CVR-2011-134R3
Supplementary Material to:
Monomeric CRP is prothrombotic and dissociates from circulating pentameric CRP on
adhered activated platelets under flow
B. Molins*, E. Peña*, R. Torre* and L. Badimon*†
*Cardiovascular Research Center, CSIC-ICCC, Institut Investigacions Biomèdiques Sant Pau
(UAB), Barcelona, Ciber Patofisiologia de la Obesidad y Nutrición, Institute Carlos III,
†Càtedra
Recerca Cardiovascular (UAB), Barcelona, Spain
CVR-2011-134R3
MATERIALS AND METHODS
CRP isoforms
High purity human pCRP (Calbiochem) was stored in 10 mmol/L Tris, 140 mmol/L NaCl buffer
(pH 8.0) containing 2 mmol/L CaCl2 to prevent spontaneous formation of mCRP from the
native pentamer. mCRP was obtained by urea chelation from purified human CRP as
previously described.15
Blood collection
Freshly drawn venous blood from non-smoking healthy volunteers with informed consent was
collected and kept at 20 ºC to be used within 2 hours of withdrawal. Platelet count, leukocyte
count, and hematocrit were all within normal ranges. Donors claimed not to have been taking
any medication during 2 weeks prior to blood extraction. The study complied with the tenets
of the Declaration of Helsinki and was reviewed and approved by our institutional Clinical
Research Committee.
In some cases, after the first extraction, donors were given a loading dose of 150 mg
clopidogrel in order to test the effect of P2Y12 blockade on mCRP-induced platelet activation.
12 h after clopidogrel treatment a second blood extraction was then performed and
successful P2Y12 inhibition was tested by ADP-induced platelet aggregation analysis.
Obtention of CD36 Fab fragments
Fab fragments of CD36 were obtained from a mouse IgG1 CD36 antibody (clone FA6-152,
Beckman Coulter) using a commercial kit (Thermo Scientific Pierce) following the
manufacturer’s instructions. Briefly, the kit uses immobilized ficin to prepare fragments from
mouse IgG1 antibodies in the presence of cysteine. The kit also contains the necessary
components for the purification of the obtained Fab fragments. Obtained Fab fragments were
analyzed by non-reducing and reducing SDS-PAGE (10 %).
Flow cytometry
For flow cytometry studies of P-selectin, CD63, and conformational change of GPIIb-IIIa,
citrate (3,8 %), 40 μM PPACK (DPhe- Pro-Arg-chloromethyl ketone) and heparin (10 IU/mL)
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anticoagulated blood samples were diluted 1:10 in modified Tyrode Buffer. 25 µl aliquots were
incubated with CRP isoforms or ADP (1 µmol/L) as positive control for 5 min at 37 ºC. In
additional experiments, the responses to CRP (50 g/mL) in heparinized (10 IU/mL) blood
were studied in the presence of mitogen-activated protein kinase (MAPK) inhibitors.
SB302580 (20 µM) was used to inhibit p38 MAPK, U0126 (10 µM) was used to inhibit MEK
½, and the inhibitor SP600125 (10 µM) was used for the inhibition of JNK. Additionally, the
response to CRP was also studied after blocking the platelet receptors CD36 (4 µg/mL CD36Fab antibody), FcRIII (2.5 µg/mL of function-blocking anti-CD16 antibody, clone 3G8,
Pharmingen), GPIIb-IIIa (5 µg/mL abciximab), and P2Y12 (oral treatment with clopidogrel).
A fluorescein isothiocyanate (FITC) conjugated monoclonal antibody (mAb) to CD61
(Pharmingen) was used as an activation-independent marker of platelets for CD62P and
CD63 analysis. P-selectin and CD63 surface expression were assessed with a phycoerytrin
(PE) conjugated anti-CD62P mAb (Pharmingen) and a PE conjugated anti-CD63 mAb
(Pharmingen), respectively. GPIIb-IIIa conformational change was assessed with a FITC
conjugated PAC-1 mAb (Beckton Dickinson) and a PE conjugated mAb to CD61 was used as
activation-independent marker. The reaction mixture was incubated in the dark at room
temperature for 10 minutes. To assess the extent of non-specific association of proteins with
platelets, blood was added to control tubes with FITC-labeled and PE-labeled non-immune
immunoglobulins. The platelet population was identified based on its forward and side scatter
and the association with CD61 antibody. A total of 10000 events were analyzed for
percentage of positive platelets using Expo32 ADC XL 4 Color software. Fluorescence was
measured with a Beckman Coulter Epics XL instrument. The fractions of the specific
fluorescence-positive platelets were obtained after subtraction of non-specific fluorescence in
samples labeled with non-immune immunoglobulins. All measurements were fluorescencecompensated on a daily basis for each set of measured samples using calibration beads.
