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Title: Both Platelet and Endothelial Cell Derived ERp5 Supports Thrombus Formation in a Laser-induced Mouse Model of Thrombosis
Supplementary Materials and Methods
Materials Recombinant His-tagged ERp72 (endoplasmic reticulum protein 72) was purchased from Enzo (Farmingdale, NY), and His-tagged PDI from Prospec (Rehovot, Israel).. Full length native integrin αIIbβ3 purified from activated platelets was purchased from AbCam. Mouse thrombin was from Haematologic Technologies Inc, (Essex Junction,
VT).
Reduced
glutathione,
GSSG
(oxidized
glutathione),
eosin
isothyocyanate, dithiothreitol, cCMP, oxidized RNAse were purchased from Sigma Aldrich (St. Louis, MO). pET-15b vector and E. coli Origami B (DE3)-competent cells were from EMD Chemicals (Gibbstown, NJ). Mouse monoclonal antibodies against PDI, ERp72 and ERp57 were from AbCam. Mouse monoclonal antibody against ERp5 was from Abnova (Taipei, Taiwan). Anti-GAPDH rabbit polyclonal antibody was from Rockland (Gilbertsville PA). Hepes Tyrode’s buffer: 119 mM NaCl, 5 mM KCl, 25 mM HEPES, 2 mM CaCl2, 2 mM MgCl2, glucose (6 g/L). Human umbilical vein endothelial cells (HUVEC) and human aortic endothelial cells (HAEC) were from Sciencell (Carlsbad, CA). Anti-β3 clone VI-PL2 and isotype control were from BD Biosciences (San Jose, CA). Goat anti-rabbit IgG conjugated to horse radish peroxidase (HRP) was from AbCam and goat anti mouse IgM was from Santa Cruz. Anti-platelet antibody CD42b labeled with Dylight 649 (Emfret Analytics, Eibelstadt, Germany) was used for intravital detection of the platelet 1
thrombus. An anti-fibrin monoclonal antibody purified from cell supernatant from the 59D8 hybridoma cell line and labeled with Alexa 488 was used to detect fibrin. The latter antibody is specific for fibrin and does not bind to fibrinogen.
Recombinant wild type ERp5, ERp5-AGHA, ERp57 and β3 integrin The cDNA for human ERp5 was obtained from a GST-tagged clone in pGEX4T2 kindly provided by J. Gibbins, University of Reading, UK. The plasmids for human ERp57 and inactive variant ERp5, ERp5-AGHA, with the a and a’ catalytic site cysteines, C55, C58, C190 and C193 mutated to alanine, were kindly provided by S. Schulman, Harvard University. The full length wild type (WT) ERp5 cDNA and ERp57 cDNA were cloned into the pET15b vector (EMD Chemicals, Gibbstown, NJ) containing a His tag and transformed into Origami E. coli. The cDNA for an inactive variant ERp5, ERp5-AGHA, was cloned into a pT7-FLAG-SBP-1 vector (Sigma #P3871) and transformed into BL21 E. coli. DNA was extracted and purified, and the sequence of each plasmid was verified through DNA sequencing. Sterile LB-Amp media was inoculated with E. coli and allowed to grow at 37oC until log phase (OD600 = 0.4-0.6). When induction of expression was required, cells were divided into induced and uninduced aliquots and IPTG was added to a final concentration of 1.0 mg/mL to the induced aliquot. The uninduced aliquot provided a control for leaky expression. Cultures were allowed to grow for 12-18 hr at 25 oC. His tagged ERp5 and ERp57 were produced as soluble protein and isolated by chromatography on a cobalt and nickel column respectively (Thermo Scientific, Rockford, IL) as described by the manufacturer. ERp5-AGHA was isolated using high capacity streptavidin beads (Thermo Scientific) and washed with 5-10 column volumes of wash buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 2 2
mM EDTA, 5 mM DTT).
