Supplemental Material
Methods
Reagents and Antibodies
Low molecular weight Lovenox (enoxaparin sodium; Sanofi-Aventis, Bridgewater,
NJ), heparin-coated capillaries (VWR, West Chester, PA), bovine serum albumin
(BSA, fraction V), prostacyclin (PGI2), and human fibrinogen (type I) (all from
Sigma-Aldrich, St Louis, MO), calcium sensing dye Fluo-4 (Invitrogen, Carlsbad,
CA), 2-methylthio-AMP triethylammonium salt hydrate (2-MesAMP, P2Y12
inhibitor, BioLog, Bremen, Germany), PAK-1-PBD and RalGDS-RBD coupled to
agarose beads and polyvinylidene fluoride (PVDF) membranes (Millipore,
Billerica, MA), Rac G-LISATM (Absorbance Based) Biochem kit (Cytoskeleton,
Denver, CO), human alpha thrombin (Enzyme research laboratories, South
Bend, IN),
3
H-serotonin (PerkinElmer Life, Shelton, CT), were purchased.
Convulxin was purchased from Kenneth Clemetson (Theodor Kocher Institute,
University of Berne, Switzerland). Collagen related peptide (CRP) was purchased
from Richard Farndale (University of Cambridge, UK). The Rac inhibitor EHT
1864 was provided by Exonhit Therapeutics (Paris, FR). Monoclonal antibodies
directed against GPIX or the activated form of murine αIIbβ3, JON/A-PE, were
purchased from Emfret Analytics (Wuerzburg, Germany). Antibodies against Pselectin conjugated to fluorescein iso-thiocyanate (BD Biosciences, Rockville,
MD), Rap1 and Rap2 (Santa Cruz Biotechnology, Santa Cruz, CA), and Rac1
(Millipore, Billerica, MA) were purchased.
1 Platelet preparation
Blood was drawn from the retro-orbital plexus into heparinized tubes. Plateletrich plasma (PRP) was obtained by centrifugation at 100g for 5 min. PRP was
centrifuged at 700g in the presence of PGI2 (2 µg/ml) for 5 min at room
temperature. After two washing steps, pelleted platelets were resuspended in
modified Tyrode’s Buffer (137 mM NaCl, 0.3 mM Na2HPO4, 2mM KCl, 12 mM
NaHCO3, 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 5 mM
glucose, pH 7.3) containing 1 mM CaCl2.
Rap1 and Rac1 pull-down assay
Washed platelets (4 x 108 platelets/sample) were stimulated for 1 or 10 minutes
in non-stirring conditions at 37°C. Reactions were stopped with ice-cold 2x lysis
buffer (100 mM Tris/HCl pH 7.4, 400 mM NaCl, 5 mM MgCl2, 2% Nonidet P-40,
20% glycerol and protease inhibitor cocktail lacking ethylenediaminetetraacetic
acid). Cell lysis was completed on ice for 15 minutes. The cell lysates were
incubated sequentially, for 45 minutes each, with PAK-1-PBD beads and
RalGDS-RBD beads (Millipore, Billerica, MA) to pull-down Rac1-GTP and Rap1GTP, respectively. After 3 washing steps the pellets were solubilized in sample
buffer (75 mM Tris/HCl pH 6.8, 10% Sodium dodecyl sulfate, 10% glycerol, 5% 2Mercaptoethanol, 0.004% Bromophenol blue) for the detection of Rap1 and Rac1
by immunoblot. Small aliquots of each sample were saved to control that each
sample contained equal amounts of proteins. Rap2-GTP was precipitated from
separate cell lysates with GST-Sepharose 4B beads coupled to RalGDS-RBD
construct prepared as described previously29.
