von Willebrand factor (vWF) exists as a multimeric glycoprotein, consisting of a series
of multimers with varying number of subunits that play a major role in hemostasis,
specifically in platelet adhesion and aggregation.[1] The activity of vWF is proportional to the size of the multimers, with high-molecular-weight
multimer (HMWM) having increased platelet binding and platelet aggregation capacity
compared with the low- and intermediate–molecular-weight multimers (LMWM and IMWM).[2]
Previous studies have shown that the overall vWF concentration increases with age
in healthy children and adults, as well as in individuals with von Willebrand disease.
However, there is little known about the changes in the vWF multimer composition across
the age spectrum from neonates to adults.[3]
An understanding of the effect of age on vWF and its multimers is important for accurate
diagnosis and management of neonates and children with hematological complications.
This study aimed to investigate age-specific changes in vWF concentration and function,
as well as the composition of the vWF multimers in a healthy population spanning the
age spectrum from neonates to adults.
Plasma samples were obtained from healthy neonates, children, and adults, without
previous thromboembolic events (e.g., thrombosis and hemorrhage) and not subjected
to any form of anticoagulant therapy, according to the previously established protocol.[4] This study was approved by the Royal Children's Hospital, Human Research Ethics
Committee (HREC #34183) and the Royal Women's Hospital Human Research Ethics Committee
(#2/08). Written informed consent was obtained from parents/guardians of children
and from adult participants themselves.
Blood samples were collected in S-Monovette tubes (Sarstedt, Australia), containing
1 volume of citrate per 9 volumes of blood, and were centrifuged at 3,000 rpm for
10 minutes at 10°C (Megafuge 1.0R, Heraeus) to obtain plasma.
vWF concentration and activity were measured using the STA R Max analyzer and Stago
reagents, STA vWF antigen (vWF:Ag) and STA ristocetin cofactor activity (vWF:RCo)
(Diagnostica Stago, France). vWF collagen binding (vWF:CB) was measured using a commercially
available enzyme-linked immunosorbent assay (Diagnostica Stago, France). vWF multimers
were analyzed using the Hydragel 11 vWF Multimer assay (Sebia, France), as previously
described.[5]
Data analysis was performed using GraphPad Prism (Version 9.0). Results from the neonatal
and pediatric age groups were compared with the results for adults using a one-way
ANOVA (analysis of variance), followed by Dunnett's test to correct for multiple comparisons.
p < 0.05 was considered to be statistically significant.
A representative image of the analyzed Hydragel vWF multimer gels is shown in [Fig. 1]. vWF activity and its associated multimers were assessed in 80 children and 20 adults,
with participant characteristics described in [Table 1]. While vWF concentration and activity, as represented by vWF:RCo, vWF:Ag, and vWF:CB,
in neonates and children were comparable to adults, the proportions of LMWM were significantly
higher in neonates compared with adults, before reaching and maintaining adult levels
at approximately 6 to 10 years of age ([Table 2]). In addition, IMWMs were significantly lower in neonates, and in children up to
5 years of age compared with adults.
Fig. 1 Representative electrophoresis gel demonstrating vWF multimer patterns in a healthy
adult (lane 1), <2 year old (lane 2), and a neonate (lane 3). Representative densitograms
for lane 1(B), lane 2(C), and lane 3(D) are also shown. vWF, von Willebrand factor.
