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Keywords:

  • von Willebrand factor;
  • ADAMTS-13;
  • desmopressin;
  • factor VIII concentrate;
  • von Willebrand disease

Summary

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

ADAMTS-13, the metalloprotease that disposes physiologically of the most thrombogenic multimers of von Willebrand factor (VWF), tends to be low in plasma when VWF is high. We evaluated the behaviour of these two proteins in naturally occurring, experimental and clinical situations associated with VWF levels spanning from undetectable to supranormal. ADAMTS-13 was approximately 10% higher (and VWF 35% lower) in 65 healthy individuals of blood group O than in 65 individuals of groups A, B and AB. Thirty-three patients with type 3 von Willebrand disease (VWD) with undetectable plasma VWF had approximately 35% higher levels of ADAMTS-13 than a comparable group of healthy individuals with normal VWF. When VWF was raised to supranormal levels by desmopressin (DDAVP) in 10 healthy volunteers, ADAMTS-13 decreased by approximately 20%, with no change of the protease in three patients with severe VWD who had no post-DDAVP VWF rise. When VWF was raised from very low to normal levels by the infusion of VWF-containing plasma concentrates in four patients with type 3 VWD and one with type 1 VWD plasma, ADAMTS-13 decreased in parallel. These data show that throughout a large spectrum of plasma VWF levels there is a negative association between this protein and the activity of its major cleaving protease.

ADAMTS-13, the 13th member of the ADAMTS family of metalloproteases characterized by the combination of A disintegrin-like and metalloprotease with ThromboSpondin type 1 motif (Hurskainen et al, 1999), is the main physiological modulator of the size of von Willebrand factor (VWF) in plasma. VWF, synthesized in endothelial cells and megakaryocytes and circulating in plasma as a series of multimers of up to 20 000 kDa in size, is essential for platelet adhesion and thrombus formation (Sadler, 1998). Its deficiency or dysfunction causes the inherited bleeding disorder, von Willebrand disease (VWD), whereas high plasma levels are associated with an increased risk of myocardial infarction and sudden death (Jansson et al, 1991; Thompson et al, 1995). That the functions of VWF and ADAMTS-13 in platelet thrombus formation are intimately intertwined is epitomized by the observation that, in patients with thrombotic thrombocytopenic purpura, the congenital or acquired deficiency of ADAMTS-13 impairs the physiological process of cleavage of the largest, highly thrombogenic multimers of VWF which, when present in plasma and/or on the endothelial cell surface, lead to massive intravascular aggregation and platelet thrombus formation in the terminal circulation of multiple organs (Moake et al, 1982; Furlan et al, 1998; Tsai & Lian, 1998).

In the last few years, ADAMTS-13 has been purified from plasma to homogeneity (Fujikawa et al, 2001; Gerritsen et al, 2001; Zheng et al, 2001), the coding gene has been located on chromosome 9 and several DNA mutations have been identified in patients with inherited deficiency (Levy et al, 2001; Kokame et al, 2002). Relatively little information is available on the conditions that maintain and regulate ADAMTS-13 levels in human plasma. It is known from in vitro studies and liver transplantation that ADAMTS-13 is mainly synthesized in the liver (Matsumoto et al, 2000; Levy et al, 2001) and that in several conditions associated with an increase in plasma of its substrate VWF, ADAMTS-13 levels are low (Mannucci et al, 2001; Reiter et al, 2003). This relationship was observed both when the increase of plasma VWF was sustained – as in chronic inflammation and during the postoperative state (Mannucci et al, 2001) – and when it was short-lasting – as after the infusion of desmopressin (DDAVP) (Reiter et al, 2003).

A negative association between VWF and ADAMTS-13 may have clinical implications. Because high levels of plasma VWF are a risk factor for cardiovascular disease (Jansson et al, 1991; Thompson et al, 1995), their association with low levels of the main VWF-cleaving protease may contribute to increase this risk. While this hypothesis can only be validated by measuring ADAMTS-13 in patients with both prevalent and incident atherothrombotic disease, we chose to investigate the relationship between VWF and ADAMTS-13 by measuring the protease in natural, experimental and clinical conditions known to be associated with a large spectrum of plasma VWF levels, spanning from undetectable to higher than normal.

Blood collection and plasma preparation

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Nine volumes of venous blood were collected from patients or healthy individuals into one volume of 0·129 mol/l sodium citrate, platelet-poor plasma was obtained by centrifugation at 1500 × g for 20 min, snap frozen and stored at −80°C until tested (see below). This research protocol was approved by the Institutional Review Board of the University of Milan.

