VWD to DDAVP using such a trial dose is approximately 90% [26]. If the results of such a trial dose are deemed satisfactory, DDAVP is then employed as hemostatic cover and given at 12–24-hourly intervals. There are very few studies that address the clinical efficacy of DDAVP [27], especially as a single therapeutic agent with no concomitant administration of antifibrinolytics. The half-life data presented here would suggest that, dependent on the necessity to maintain normal VWF levels in order to prevent or stop bleeding, DDAVP given 12-hourly is potentially an ineffective therapy in some Type 1 VWD patients who require repeated doses of DDAVP. However, the rapid fall in VWF levels post-DDAVP in some individuals with Type 1 VWD may be compensated by other hemostatic effects of DDAVP [28]. Large clinical studies analyzing the patient's clinical response and relating this to the VWF half-life would answer this question. However, these data do question the validity of the ‘accepted’ method of assessing the response to DDAVP.

In conclusion, some individuals with Type 1 VWD have a shortened t½ VWFAg compared to individuals with normal VWF levels. This may reflect an increased clearance of VWF, which would therefore be one potential pathogenic mechanism in Type 1 VWD. Because a partial quantitative deficiency of VWF appears to be a complex disorder, this study could not address the mechanism of the increased clearance observed. The accumulating data on t½ VWF:Ag in Type 1 VWD underline the need for further studies and a better understanding of the in vivo mechanisms of VWF clearance both intrinsic and extrinsic to VWF.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and methods
  5. Results
  6. Discussion
  7. References
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