Table 1
Participant demographic data
|
Neonates
|
≤2 y
|
>2–5 y
|
6–10 y
|
11–17 y
|
Adults
|
|
Participants (n =)
|
20
|
22
|
11
|
8
|
19
|
20
|
|
Median age
|
48.0 h
|
0.9
|
3.5
|
9.5
|
13.9
|
32.7
|
|
Age range
|
24.0–96.0
|
0.3–1.4
|
2.3–4
|
8.8–10.3
|
11.7–17.6
|
20.5–53.9
|
|
Sex
|
10 M/10 F
|
18 M/4 F
|
5 M/6 F
|
5 M/3 F
|
10 M/9 F
|
7 M/13 F
|
|
Blood group
|
|
O (n =)
|
5
|
6
|
2
|
2
|
4
|
Not available
|
|
A/B/AB (n =)
|
7
|
8
|
5
|
4
|
8
|
Table 2
Age-specific results for vWF:RCo, vWF:Ag, vWF:CB, and vWF:CB/vWF:Ag ratio, vWF low-molecular-weight
multimer (LMWM), vWF intermediate-molecular-weight multimer (IMWM), and vWF high-molecular-weight
multimer (HMWM)
|
Neonates (h)
|
≤2 y
|
>2–5 y
|
6–10 y
|
11–17 y
|
Adults
|
|
vWF:RCo
|
|
Mean
|
102.1
|
74.0
|
88.9
|
86.1
|
97.2
|
92.7
|
|
95% CI
|
83.1–121.0
|
60.7–87.2
|
71.5–106.3
|
68.3–103.9
|
81.2–113.2
|
78.1–107.3
|
|
vWF:Ag
|
|
Mean
|
112.6
|
83.8
|
98.8
|
91.3
|
89.8
|
101.9
|
|
95% CI
|
96.3–128.9
|
71.0–96.5
|
82.2–115.4
|
70.2–112.3
|
68.9–110.5
|
88.6–115.2
|
|
vWF:CB
|
|
Mean
|
113.6
|
89.1
|
90.5
|
112.9
|
78.8
|
72.6
|
|
95% CI
|
81.9–145.6
|
64.9–113.3
|
35.9–145.2
|
43.5–182.4
|
63.1–94.6
|
51.7–93.5
|
|
vWF:CB/vWF:Ag
|
|
Mean
|
1.0
|
1.3
|
0.9
|
1.2
|
1.3
|
0.8
|
|
95% CI
|
0.7–1.3
|
0.7–1.9
|
0.4–1.4
|
0.5–1.8
|
0.3–2.3
|
0.5–1.0
|
|
vWF LMWM
|
|
Mean
|
19.9
[a]
|
17.7
|
18.3
|
15.5
|
15.4
|
15.5
|
|
95% CI
|
17.1–22.7
|
15.7–19.7
|
14.5–22.1
|
13.9–17.1
|
12.7–18.1
|
14.0–17.0
|
|
vWF IMWM
|
|
Mean
|
27.5
[a]
|
27.7
[a]
|
26.9
[a]
|
30.5
|
28.4
|
30.5
|
|
95% CI
|
25.5–29.6
|
26.5–28.8
|
24.7–29.1
|
28.3–32.7
|
26.9–29.8
|
29.1–31.8
|
|
vWF HMWM
|
|
Mean
|
52.6
|
54.6
|
54.8
|
54.0
|
56.2
|
54.0
|
|
95% CI
|
48.3–56.8
|
52.6–56.7
|
49.7–59.8
|
51.4–56.6
|
53.1–59.3
|
51.8–56.3
|
Abbreviations: CB, collagen binding; CI, confidence interval; vWF, von Willebrand
factor.
Note: Test results are expressed as percentage (%).
a
p-Value significantly different from adults (p < 0.05).
The results of our study demonstrate significant age-specific differences in proportions
of some of the vWF multimers, with no age-specific differences observed when considering
the overall vWF activity and concentration.
Our vWF concentration and activity findings are comparable with previously published
work,[6]
[7]
[8]
[9]
[10] with no change across the age spectrum. vWF multimers have been previously investigated
in critically ill children with varying comorbidities such as febrile seizures and
in the setting of extracorporeal membrane oxygenation.[11]
[12]
Previous studies investigating vWF multimers have shown that neonates have increased
amounts of HMWM.[13]
[14] Conversely, we demonstrated that in healthy neonates and children, proportions of
LMWM are increased and proportions of IMWM are decreased in neonates and young children
compared with adults. However, proportions of HMWM were relatively the same across
the age spectrum. Differences between our findings and previous studies could be explained
by: (1) differences in the age (e.g., neonates born <36 weeks' gestation) and health
status (e.g., thrombotic thrombocytopenic purpura, necrotizing enterocolitis) of the
participants used across studies, and (2) different methodological techniques used
to investigate vWF multimers. Specifically, the vWF multimer analysis techniques utilized
in the studies conducted by Katz et al[13] and Weinstein et al[14] are in-house, labor-intensive, manual laboratory tests that are operator-dependent.
On the other hand, the semi-automated method produced by Sebia reduces operator dependence
and has been shown to be reliable and successful in correctly visualizing and interpreting
vWF multimers.[15]
Differences were seen in this study, across the pediatric age groups when vWF was
characterized. Further research is required to gain a better understanding of the
effect and interaction of the specific vWF multimers and current therapeutics.
In conclusion, while there are no changes in the overall activity and function of
vWF, the sizes specific vWF multimers vary significantly with age in healthy neonates
and children up to 5 years of age. Our findings highlight that physiological age-specific
differences in hemostatic proteins such as vWF can only be detected using sensitive
assays that account for the specific forms of proteins, such as the vWF multimers.
