The Effect of Shear Stress on the Size, Structure, and Function of Human von Willebrand Factor

Abstract

Clinical outcomes from ventricular assist devices (VADs) have improved significantly during recent decades, but bleeding episodes remain a common complication of long-term VAD usage. Greater understanding of the effect of the shear stress in the VAD on platelet aggregation, which is influenced by the functional activity of high molecular weight (HMW) von Willebrand factor (vWF), could provide insight into these bleeding complications. However, because VAD shear rates are difficult to assess, there is a need for a model that enables controlled shear rates to first establish the relationship between shear rates and vWF damage. Secondly, if such a dependency exists, then it is relevant to establish a rapid and quantitative assay that can be used routinely for the safety assessment of new VADs in development. Therefore, the purpose of this study was to exert vWF to controlled levels of shear using a rheometer, and flow cytometry was used to investigate the shear-dependent effect on the functional activity of vWF. Human platelet-poor plasma (PPP) was subjected to different shear rate levels ranging from 0 to 8000/s for a period of 6 h using a rheometer. A simple and rapid flow cytometric assay was used to determine platelet aggregation in the presence of ristocetin cofactor as a readout for vWF activity. Platelet aggregates were visualized by confocal microscopy. Multimers of vWF were detected using gel electrophoresis and immunoblotting. The longer PPP was exposed to high shear, the greater the loss of HMW vWF multimers, and the lower the functional activity of vWF for platelet aggregation. Confocal microscopy revealed for the first time that platelet aggregates were smaller and more dispersed in postsheared PPP compared with nonsheared PPP. The loss of HMW vWF in postsheared PPP was demonstrated by immunoblotting. Smaller vWF platelet aggregates formed in response to shear stress might be a cause of bleeding in patients implanted with VADs. The methodological approaches used herein could be useful in the design of safer VADs and other blood handling devices. In particular, we have demonstrated a correlation between the loss of HMW vWF, analyzed by immunoblotting, with platelet aggregation, assessed by flow cytometry. This suggests that flow cytometry could replace conventional immunoblotting as a simple and rapid routine test for HMW vWF loss during in vitro testing of devices.

Full text

The Effect of Shear Stress on the Size, Structure, and
Function of Human von Willebrand Factor
*Chris Hoi Houng Chan, *Ina Laura Pieper, *Scott Fleming, *Yasmin Friedmann,
†Graham Foster, *Karl Hawkins, *Catherine A. Thornton, and
*Venkateswarlu Kanamarlapudi
*Institute of Life Science, College of Medicine, Swansea University; and †Institute of Life Science, Calon
Cardio-Technology Ltd, Swansea, Wales, UK
Abstract: Clinical outcomes from ventricular assist devices
(VADs) have improved significantly during recent
decades, but bleeding episodes remain a common compli-
cation of long-term VAD usage. Greater understanding of
the effect of the shear stress in the VAD on platelet aggre-
gation, which is influenced by the functional activity of high
molecular weight (HMW) von Willebrand factor (vWF),
could provide insight into these bleeding complications.
However, because VAD shear rates are difficult to assess,
there is a need for a model that enables controlled shear
rates to first establish the relationship between shear rates
and vWF damage. Secondly, if such a dependency exists,
then it is relevant to establish a rapid and quantitative assay
that can be used routinely for the safety assessment of new
VADs in development. Therefore, the purpose of this
study was to exert vWF to controlled levels of shear using
a rheometer, and flow cytometry was used to investigate
the shear-dependent effect on the functional activity of
vWF. Human platelet-poor plasma (PPP) was subjected to
different shear rate levels ranging from 0 to 8000/s for a
period of 6 h using a rheometer. A simple and rapid flow
cytometric assay was used to determine platelet aggrega-
tion in the presence of ristocetin cofactor as a readout for
vWF activity. Platelet aggregates were visualized by confo-
cal microscopy. Multimers of vWF were detected using gel
electrophoresis and immunoblotting. The longer PPP was
exposed to high shear, the greater the loss of HMW vWF
multimers, and the lower the functional activity of vWF for
platelet aggregation. Confocal microscopy revealed for the
first time that platelet aggregates were smaller and more
dispersed in postsheared PPP compared with nonsheared
PPP. The loss of HMW vWF in postsheared PPP was dem-
onstrated by immunoblotting. Smaller vWF platelet aggre-
gates formed in response to shear stress might be a cause of
bleeding in patients implanted with VADs. The method-
ological approaches used herein could be useful in the
design of safer VADs and other blood handling devices. In
particular, we have demonstrated a correlation between
the loss of HMW vWF, analyzed by immunoblotting, with
platelet aggregation, assessed by flow cytometry. This sug-
gests that flow cytometry could replace conventional
immunoblotting as a simple and rapid routine test for
HMW vWF loss during in vitro testing of devices. Key
Words: von Willebrand factor—Shear stress—Platelet
aggregation—Multimer analysis.
