Area of spread platelets [µm2]
80
70
60
50
40
30
20
10
0
0
2
4
6
8
10
12
∞
14
Microdot diameter [μm]
Figure S1. Platelet spreading is spatially regulated at the microscale. Platelets
adhere and spread onto fibrinogen microdots fabricated via protein microcontact
printing. On larger fibrinogen microdots (e.g., 7 and 10 µm in diameter; “∞” denotes
no geometric boundary), platelet spreading conforms to the microenvironmental
geometric boundaries of the microdots with high fidelity. Decreasing the fibrinogen
microdot size (e.g., diameters of 5 µm and below), however, leads to platelet
spreading beyond the geometric boundaries of the microdots. This phenomenon is
quantified by measuring the surface area of spread platelets on fibrinogen (blue) and
collagen (green) microdots of different diameters. Gray line denotes the protein
microdot area and diameters for reference. Scale bar = 5 µm. Error bars indicate SE.
15 min
30 min
60 min
90 min
105 min
Figure S2. Two hour time course of P-selectin expression on platelets that
adhere and spread onto protein micropatterns. Platelets incubated on 5 µm
diameter fibrinogen (blue) microdots were fixed at different time points and stained
with anti-P-selectin (green) and plasma membrane staining (red). Samples were not
permeabilized.
Flow
Flow
Figure S3. Platelet α-granule secretion is spatially regulated at the microscale
under physiologic flow conditions. Via live cell imaging, anti-P-selectin (green)
staining was visualized on washed platelets (red) adhered onto fibrinogen microdots
(blue) under physiologic flow conditions in a microfluidic device. Immediately upon
initial adhesion of platelets on the microdots, buffer containing P-selectin antibodies
was perfused at a shear rate of ~100 s-1. Platelets adhered on 5 µm diameter
microdots extended filopodia beyond the micropattern boundaries which co-localized
with strong anti-P-selectin staining. Scale bars = 5 µm.
407 nm
622 nm
858 nm
1002 nm
Figure S4. Protein micropatterns comprising submicron “holes” revealed that
α-granule distribution is spatially regulated at the nanoscale. Protein
micropatterns comprising submicron “holes” (blue, fibrinogen, average hole sizes
are shown above) revealed that α-granule secretion (via anti-P-selectin staining,
green) is spatially regulated at the nanoscale within platelets co-localizing at the
patterned holes (sizes shown). Averaged (n=5 platelets) fluorescence intensity line
plots of platelets (membrane: red) spread over fibrinogen micropatterns (blue) with
858 nm holes show alternating peaks of P-selectin (green) and fibrinogen intensity.
Scale bar= 5 µm.
Non-permeabilized Permeabilized
A
B
C
D
P-selectin
No primary Ab
P-selectin
No primary Ab
Figure S5. Spatial regulation of platelet α-granule secretion occurs both apical
and basal side of adhered platelets. (A) Permeabilization of adherent platelets spread
on fibrinogen with 0.5 % Triton X treatment remove some P-selectin expression as
compared to non-permeabilized platelets. (B) P-selectin is predominantly co-localized
with the regions of the platelet spread beyond the protein microdot boundaries, which is
similar to that in non-permeabilized conditions. (C) However, when platelets are
adhered and spread onto micropatterned holes, P-selectin co-localization with those
regions only occurred under permeabilized conditions, suggesting that micropatternregulated P-selectin distribution occurs either inside or at the basal surface of the
platelets. (D) Platelets (permeabilized) stained with membrane dye (red) and spread
onto fibrinogen microdots or micropatterned holes were immunostained with anti-Pselectin (Left, anti-P-selectin, mouse monoclonal, clone AK4) as compared with a
negative control (Right, secondary antibody only - Alexa Fluor 488 conjugated goat antimouse IgG). Scale bars = 5 µm.
P-selectin
PAC-1
CD41(αIIb)
CD42b (Ib)
P-selectin
PAC-1
CD41(αIIb)
CD42b (Ib)
Figure S6. α-granules and activated αIIbβ3 integrins distribute beyond the
protein micropattern boundaries. The distribution of various membrane proteins
(green) were examined by immunostaining (not permeabilized) on platelets (red)
that spread beyond fibrinogen microdots (blue). α-granules, via-P selectin binding,
and activated integrin αIIbβ3 (via PAC-1 binding) were distributed beyond the
microdot boundaries, whereas the distribution of other membrane proteins such as
CD41, which stains all αIIbβ3 molecules regardless of activation state, and CD42b,
which stains gpIb, did not exhibit any spatial relationship with the micropattern
geometry. Scale bars = 5 µm.
2 µm
3 µm
4 µm
5 µm dots
Control
LA
Figure S7. The actin cytoskeleton controls spatial regulation of platelet
spreading and α-granule secretion. Different sizes (diameters shown) of
protein microdots indicate that exposure to lactrunculin A (LA) primarily inhibit
platelet spreading (red, cell membrane stain) and α-granule secretion (anti-Pselectin binding, green) beyond the geometric boundaries of the protein
micropattern, but not adhesion or spreading within the micropattern. All
samples are permeabilized during staining.
3 µm dot
5 µm dot
∞
control
NSC23766
Figure S8. Rac-1 mediates spatial regulation of platelet spreading and αgranule secretion. Different sizes (diameters shown) of protein microdots
indicate that exposure to NSC23766 inhibits platelet spreading beyond the
geometric boundaries of the protein micropattern, but not adhesion or spreading
within the protein micropattern itself. Red = membrane stain, blue = fibrinogen,
green = F-actin. All samples are permeabilized during staining. Scale bar= 5 µm.
Control
Y27632
NSC23766
Figure S9. Effect of ROCK and Rac-1 inhibition on f-actin distribution in
geometrically non-constrained platelets. The ROCK inhibitor Y27632 and Rac-1
inhibitor NSC23766 differentially affected filamentous actin (f-actin) rearrangement
in spread platelets. Given a large enough surface area to spread fully, control
platelets (red, cell membrane stain) formed f-actin detected by phalloidin (green) as
thick stress fibers throughout the cytoplasm and margins of the platelet (left).
Y27632 treatment reduced stress fiber formation (middle) whereas NSC23766
treatment reduced filamentous actin formation at the margins (right). All samples
are permeabilized during staining. Scale bar= 5 µm.