Platelet Aggregation
Blood collected into 0.1 volume 138 mM trisodium citrate was centrifugated (15 min, 150 x g,
20 ºC) to obtain platelet rich plasma (PRP). Platelet aggregation was performed as previously
reported.18 Extent of aggregation was determined in PRP with an optical aggregometer
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(Aggrecorder-II). A volume of 200 µL of PRP adjusted to 250x106 platelets/mL was incubated
with 200 µL solution containing CRP isoforms or control buffer for analysis at indicated
concentrations.
Additionally, the response to CRP was also studied in the presence of SB302580 (20 µM) to
inhibit p38MAPK and a blocking function antibody anti-CD36 (4 µg/mL CD36-Fab antibody).
Thrombelastographic coagulation analysis
Dynamic whole blood clot formation was performed with the ROTEM coagulation analyzer
(Pentapharm, Munich, Germany), which is based on the thrombelastograph system using the
extrinsically activated ExTEM assay (containing 20 µl CaCl2 0.2 M, 20 µl TF, 300 µl citrated
blood), the intrinsically activated InTEM assay (containing 20 µl CaCl2 0.2 mol/l, 20 µl
thromboplastin–phospholipid, 300 µl citrated whole blood), and polymerized fibrinogen/fibrin
was measured using the platelet-inactivating test FIBTEM assay (containing 20 µl CaCl2 0.2
mol/l and cytochalasin D, 70 µl TF, 300 µl whole blood). The following variables were
determined: clotting time (CT, s), clot formation time (CFT, s), clot firmness (A10, mm), and
maximum clot firmness (MCF, mm). The ROTEM device was checked for proper functioning
according to the manufacturer's recommendation using a control serum (ROTROL). Tests
were performed using ROTEM cups and pins. All reagents were purchased from Pentapharm
GmbH (Munich, Germany).
Determination of Thrombin-Antithrombin (TAT) complexes and Tissue FactorProcoagulant Activity (TF-PCA)
Citrated blood was incubated with CRP isoforms (15 min, 37ºC) and plasma was obtained by
centrifugation (15 min, 1400 g). Levels of TAT were measured by enzyme immunoassay
(Assay Pro) following the manufacturer’s instructions. TF-PCA was measured by using a
factor (F) Xa generation test previously described [Camino et al]. TF-PCA values were
obtained from a standard curve performed with FXa.
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Platelet Isolation
Blood collected into acid citrate dextrose (29.9 mM sodium citrate, 113.8 mM glucose,
72.6 mM NaCl, 2.9 mM citric acid, pH 6.4) was centrifuged (15 min, 150 x g, 20 ºC) to obtain
PRP. PRP was removed and centifuged (15 min, 1400 x g, 20 ºC) in the presence of 0.1
g/mL of the platelet adenylyl cyclase prostaglandin (PGE1). Platelet pellets were
resuspended in Hepes-Tyrode buffer (134 mmol/L NaCl, 0.34 mmol/l Na2HPO4x12H2O, 2.9
mmol/L KCl, 12 mmol/L NaHCO3, 1 mmol/L MgCl2x6H2O, 20 mmol/L Hepes, 5 mM glucose)
and the wash step was repeated in the presence of PGE1. Washed platelets were then
incubated (5 min, 37 ºC) with the different CRP isoforms (25 µg/mL). After the incubation
period platelets were centrifuged (15 min, 1400 x g), supernatants were removed and platelet
pellets stored deep-frozen (-80 ºC). The pellet was lyzed with 1 mL of cold lysis buffer (1 %
Triton X-100, 10 mM Tris, pH 7.5, 150 mM KCl, and protease inhibitors), and then centrifuged
at 10 000 g for 10 min at 4°C to obtain platelet lysates.
Western Blot Analysis
Sample extracts (25 µg protein) were resolved by 8 % SDS-PAGE and electrotransferred to
nitrocellulose membranes, as described previously.19
VASP phosphorylation and VASP were detected with the phosphorylation specific monoclonal
VASP antibody 16C2 directed against the serine 239 phosphorylation site of VASP (46 kDa)
(NanoTools) and a mouse monoclonal antibody that recognizes VASP (46 kDa) and VASP
phosphorylated at serine 157 (50 kDa) (clone IE273 Immunoglobe).
Band densities were determined with the ChemiDoc™ XRS system (Bio-Rad) in
chemiluminescence detection modus and Quantity-One software (Bio-Rad). Normalization
was performed against -actin (Abcam).