During the past few decades, ventricular assist
devices (VADs) have emerged as an increasingly
popular therapy in patients with advanced heart
failure who do not respond to medical or
resynchronization therapy. VADs are used either as a
bridge-to-transplant or as destination therapy in
patients deemed ineligible for cardiac transplanta-
tion, or those who cannot receive a transplant due to
the limited supply of donors (1). While VADs have
already benefitted many patients, VAD-related blood
damage remains a major issue. This includes
hemolysis (2,3), platelet activation (4), alteration
of the coagulation cascade and thrombosis (5),
reduced functionality of leukocytes (6), release of
doi:10.1111/aor.12382
Received April 2014; revised June 2014.
Address correspondence and reprint requests to Dr. Chris Hoi
Houng Chan, Institute of Life Science, College of Medicine,
Swansea University, Swansea, Wales SA2 8PP, UK. E-mail:
chris_houng@caloncardio.com; h.h.chan@swansea.ac.uk
Presented in part at the 21st Congress of the International
Society for Rotary Blood Pumps held September 26–28, 2013 in
Yokohama, Japan.
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Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Artificial Organs 2014, 38(9):741–750

microparticles (7), and degradation of von Willebrand
factor (vWF) (8,9).
Gastrointestinal (GI) bleeding is one complication
associated with the placement of a VAD, with rates
as high as 65% within the first year after VAD place-
ment (10,11). The mechanism underlying this
problem in patients on long-term continuous flow
mechanical support is not well understood (1,9,11–
22). This cannot be explained by the anticoagulation
regimen alone, but may be symptomatic of acquired
von Willebrand syndrome (AvWs) which could be
the result of shear stress caused by the VAD. In a
literature review of clinical studies describing patient
populations suffering from GI bleeding, two popula-
tions in particular were found to stand out: those
suffering from aortic valve stenosis and those
implanted with axial flow VADs (Table 1). In aortic
stenosis, there is a high level of shear stress and loss
of HMW vWF multimers (23,24). In contrast to axial
flow pumps, centrifugal flow VADs and total artifi-
cial hearts (TAHs) have fewer reported events of GI
bleeding. The centrifugal pump with the most reports
of GI bleeding is the novel HeartWare device (20,25–
28). In patients implanted with VentrAssist and
EVAHEART, there have been few reports of bleed-
ing, although they have been diagnosed with AvWs,
showing a loss of high molecular weight (HMW)
vWF multimers and decreased ratios of collagen
binding capacity and ristocetin cofactor activity to
vWF antigen (17). Comparing the shear stress in
these devices provides a partial explanation as the
characteristic hydraulic performance of axial flow
usually involves high rotational pump speed com-
pared with centrifugal flow resulting in higher shear
stress (29). Furthermore, TAHs typically have lower
shear stress than centrifugal pumps due to their pul-
satile operation (11). Thus, the hypothesis is that the
higher the shear stress, the more damage caused to
vWF, resulting in decreased platelet aggregation and
thus bleeding.
In light of this, it is important to understand the
point at which shear stress becomes damaging, and
why centrifugal VAD patients do not suffer GI
bleeding to the same extent, although obviously suf-
fering impairment of vWF. It is difficult to calculate
shear stress in VADs both in vitro and in vivo.