2 Western blotting
Proteins were separated by sodium dodecyl sulfate–polyacrylamide gel
electrophoresis on 10-20% gradient gels and transferred to polyvinylidene
fluoride membranes. Standard western blotting procedures were used. Rap1 and
Rap2 were detected and band intensity was quantified with the Odyssey Infrared
Imaging System (Li-Cor Biosystems). Rac1 was detected by Western Lightning
enhanced chemiluminescence. Band densitometric analysis was performed with
ImageJ software.
Quantitative Rac activation assay
Washed platelets (100 µl samples at 109 platelets/ml) were stimulated with 500
ng/ml of convulxin for increasing time periods (0, 15, 30, 60, 300 seconds) in
non-stirring conditions (at 37°C). The reaction was terminated by addition of an
equal volume of 2x lysis buffer (Cytoskeleton, Denver, CO), vortexed and
immediately clarified by centrifugation at 14,000 rpm for 2 minutes at 4°C.
Lysates were snap frozen and stored at -80°C. 20 µl of each lysate were saved
for protein quantitation with Precision Red protein assay reagent (Cytoskeleton,
Denver, CO). Samples were thawed on ice and the protein concentration in the
sample was adjusted to 0.5 mg/ml. The amount of Rac1-GTP (ng) in each lysate
was determined with the Rac G-LISATM (Absorbance Based) Biochem kit
(Cytoskeleton, Denver, CO) according to the instructions of the manufacturer.
Spreading assay
Glass-slides were coated for 1 hour with a fibrinogen suspension (1 mg/ml) and
blocked with 3% BSA. Washed platelets (2 x 107 platelets/ml) were plated in the
3 presence or absence of 500 ng/ml of the GPVI agonist convulxin. Activated
platelets were allowed to adhere to the fibrinogen-coated glass slides for 3, 10 or
30 minutes at 37°C. Non-activated platelets were allowed to settle for 5 minutes
before fixation. The reaction was stopped by fixing the cells with 3.7%
formaldehyde. Samples were blocked for 30 minutes with 3% BSA solution,
stained with Alexa488-labeled antibodies to glycoprotein IX (GPIX), and analyzed
on a Nikon Ti-U inverted microscope (Nikon Instruments Inc., Melville, NY)
equipped with a Retiga EXL monochrome camera (QImaging, Surrey, Canada).
Images were acquired using Nikon NIS Elements software (NIS-Elements
Advanced Research; Melville, NY, USA) and a 100x oil objective. The surface
area of the cells was measured using the Nikon NIS Elements software.
Dense granule secretion
PRP was incubated for 30 minutes at 37°C with 3H-serotonin (2 µCi [0.074
MBg]/ml). After one washing step, platelets were re-suspended (4 x 108
platelets/ml) in modified Tyrode’s Buffer containing 1 µM imipramine and 1 mM
CaCl2. Platelets were stimulated in non-stirring conditions with increasing doses
of convulxin. The reaction was stopped after 10 minutes with 0.1M EDTA / 4%
formaldehyde solution. The samples were then centrifuged for 5 minutes at
10,000g and the supernatants were used for scintillation counting of
3
H-
serotonin. Total or 100% dense granule secretion was defined as the 3Hserotonin in samples lysed with 0.5% Triton X-100.
4 Aggregometry
Washed platelets were re-suspended at a concentration of 3 x 108 platelets/ml in
modified Tyrode’s Buffer containing 0.35% BSA and 1 mM CaCl2. The
experiment was performed at 37°C in the presence of 50 µg/ml fibrinogen and
under stirring conditions (1200 rpm). Increasing concentrations of convulxin were
added and light transmission was recorded over 5 minutes on a Chrono-log 4channel optical aggregation system (Chrono-log, Havertown, PA).
5 Figure Legends
Supplemental Figure I. Rap1 regulates Rac1 activation independently of
integrin aIIbb3 outside-in signaling. Rac1 activation in WT or CalDAG-GEFI-/(CDI-/-) platelets stimulated with 500 ng/ml convulxin (HD Cvx) in the absence or
presence of 30 mg/ml of the aIIbb3-blocking antibody, Leo.H4. The bottom panel
shows total Rac1 as loading control. Representative of 4 independent
experiments.
