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) CVR-2011-134R3 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), FcRIII (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 CVR-2011-134R3 (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. CVR-2011-134R3 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 CVR-2011-134R3 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 CVR-2011-134R3 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.