Therefore, it is important to establish an in vitro
laboratory model with greater sensitivity and the
ability to better quantify HMW vWF multimer
loss while furthering our understanding of the rela-
tionship between HMW vWF abundance and
activity.
Research focusing on the impact of wall shear rate
caused by perfusing normal plasma through long cap-
illary tubing, and that of pumping plasma in a mock
circulatory loop driven by a VAD has shown a rela-
tionship between the shear rate and the loss of HMW
vWF multimers (8,30,31). However, neither of these
models provide a way to subject the plasma to accu-
rate and controlled levels of shear rate environment.
A rheometer can be used to apply controlled levels of
shear rate during timed intervals. Therefore, the
purpose of this study was to: (i) quantify HMW vWF
breakdown at specified levels of shear rate using a
rheometer; (ii) correlate the loss of HMW multimers
to vWF functionality; and (iii) investigate the effect
of mechanical shear on platelet aggregates. This
information would be of value to VAD developers
who could modify device design to avoid damaging
shear stress levels.
TABLE 1. Literature review describing patient populations suffering from GI bleeding and vWF diagnostic methods
Device or medical condition
GI
bleeding
vWF diagnostic methods
Reference
HMW vWF
loss (multimer
analysis)
Decrease in
vWF : RCo /
vWF : Ag activity
Decrease in
vWF:CB/
vWF : Ag activity
CF-VAD Axial flow HeartMate II Yes Yes Yes Yes 9,11–13,16–19
Jarvik 2000 Yes N/A N/A N/A 15
MicroMed DeBakey Yes N/A N/A N/A 11
Thoratec BVAD Yes Yes Yes Yes 18,19
Centrifugal
flow
VentrAssist No Yes Yes Yes 11,18
CentriMag BiVAD N/A Yes N/A N/A 16
HeartWare Yes N/A N/A N/A 20,25–28
EVAHEART No No Yes N/A 29
Duraheart N/A No N/A N/A 16
Pulsatile
TAH
CardioWest No No No No 18
HeartMate XVE No No N/A N/A 11,16
No device Aortic valve stenosis Yes Yes N/A Yes 23,24
C.H.H. CHAN ET AL.742
Artif Organs, Vol. 38, No. 9, 2014

MATERIALS AND METHODS
Blood preparation
Peripheral blood (72 mL) was collected from six
healthy volunteers in vacutainers containing 9NC
coagulation sodium citrate 3.2% (455322, Greiner
Bio-one, Wemmel, Belgium). Platelet-rich plasma
(PRP) was prepared by centrifuging whole blood for
7 min at 500 ×gat room temperature. Platelet-poor
plasma (PPP) was prepared by centrifuging PRP for
5 min at 13 000 ×gat room temperature. The PPP
was extracted and analyzed using the automated
hematology analyzer CELL-DYN Ruby (Abbott
Diagnostics, Abbott Park, IL, USA) to ensure that
the plasma was void of blood cells. PPP was used
instead of whole blood to ensure that any potential
effect of shear on vWF was not a result of cellular
interactions. This study was approved by the South
Wales Research Ethics Committee and all donors
gave informed written consent.
Rheometry
PPP was subjected to shear stress rates of 4000 and
8000/s in an AR-G2 rheometer (TA Instruments,
New Castle, DE, USA) at 37°C. The rheometer was
equipped with double concentric geometry in which a
rotating inner cylinder cup allows generation of uni-
formly established shear flow at a well-defined shear
rate. The PPP was sampled at hourly intervals (6 h in
total). Static PPP at 37°C was used as a control. The
sheared PPP was analyzed by flow cytometry imme-
diately after the shear tests and the remaining plasma
samples were stored at −80°C for ≤5 days prior to
analysis for the vWF multimer by immunoblotting.