Patients with type 3 VWD

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Thirty-three patients were previously diagnosed on the basis of an autosomal recessive pattern of inheritance, undetectable levels of VWF antigen (VWF:Ag) in plasma (<1% of average normal plasma, normal laboratory range 51–154%) and undetectable or low levels of factor VIII coagulant activity (ranging from <1 to 10%, normal range: 48–161%). Fourteen were men, 19 women with a median age of 20 years (range: 1–59). They had a history of moderately severe clinical symptoms (haemathroses, haematomas, epistaxis, gastrointestinal bleeding, menorrhagia and postoperative bleeding) that in the past had often demanded treatment with plasma fractions containing factor VIII and VWF. The majority of these multitransfused patients were positive for anti-hepatitis C virus (HCV) but none had a significant impairment of the synthetic function of the liver. At the time of blood sampling for this study no patient had been treated with replacement therapy for at least 1 week.

Treatment with DDAVP

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Ten healthy volunteers (eight men and two women, median age 39, range: 26–50 years) and three female patients, two with type 3 VWD and one with type 1 VWD and very low levels of VWF:Ag (2%), were infused with DDAVP, 0·3 μg/kg body weight in 50 ml saline over 30 min. Blood samples were obtained before and 1, 2, 6 and 24 h after the start of the infusion.

Treatment with plasma fractions

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Five patients needed replacement therapy with plasma concentrates of factor VIII and VWF to manage spontaneous bleeding episodes. Four had type 3 VWD, one had type 1 VWD with very low levels of VWF:Ag (2%) and she had had an inadequate response to DDAVP at the time of a test dose. Citrated blood samples collected and processed as described above for VWF:Ag and ADAMTS-13 were obtained before concentrate infusion at 15, 30 and 60 min postinfusion and then at various time intervals during the first 24–48 h postinfusion. The virus-inactivated concentrates containing factor VIII and VWF used for these patients were Hemate-P (Aventis Behring, Milano, Italy) in four cases and Fanhdi (Grifols, Pisa, Italy) in the remaining case. The latter concentrate has features similar to that previously evaluated in a prospective clinical study (Mannucci et al, 2002).

Data expression and statistical analysis

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

The VWF:Ag and ADAMTS-13 values were expressed as a percentage of average normal plasma, i.e. citrated plasma pooled from at least 50 healthy individuals of both sexes or, for DDAVP experiments, as a percentage of baseline values, taken as 100%. The results of both measurements were not normally distributed, according to a Kolmogorov–Smirnov test. To compare values before and at various time intervals after treatment with plasma fractions or DDAVP, anova according to Kruskal–Wallis and the non-parametric Mann–Whitney U-test were employed. For descriptive purposes, results are presented as mean ± SD. P-values of <0·05 were considered statistically significant.

Blood groups

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Healthy individuals of blood group O had, on average, significantly lower plasma levels of VWF:Ag than individuals with ABO blood groups other than O (mean ± SD: 99 ± 35 vs. 134 ± 48, P < 0·0001). Mean ADAMTS-13 levels were slightly but statistically significantly higher in individuals with blood group O than in those with blood groups non-O (102 ± 29 vs. 91 ± 28, P-value 0·022) (Fig 1).

image

Figure 1. Values of von Willebrand factor antigen (VWF:Ag) (top) and ADAMTS-13 (bottom) in 65 healthy individuals of blood group O compared with an equal number of individuals of blood groups non-O (A, B and AB). Values are expressed as a percentage of average normal plasma levels (taken as 100%) and the horizontal lines indicate the median values (see also Results for more details).

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Type 3 VWD patients

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

At baseline conditions, 33 patients with undetectable plasma levels of VWF:Ag had significantly higher mean plasma levels of ADAMTS-13 than a control group of 33 healthy individuals with normal levels of VWF:Ag (103 ± 33) matched with patients for sex, age and blood group (136 ± 49 vs. 98 ± 31, P = 0·0006) (Fig 2).

image

Figure 2. Values of ADAMTS-13 (expressed as a percentage of average normal plasma levels) in 33 patients with type 3 von Willebrand disease (VWD) and unmeasurable levels of von Willebrand factor antigen (VWF:Ag) and in 34 healthy individuals with normal VWF:Ag comparable for age, sex and blood group. The horizontal bars show median values (see also Results for more details).