Immunofluorescence
Washed platelets were fixed with 3.8 % paraformaldehyde for 30 min at room temperature,
centrifuged at 150 x g and resuspended in PBS. Washed platelets were then immobilized on
poly-L-lysine-coated coverslips overnight, permeabilized with Tween 0.5 %, incubated with
blocking buffer and afterwards incubated with the appropriate indicated VASP antibody and
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with Alexa Fluor 633 phalloidin (Molecular Probes). Coverslides were washed and incubated
with Alexa Fluor 488 donkey anti-mouse IgG (H+L) (Molecular Probes). Immunostained
coverslides were washed and covered with Prolong Gold antifade reagent. Images were
recorded by fluorescence confocal microscopy (HCX PL APO 63x/1.2 W Corr/0.17 CS).
Controls with no primary antibody showed no fluorescence labelling. Mean fluorescence
intensity was analyzed with Leica TCS SP2 software.
Measurement of cGMP activity
Washed platelets (3x108 platelets/ mL) were treated with 3-isobutyl-1-methylxanthine (IBMX)
1mM, a non selective phosphodiesterase inhibitor that promotes cAMP and cGMP
accumulation, prior mCRP (25 g/mL) stimulation. Reactions were stopped by adding an
equal volume of ice-cold solution containing ethanol 90% and HCl (0,1N) 10%. Samples were
vortexed for 30 s and kept in ice before centrifugation (1500 x g for 30 min at 4°C).
Supernatants were collected and evaporated before being re-suspended in assay buffer.
cGMP levels were measured using commercially available cyclic GMP XP Assay Kit (Cell
Signaling) following the manufacturer’s instructions. As detailed by the manufacturer, the
percentage of activity was calculated as follows: % activity=100x[(A-Amin)/(Amax-Amin)],
where A was the sample absorbance, Amax was the absorbance at maximum stimulation
(control in the presence of IBMX), and Amin was the lowest absorbance (mCRP without
IBMX). Experiments were performed three times in duplicate.
Dissociation of pCRP under flow conditions on adhered platelets
Type I collagen-coated slides were prepared and placed in the flow chambers and flow
experiments were performed as described in detail elsewhere, 15, 20 with small modifications.
Two parallel-plate perfusion chambers were placed in a series mode to analyze pCRP in the
proximal chamber and mCRP in the distal flow-chamber. mCRP and pCRP can not be
analyzed simultaneously in the same chamber due to technical limitations. Heparinized (10
IU/mL) blood containing 5 g/mL of pCRP was perfused at constant shear rates of 250 s-1 and
1500 s-1. After perfusions, collagen-coated slides were rinsed with PBS pH 7.4 and fixed with
3.8 % paraformaldehyde and incubated in blocking buffer. Immunodetection of CRP isoforms
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was performed with a mAb against pCRP (clone 1D6) and mCRP (clone 8C10), kindly
provided by Dr LA Potempa.11 Coverslides were then washed and incubated with Alexa Fluor
488 donkey anti-mouse IgG (H+L). Immunostained coverslides were washed and covered
with Prolong Gold antifade reagent. Images were recorded by fluorescence confocal
microscopy (HCX PL APO 20X / 0,7 IMM CORR). Excitation was produced via the 488 laser
line and emission was measured along with interference contrast images on a separate
photomultiplier for overlay. Controls without primary antibody showed no fluorescent labeling.
Additionally, resting and effluent sheared platelets were collected and immobilized on poly-Llysine-coated coverslides as described elsewhere.21 Afterwards, immunodetection of CRP
isoforms was performed as described above.
Platelet adhesion experiments under flow conditions
Type I collagen-coated slides were placed in a flow chamber and flow experiments were
performed as described elsewhere.15, 20 Platelets were rendered fluorescent by the addition of
mepacrine 10 μmol/L (Sigma). Blood with or without pCRP (5 g/mL) was recirculated
through the flow chamber at a constant shear rate of 1500 s -1 for 10 min in order to dissociate
pCRP into mCRP in the surface of adhered platelets. Immediately afterwards, flow was
switched to blood without CRP (also in the presence of mepacrine) that was further perfused
through the chamber with the preformed thrombi for 3 min at 1500 s-1. Platelet adhesion was
scanned with a Leica TCS SP2 confocal laser scanning microscope and surface covered by
platelets was calculated using NIH Image software (by Dr Wayne Rasband, National
Institutes of Health).22
Statistical Analysis
Results were expressed as mean±SEM. After testing for normal distribution and equality of
variances with Levene’s F test, Student’s t-test or ANOVA as appropriate were used to
determine statistical significance between treatments. A value of P<0.05 was considered
significant.