Plasma vWF multimer analysis by immunoblotting
Sheared and nonsheared PPP was subjected to
electrophoresis on high gelling temperature agarose
(0.6% agarose, w/v, Sea Kem, FMC Bioproducts,
Rockland, ME, USA) in a horizontal gel apparatus
(81–2325, Galileo Bioscience, Cambridge, MA,
USA) at 4°C. Electrophoresis was performed at
30 mA for 30 min and then at 50 mA until the dye
front had migrated 10–12 cm from the origin (total
gel running time was 6 h). vWF multimers separated
on agarose gel were transferred to polyvinylidene
difluoride (PVDF) 0.45 μm membrane (IPVH304F0,
Immobilon-P, Millipore Corporation, Billerica, MA,
USA) for 15–17 h at 70 mA in the electroblotting
tank (91–2020-TB, Galileo Bioscience). vWF detec-
tion using anti-human vWF primary (ab6994,
Abcam, Cambridge, UK) and horseradish peroxidise
(HRP)-conjugated secondary (ab6721, Abcam) anti-
bodies, and chemiluminescence substrate (170–5060,
Bio-Rad, Hercules, CA, USA) was performed as
described by Krizek et al. (32). The shear-induced
changes in the size distribution of plasma vWF
multimers were studied by densitometric scanning
initiated from the origin (Quantity One software
v4.6.8, BioRad, Hertfordshire, UK). The loss of
HMW vWF multimers was quantified by the rate of
change in gradient of HMW vWF.
vWF : Ristocetin (vWF : RCo) assay
vWF functionality was assessed using the
vWF : Ristocetin (vWF : RCo) assay developed by
Chen et al. (33). The assay measures the ability of
vWF (in PPP) to bind to platelets in the presence of
ristocetin resulting in aggregation of the platelets.
Human platelets (50 000 platelets per μL) were
stained either green (CellTracker Green CMFDA,
Life Technologies, Glasgow, UK) or red
(MitoTracker Red FM, Life Technologies), and
incubated with 2 μL PPP and 1 mg/mL ristocetin solu-
tion for 45 min at room temperature in the dark rotat-
ing at 30 rpm (SB3 Rotator, Stuart, Bibby Scientific,
Staffordshire, UK). Nonaggregated platelets (green
or red events) and aggregated platelets (double-
labeled events) were quantified by flow cytometry
(FACSAria I, BD Biosciences, Oxford, UK) and the
percentage of double-labeled events was measured
(FACS Diva 6.1.3, BD Biosciences). The greater the
percentage of double-labeled events, the higher the
platelet aggregation and hence the vWF functionality.
Confocal microscopy of platelet aggregates
vWF : RCo assays were performed as described
above. Post 24 h incubation at room temperature,
70 μL reaction mixture samples containing vWF and
red/green platelets were stained with anti-human
vWF antibody (ab6994, Abcam) labeled with Pacific
Blue (Z25041, Zenon Pacific Blue mouse IgG1 label-
ling kit, Life Technologies) in an 8-well borosilicate
chamber slide (155411, Thermo Scientific, New York,
NY, USA). Individual and aggregated fluorescent
platelets adhered by vWF were fixed by Vectashield
hardset mounting medium (H-1400, Vector Labora-
tories, Peterborough, UK) and examined with a con-
focal microscope (LSM 710, Zeiss, Jena, Germany).
Images of green and red fluorescent platelets and
blue fluorescent-stained vWF were captured and
analyzed using ZEN imaging software 2012.
vWF ELISA
The concentration of vWF : antigen (vWF : Ag) in
the sheared PPP was quantified by a vWF specific
enzyme-linked immunosorbent assay (ELISA) kit
(ab108918, Abcam) according to manufacturer’s
VON WILLEBRAND FACTOR AND SHEAR STRESS 743
Artif Organs, Vol. 38, No. 9, 2014

instructions and absorbance at 450 nm measured
(POLARstar Omega, BMG LABTECH, Ortenberg,
Germany).
Statistical analysis
Due to multiple measurements made per PPP
sample at different time points and at different shear
rates, a repeated measures analysis of variance on the
data sets was conducted. The shear rates and the time
points were treated as fixed effects and the sample as
a random effect. Once significant effects were found,
pairwise comparison tests were conducted, adjusted
for the multiple comparisons using Bonferroni’s
method. Analysis was performed using the RStudio v
0.97.551 (RStudio, Boston, MA, USA) and R statis-
tical environment, v3.0.2 (R Core Team, Vienna,
Austria).