Supplemental Figure II. Rap2 activation downstream of GPVI is regulated
by CalDAG-GEFI and P2Y12. A) Time course of Rap2 activation (top panel)
upon stimulation of wild type (WT), CalDAG-GEFI-/- (CDI-/-), P2Y12-/- or
CalDAG-GEFI-/- x P2Y12-/- (DKO) platelets with 1 mg/ml convulxin (Cvx) for 1
and 10 minutes. The bottom panel shows total Rap2 as loading control. B)
Densitometric analysis of Rap2-GTP shown as percentage of maximal activation
(mean ± SD, n = 3).
Supplemental Figure III. Inhibitory effect of EHT 1864 on Rac1 activation in
murine platelets. Wild type platelets were treated with DMSO (-) or with different
doses of the Rac1 inhibitor EHT 1864 (50 or 100 µM) for 5 minutes at 37°C,
stimulated (+) with high dose convulxin (HD Cvx, 500 ng/ml) for 1 minute, and
formation of Rac1-GTP was determined. The bottom panel shows total Rac1 as
loading control. Representative of 3 independent experiments.
Supplemental Figure IV. Role of Rap1 and Rac1 signaling in platelets
stimulated with collagen-related peptide (10 µg/m CRP). A) Rac1 (top panel)
and Rap1 (middle panel) activation in resting (0) and stimulated conditions (5’).
6 The bottom panel shows total Rap1 as loading control. B) Spreading on
fibrinogen for 30’. C) Flow cytometric analysis of P-selectin exposure as measure
of α-granule secretion. D) Calcium influx in the presence of 2.5 mM EDTA. The
platelets analyzed were wild type (WT, black) and CalDAG-GEFI-/- x P2Y12-/(DKO, grey) in the absence (solid) or presence (checkered) of 100 µM EHT
1864. Representative of 3 independent experiments (mean±SD).
Supplemental Figure V. Quantitative analysis of Rac1 activation by G-Lisa.
Wild type (WT, solid black bar), CalDAG-GEFI-/- x P2Y12-/- (DKO, solid grey
bar) or WT treated with 100 µM EHT 1864 (WT/EHT, checkered bar) platelets
were stimulated with 500 ng/ml convulxin for 0, 15, 30, 60, 300 seconds, lysed
and equalized for total protein concentration. The graph shows ng (mean ± SD)
of Rac-GTP in 1 mg of platelet proteins (n = 6; *p<0.05, **p<0.01, ***p<0.001).
7 Supplemental Figure I
Rac1-GTP
Rac1
HD Cvx
Leo.H4
WT
+
-
+
-
+
+
+
+
+
WT CDI-/- WT CDI-/- WT
Supplemental Figure II
A
CDI-/- P2Y12-/- DKO
WT
Cvx (min)
0
1
10
1
10
Rap2-GTP
B
Rap2-GTP (%)
Rap2
Time (min)
8 1
10
1
10
Supplemental Figure III
Rac1-GTP
Rac1
HD Cvx
-
EHT 1864
+
-
+
+
50µM 100µM
Supplemental Figure IV
C
DKO
0 5
Rac1-GTP
Rap1-GTP
Rap1
ns
2 µm
9 ***
*
ns
*
MFI (Fluo-4)
ns
ns
Ca2+ flux
(% of maximal)
ns
***
*
***
D
*** ***
platelet surface
area (µm2)
B
*
CRP
CRP
WT
0 5
MFI
(α-P-selectin-FITC)
A
Time (sec)
*
Rac-GTP (ng/mg protein)
Supplemental Figure V
25
WT
DKO
WT/EHT
20
*
ns
15
ns
*
ns
**
**
10
0
ns
15
30
Time, sec
10 ***
***
60
ns
*
**
300