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Treatment with DDAVP

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Ten healthy individuals infused with DDAVP showed a highly significant two- to threefold relative increase of VWF:Ag, peaking at 1–2 h postinfusion and tending to return to baseline values thereafter (Table I). At the same postinfusion times there was a statistically significant relative decrease of ADAMTS-13 (P = 0·0025 at 1 h, 0·002 at 2 h), with a progressive return to baseline values at 6 (P = 0·45) and 24 h postinfusion (P = 0·45). Similar patterns of changes and degrees of statistical significance were observed when absolute values of both measurements were analysed instead of values relative to baseline values taken as 100% (data not shown). Table II shows that in three patients with VWD-treated as above with DDAVP ADAMTS-13 levels had small fluctuations around baseline values and no change in VWF:Ag levels (data not shown).

Table I.  Changes in ADAMTS-13 and von Willebrand factor antigen (VWF:Ag) plasma levels in 10 healthy volunteers examined at baseline conditions and at various time intervals for 24 h after infusion of 0·3 μg/kg of desmopressin (DDAVP).
Time after DDAVP (h)Mean (%)ADAMTS-13 (SD)P-valueMean (%)VWF:Ag (SD)P-value
  1. Values of both measurements are expressed as a percentage relative to baseline values (taken as 100%).

Baseline100  100  
179130·002523042<0·0001
282110·002212390·0002
695240·45170410·0002
24102130·45110220·26
Table II.  Changes in ADAMTS-13 plasma levels in three patients with severe von Willebrand disease (VWD) examined at baseline and at various time intervals for 24 h after infusion of 0·3 μg/kg of desmopressin (DDAVP).
Patients withBaseline levels (%)After 1 h (%)After 2 h (%)After 6 h (%)After 24 h (%)
  1. Values of ADAMTS-13 are expressed as a percentage of average normal plasma levels.

Type 3 VWD11398109106110
Type 1 VWD10111490104101
Type 3 VWD134126131110125

Treatment with plasma concentrates

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

Figures 3 and 4 show the absolute changes of VWF:Ag and ADAMTS-13 levels after the infusion of plasma concentrates in four patients with type 3 VWD and in one with type 1 VWD, the latter with detectable but very low levels of VWF:Ag. In the first three postinfusion plasma samples taken within 1 h postinfusion, the rise of VWF:Ag to normal levels attained after replacement therapy was paralleled by a concomitant decrease of ADAMTS-13. Subsequently, ADAMTS-13 levels tended to return to baseline values, paralleling the progressive decrease of VWF:Ag levels (Figs 3 and 4).

image

Figure 3. Changes in von Willebrand factor antigen (VWF:Ag) (expressed as a percentage and indicated by circles) and ADAMTS-13 plasma levels (indicated by triangles) in two patients with type 3 von Willebrand disease (VWD) (panel A and C) and in a patient with severe type 1 VWD (panel B). Patients were treated at the time indicated by arrows with the concentrate Hemate-P, at dosages of 60 IU/kg (patient A), 60 IU/kg (patient B) and 80 IU/kg (patient C).

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image

Figure 4. Changes in von Willebrand factor antigen (VWF:Ag) (indicated by circles) and ADAMTS-13 (indicated by triangles) in two patients with type 3 von Willebrand disease (VWD) treated with the concentrates Fanhdi® (panel A) and Hemate-P® (panel B), at dosages of 70 IU/kg and 60 IU/kg respectively. The patients shown in panel B received an additional dose of 20 IU/kg at 24 h.

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Discussion

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References

In this study, four situations characterized by VWF levels ranging from undetectable (type 3 VWD) to higher than normal (post-DDAVP) were chosen to establish whether or not ADAMTS-13 changed in the opposite direction. Immunoreactive VWF (VWF:Ag) was chosen as measurement of the total mass of this moiety, whereas ADAMTS-13 was measured by a functional assay as no immunoassay of the protease was available at the time of this study. We have previously shown that this functional assay is not influenced by high levels of endogenous VWF:Ag in test plasmas, because similar protease values were obtained before and after cryoprecipitation, which markedly reduces VWF:Ag levels (Mannucci et al, 2001).

As expected, in persons with blood group O, the plasma levels of VWF:Ag were approximately 35% lower than in individuals with blood groups non-O. In contrast, levels of ADAMTS-13 activity were approximately 10% higher than in persons of blood groups non-O, although there was much overlap of values. To corroborate the existence of an inverse relationship between VWF and ADAMTS-13, patients with type 3 VWD were chosen as a natural model of undetectable VWF. They had approximately 35% higher mean protease levels than a group of healthy persons with normal VWF that were matched with patients for features that may influence plasma protease levels (gender, age and blood group). Five patients had an ADAMTS-13 value of 200% or higher (Fig 2), and were the highest values ever measured by us in clinical samples (Mannucci et al, 2001).