RESULTS
Quantification of the rate of change of HMW and
LMW vWF using densitometry
The rate of change of HMW and low molecular
weight (LMW) multimers was quantified using
densitometry (Fig. 1A–C, right panel) and the results
are summarized in Fig. 2A (HMW vWF) and 2B
(LMW vWF).
HMW vWF
Repeated measures analysis of variance shows a
very significant dependence of the densitometry
values on the shear rate (P<10−6) and on time
(P<10−6). The interaction between time and shear
rate is also significant (P<0.002). Pairwise com-
parisons (with Bonferroni correction) confirm the
FIG. 1. Measurement of vWF multimers
by immunoblotting. HMW and LMW vWF
multimer immunoblot images (left panel)
and densitometric analysis (right panel)
for human PPP under different shear
rates: (A) static controls 0/s, (B) 4000/s,
and (C) 8000/s at hourly intervals during
6 h at 37°C.
C.H.H. CHAN ET AL.744
Artif Organs, Vol. 38, No. 9, 2014

significant difference between any two levels of shear
rate and show that significant differences are
achieved between measurements that are at least 4 h
apart. This is summarized in Fig. 2A which shows
three distinct curves. The mean densitometry values
in the control sample remain high while there is a
decrease with time in the sheared samples, with a
stronger decrease in the sample subjected to the
higher shear.
LWM vWF
Repeated measures analysis of variance showed a
significant dependence of the densitometry values on
the shear rates (P<0.002) and on time (P<10−5).
There was no significant interaction between shear
rate and time in this case. Pairwise comparisons with
Bonferroni correction show that the significant dif-
ferences are between the static and high shear values,
and that measurements need to be at least 5 h apart
to show significant changes. This is summarized in
Fig. 2B, which shows three distinct curves describing
mean densitometry values of the three shear rates.
The curves representing sheared samples exhibit an
upward trend with time of the densitometry values.
To summarize, the results reflect loss of HMW vWF
at 4000 and 8000/s, compared with the static control
at hourly intervals during 6 h at 37°C in Fig. 1 (left
panel). There is a positive correlation between shear
rate and degradation of vWF, with 8000/s causing the
greatest loss of HMW bands compared with the static
control. Similarly, there is a correlation between
shear rate and an increase in LMW bands, suggesting
that the HMW bands may be cleaved into smaller
fragments. This conclusion is strengthened by the
quantification of vWF antigen below.
Quantification of vWF antigen by ELISA
The total vWF antigen decreases during the 6-h
test in the static control and the sheared samples
(Fig. 3). However, as there is no difference between
the groups, we can conclude that the HMW band loss
demonstrated by immunoblotting (Fig. 1) in the
sheared samples is due to a cleavage of HMW
multimers into LMW multimers.
A
0123456
0123456
0.0 0.2 0.40.60.81.01.2
time (h)
Mean normalized HMW vWF densitometry values
Shear rate:
static
4000 1/sec
8000 1/sec
B
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0
time (h)
Mean normalized LMW vWF densitometry value
Shear rate:
static
4000 1/sec
8000 1/sec
FIG. 2. (A) Means (over the different samples) ±SE of the nor-
malized results of densitometric analysis for HMW vWF in human
PPP under different shear rates (static control 0, 4000, and
8000/s at hourly intervals during 6 h at 37°C). (B) Means (over
the different samples) ±SE of the normalized results of
densitometric analysis for LMW vWF in human PPP under differ-
ent shear rates (static control 0, 4000, and 8000/s at hourly
intervals during 6 h at 37°C).
FIG. 3. Total vWF antigen quantified by ELISA in postsheared
human PPP at different shear rates for 6 h. Human PPP sub-
jected to 0/s (static control), 4000 or 8000/s, and then analyzed
by ELISA.
VON WILLEBRAND FACTOR AND SHEAR STRESS 745
Artif Organs, Vol. 38, No. 9, 2014

vWF functionality assessed using flow cytometry
and visualized using confocal microscopy
The assessment of the vWF functionality was per-
formed using a ristocetin-dependent flow cytometry
assay of platelet aggregation. When equal concentra-
tions of green and red platelets, resolved by the FITC
and APC channels, respectively (Fig. 4A, left panel),
were mixed in the absence of PPP, single green and
red platelets dominated and double positive events
(platelet aggregates) were negligible (<3%). These
events were considered as a background effect and
removed from the counting of aggregates when PPP
was added. The number of aggregates at time 0 with
PPP added was considered as the baseline, and all
further counts were compared with this baseline.
