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Micropatterned array to assess the interaction of single platelets with platelet factor 4-heparin-IgG complexes.
[heparin-induced thrombocytopenia]
We
report
a
strategy
to
generate
by
electron
beam
lithography
high
fidelity
micropatterned
arrays
to
assess
the
interaction
of
single
platelets
with
immobilised
ligands
.
As
a
proof-of-principle
we
functionalised
the
microarrays
with
platelet
factor
4
(
PF
4
)
-
heparin-
IgG
complexes
.
We
embedded
biotinylated
water-soluble
quantum
dots
into
polyethylene
glycol
(
PEG
)
-
coated
micropatterned
arrays
and
functionalised
them
via
streptavidin
to
bind
biotinylated
ligands
,
here
biotinylated-
PF
4
/
heparin
complexes
.
The
integrity
of
the
PF
4
/
heparin-complexes
was
shown
by
binding
of
anti-
PF
4
/
heparin
antibodies
.
Ligand
density
was
quantified
by
immunofluorescence
and
immunogold
antibody
labelling
.
Real-time
calcium
imaging
was
employed
for
read-out
of
single
platelets
activated
on
micropatterned
surfaces
functionalised
with
PF
4
/
heparin-
IgG
complexes
.
With
the
smallest
micropatterns
(
0
.
5
x
0
.
5
µm
)
we
show
that
single
platelets
become
strongly
activated
by
binding
to
surface-immobilised
PF
4
/
heparin-
IgG
,
while
on
larger
micropatterns
(
10
x
10
µm
)
,
platelet
aggregates
formed
.
These
findings
that
HIT
antibodies
can
cause
platelet
activation
on
microarrays
illustrate
how
this
novel
method
opens
new
avenues
to
study
platelet
function
at
single
cell
level
.
Generating
functionalized
microarray
surfaces
to
which
highly
complex
ligands
can
be
bound
and
quantified
has
the
potential
for
platelet
and
other
cell
function
assays
integrated
into
high
-throughput
microfluidic
microdevices
.
Diseases
Validation
Diseases presenting
"platelet function"
symptom
heparin-induced thrombocytopenia
malignant atrophic papulosis
oculocutaneous albinism
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