Two models of moderate increases of VWF:Ag levels were also evaluated. Reiter et al (2003) have shown that, in healthy volunteers, the increase of VWF supranormal levels induced by DDAVP through the release of the protein from endothelial cell Weibel-Palade bodies was paralleled by an average of 20–30% decrease of ADAMTS-13. Because we had previously found no significant post-DDAVP decrease of ADAMTS-13 (Mannucci et al, 2001), perhaps because the number of individuals investigated at that time was insufficient to avoid a type 2 error, we repeated the experiments of Reiter et al (2003). In 10 healthy individuals, the pattern and degree of changes of the two moieties was in the same direction of those found by Reiter et al (2003), i.e. a 10–20% decrease of ADAMTS-13 paralleled by a much more marked, two- to threefold, rise of VWF:Ag. That the decrease of ADAMTS-13 was mediated by the increase of VWF and not by a direct effect of DDAVP was shown when this drug was administered experimentally to patients with type 3 VWD. As expected from previous knowledge that type 3 VWD is unresponsive to DDAVP because patients lack mobilizable VWF in Weibel-Palade bodies, the VWF:Ag remained undetectable after DDAVP administration. ADAMTS-13 levels were accordingly unmodified, with small postinfusion fluctuations within the degree of reproducibility of the assay.

The other model of VWF increase chosen for this study was replacement therapy with VWF-containing plasma concentrates in patients with severe VWD with undetectable or very low pretreatment levels of this moiety. This model is different from that provided by DDAVP: the source of VWF is exogenous while it is endogenous after DDAVP, and the increase of VWF from undetectable or very low to normal values is much more rapid and longer lasting. Again, VWF:Ag and ADAMTS-13 showed opposite patterns of change. Soon after infusion and the attainment of normal plasma levels of VWF, ADAMTS-13 decreased to baseline values when exogenous VWF began to be cleared from plasma. The decrease of ADAMTS-13 plasma levels occurred in spite of the fact that VWF-containing concentrates also contain small amount of ADAMTS-13 that were not specifically quantified in the batches used here.

These studies show the existence of an inverse relationship between the plasma levels of VWF and ADAMTS-13, although the absolute changes of ADAMTS-13 are smaller than those of VWF. The limited degree of manipulation of the experimental conditions that are possible in humans did not permit us to ascertain the mechanism of this phenomenon, so that the biological significance of these findings is uncertain. Low ADAMTS-13 levels might be due to the ‘sequestration’ of the protease to the vascular endothelial cells when the latter are secreting increasing amounts of VWF (Dong et al, 2002), but this hypothetical mechanism does not account for the high levels of ADAMTS-13 found in the presence of undetectable VWF in type 3 VWD, or for the protease drop observed in the same patients when plasma VWF is restored to normal levels by concentrate infusion. The changes of ADAMTS-13 occur too rapidly to postulate that the plasma levels of VWF modulate the expression of the ADAMTS-13 gene, leading to its down-regulation when they are high or to up-regulation when they are low.

To summarize; high levels of VWF, a risk factor of coronary heart disease (Jansson et al, 1991; Thompson et al, 1995), are accompanied by low levels of the main protease that cleaves the largest, highly thrombogenic forms of this protein. Whether or not there is an independent role for low ADAMTS-13 levels in determining an increased risk of cardiovascular disease can only be established by clinical association studies based on the parallel measurements of the two proteins. A preliminary study carried out in a small group of 14 elderly women with chronic coronary heart disease found that ADAMTS-13 was low and VWF:Ag high (Yoo et al, 2003). To obtain more information on the role of ADAMTS-13 in cardiovascular disease is not only of mechanistic interest, because preparations of ADAMTS-13, which are produced by recombinant DNA technology to treat patients with thrombotic thrombocytopenic purpura with low or undetectable levels of the protease (Plaimauer et al, 2002), might have a potential application in the control of this risk.

References

  1. Top of page
  2. Summary
  3. Patients and methods
  4. Blood collection and plasma preparation
  5. Healthy individuals with different blood groups
  6. Patients with type 3 VWD
  7. Treatment with DDAVP
  8. Treatment with plasma fractions
  9. ADAMTS-13 activity assay
  10. VWF:Ag assay
  11. Data expression and statistical analysis
  12. Results
  13. Blood groups
  14. Type 3 VWD patients
  15. Treatment with DDAVP
  16. Treatment with plasma concentrates
  17. Discussion
  18. Acknowledgments
  19. References
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