Repeated measures analysis of variance of the
vWF : RCo activity revealed a significant effect of
shear (P<10−6) and also a strong effect of time
(P<10−6). Interaction between shear rate and time
was also significant (P<0.0002). Pairwise compari-
sons with Bonferroni correction further showed that
there is a significant difference between any two shear
rates in the following order of significance: none :
8000/s, 4000 s : 8000/s, and none : 4000/s. Pairwise
comparisons with Bonferroni correction between
time points show that significant difference in activity
will be found between measurements that are at least
4 h apart. These results are evident in Fig. 5.
FIG. 4. Quantification of vWF functional-
ity by flow cytometry and confocal micros-
copy. Left panel: vWF functionality
quantified by a ristocetin-dependent flow
cytometry assay measuring the percent-
age of platelet agrregates. Right panel:
Platelet aggregates of individually stained
red or green platelets visualized by con-
focal microscopy. (A) No PPP negative
control, (B) static control, (C) 4000/s, (D)
8000/s at 6 h, respectively.
C.H.H. CHAN ET AL.746
Artif Organs, Vol. 38, No. 9, 2014

The vWF/platelet aggregates were then assessed
by confocal microscopy that showed single green
and red platelets in the negative control (no PPP)
sample (Fig. 4A, right panel), and platelet aggre-
gates in the samples to which static control or
sheared PPP was added (Fig. 4B–D, right panel).
The sheared samples resulted in several smaller
platelet aggregates compared with the static control.
Although there were also single platelets present in
the static and sheared samples, they stayed at the
surface whereas the aggregates sank to the bottom
of the well, which meant that isolated, light platelets
could not be visualized simultaneously (Fig. 4B–D,
right panel). The results of vWF : RCo activities,
D/G ×100% were converted into IU/dL by using
standard curves with serial dilutions of standard
normal reference plasma (CCNRP, CRYOcheck,
Precision Biologic, Halifax, NS, Canada) (not
shown).
There is a strong linear correlation between the
loss of HMW vWF multimers as measured by
immunoblotting and the reduction in functional
activity of vWF as measured by flow cytometry
(Fig. 6). In Table 2, the results show that the
vWF functionality to antigen ratio (vWF : RCo /
vWF : Ag) of the static control, 4000 and 8000/s at 6 h
was 1.01, 0.67, and 0.42, respectively. These results
prove that we were able to create an AvWs pheno-
type with human PPP in vitro. The vWF :
RCo / vWF : Ag ratio for a normal healthy donor is
close to 1.0, and for a type 2 von Willebrand disease
patient it is less than 0.43 (17,33).
Visual investigation of platelet aggregation by
confocal microscopy
vWF multimers interspersing platelets in aggre-
gates can be visualized using confocal microscopy
(Fig. 7). The structural platelet aggregations of
nonsheared PPP (static control) were less dispersed
FIG. 5. Mean (over the different PPP samples) ±SD of the vWF:
RCo activity for human plasma under different shear rates (static:
0s
–1, 4000 s–1 and 8000 s–1) normalized to the baseline values at
time 0.
TABLE 2. The overall results of vWF multimer electrophoresis, vWF : Ag (antigen), vWF : RCo (ristocetin cofactor
activity), vWF : RCo / vWF : Ag (the ratio of ristocetin cofactor activity to vWF : Ag antigen) for human PPP under
different shear rate static control 0, 4000, and 8000/s at 6 h at 37°C
Shear rate
(per s)
VWF multimer
electrophoresis at 6 h
VWF:Ag
(IU/dL) at 6 h
VWF : RCo
(IU/dL) at 6 h
VWF : RCo /
VWF:Agat6h
094.3 95.4 1.01
4000 87 58.39 0.67
8000 90.2 37.92 0.42
FIG. 6. Normalized rate of change in gradient HMW vWF
multimer densitometry values compared with normalized
vWF : RCo flow cytometry values in samples from different shear
conditions (n=4). There is a linear correlation between the
methods for the two different shear rates, 4000/s R2=0.924 :
8000/s R2=0.9697.
VON WILLEBRAND FACTOR AND SHEAR STRESS 747
Artif Organs, Vol. 38, No. 9, 2014

and bigger in size compared with those of the
postsheared PPP (at 8000/s, 6 h). The lower MW and
lower activity vWF likely led to a smaller, more dis-
persed platelet aggregate (Fig. 8).
DISCUSSION
Current blood trauma research related to VADs
often focuses on hemolysis with few reports of cleav-
age of vWF during in vitro testing (34). In this study,
we have demonstrated shear dependent degradation
of HMW vWF multimers and loss of vWF activity,
although the total amount of vWF remains pre-
served. These results show that we were able to
create an in vitro model of AvWs.
The molecular weight of vWF has also been
shown here to be a major determinant of the adhe-
sive functional activity causing platelet aggregation,
and that there is a correlation between the loss of
HMW vWF and vWF activity (Fig. 6). These results
indicate that the assessment of vWF activity using
flow cytometry, a faster and more sophisticated
analysis than the gold standard vWF immunoblot,
could be sufficient for the evaluation of VADs with
regard to AvWs in comparative in vitro studies. In
addition, the structural aggregation confocal micros-
copy analysis could provide more insight with regard
to AvWs.
In this study, relatively low shear rates were used
to establish the model. The maximum shear rate will
be dependent on the rheometers used, and the
researcher may want to select a model that spans a
high range because the estimated shear condition in a
centrifugal pump is >20 000/s (8). Another limitation
was the residence time (6 h) of the PPP which is
continuously sheared in the rheometer, whereas the
blood passes through the VAD in a period of milli-
seconds. However, this work provides a simple and
accurate way to expose the blood plasma samples to
controlled shear rates ranging from physiological to
pathological levels which allow evaluation of the
impact of shear rate on the vWF. This is of value to
provide further insight into bleeding complications in
patients with VAD. Our future work will focus on
FIG. 7. Platelet aggregation visualized by
confocal microscopy. vWF multimers
were stained with Pacific blue conjugated
antibody to demonstrate how they are
positioned relative to platelets in the
aggregates.
FIG. 8. Confocal microscopy of platelet
aggregates stained with red or green in
the presence of human PPP form the
static control (A) and sheared at 8000/s
(B) after 6 h. The nonsheared PPP
sample results in less dispersed and
larger platelet aggregates compared with
the sheared vWF sample.
C.H.H. CHAN ET AL.748
Artif Organs, Vol. 38, No. 9, 2014

further exploration of the shear stress and time
domains of vWF mechanoenzymatic stability, espe-
cially at higher ranges of shear stress and shorter
exposure times. In addition, the use of whole blood
instead of PPP is the next step to mimic physiological
conditions, although shear stress might also damage
or activate platelets causing release of ADP and vWF
multimers into the plasma. Furthermore, the role of
ADAMTS13, the protease known to break down
vWF (31), in HMW vWF destruction during high
shear stress will be considered.
CONCLUSIONS
The results shown here confirmed that the longer
human platelet-poor plasma is exposed to high shear
rate, the greater the loss of the high molecular weight
von Willebrand factor multimers and decline in the
functional activity of vWF will be. There is a strong
linear correlation between HMW vWF multimer loss
and the lower functional activity of vWF. Therefore,
this study indicates that a simple, fast, accurate flow
cytometric evaluation of the vWF activity is sensitive
enough for a comparative acquired von Willebrand
syndrome study for in vitro testing of different shear
conditions. This work could improve in vitro ventricu-
lar assist device evaluation and bring it a step closer to
assessment of total blood trauma. It is recommended
that the effort of blood pump design should not only
focus on mechanical stability and hemolysis, but also
on the other blood components that could be
damaged such as leukocytes, platelets, and soluble
factors such as vWF and factor VIII. Future in vitro
device evaluation could benefit from inclusion of
assays such as this to provide a more complete picture
of the overall effect on blood function.
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