Protein was eluted by adding 12 mls of wash buffer
containing 2 mM Biotin. The eluted ERp5 and ERp5-AGHA were dialyzed extensively against 1X PBS. ERp57 was dialyzed against 20 mM Tris, 150 mM NaCl, 1 mM EDTA pH 8. Dialysate was then concentrated and purity verified by SDS-PAGE. Recombinant calmodulin-tagged integrin β3 expressing cells were kindly provided by Professor Willem Ouwehand, University of Cambridge, UK. The protein was expressed in Drosophila Schneider S2 cells and purified on a W7 resin affinity column. Detection of ERp5 in platelets and endothelial cells with anti-ERp5 antibody Washed platelets were prepared at a concentration of 200 x 109/L. Platelets (100 x 106) were stimulated with or without thrombin at 0.5 U/ml. A suspension of 100 x 103 platelets in 10 μl (with or without thrombin) was directly added to 10 μl of SDS loading buffer containing 5% β-mercaptoethanol, and heated at 90oC for 5 min. The sample was run on SDS PAGE and blotted for ERp5 and GAPDH. Releasate (10 μl) separated from the suspension after centrifugation of 20 x 106 platelets (with or without thrombin) was directly added to reducing SDS loading buffer as above and probed for ERp5. Anti-ERp5 antibody (6.6 nM) was used for detection. HUVEC from passages 2-4 were seeded at 1x105 cells/well of a 6 well plate and cultured overnight. HUVEC were serum starved for 6 h, washed and fresh (serum free) media added (250 μl). Thrombin was added at 0.5 U/ml. Supernatant was collected at the following time points: 5, 10, 30 and 60 min. A volume of 20 μl supernatant was applied to SDS as described previously for platelet supernatants. Lysis buffer (RIPA) (0.5 ml) was directly added to HUVECs on the wells after the
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supernatant (with or without thrombin) was removed. Equal volume (2 μl) of HUVEC lysate was separated on SDS-PAGE and probed for ERp5 and GAPDH. Experiments were performed in triplicate. Densitometry was performed on ERp5 and GAPDH bands using ImageQuant LAS 4000 (GE Healthcare). The amount of ERp5 secreted was compared to premeasured amount of recombinant ERp5 (0.2 and 1 ng) run simultaneously on SDS-PAGE with platelet and HUVEC preparations. Quantitation of the amount of ERp5 in plasma, lysates and releasates of platelets and HUVEC was performed by sandwich ELISA in 96 well plates. Wells were coated with immunoaffinity-purified rabbit antiERp5 at 6.6 nM. A standard curve was produced by adding serial dilution of recombinant ERp5 to the coated wells. Plasma (1:10, 100 μl), platelet and endothelial cell lysates prepared by RIPA treatment as above (10 μl), platelet and endothelial cell releasates after stimulation (100 μl) were incubated in antiERp5 coated wells for 1 h, RT. After washing, sheep anti-ERp5 was added at 1 µg/ml. Detection was with HRP-conjugated bovine antibody to sheep IgG (0.1 µg/ml) followed by 50 μl of chromogenic TMB substrate and measurement of the optical density at 650 nm.
Intravital microscopy Mice were preanesthetized with intraperitoneal Ketamine (125 mg/kg), Xylazine (12.5 mg/kg) and atropine (0.25 mg/kg). A tracheal tube was inserted and the mouse maintained at 37°C on a thermo-controlled rodent blanket. To maintain anesthesia, Nembutal was administered through a cannulus placed in the jugular vein. After the scrotum was incised, the testicle and surrounding cremaster muscle were exteriorized onto an intravital microscopy tray. The cremaster preparation was superfused with thermo-controlled (37°C) and aerated (95% N2, 5% CO2) 4
bicarbonate-buffered saline throughout the experiment. Microvessel data were obtained using an Olympus AX microscope with a 60X 1.0 NA water immersion objective. The intravital fluorescence microscopy system has previously been described in detail.31 Digital images were captured with a Cooke Sensicam CCD camera (The Cooke Corporation, Auburn Hills, MI) in 640 x 480 format connected to a VS4-1845 Image Intensifier GEN III (Video Scope International, Dulles, VA).
Laser-induced injury Injury to a cremaster arteriolar (30-50 μm diameter) vessel wall was induced with a Micropoint Laser System (Photonics Instruments, Chicago, IL) focused through the microscope objective, parfocal with the focal plane and tuned to 440 nm through the dye cell containing 5 mM coumarin in methanol.
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Data were captured digitally
from two fluorescence channels, 488/520 nm and 647/670 nm. Data acquisition was initiated both prior to and following a single laser pulse for each injury. The microscope system was controlled and images were analyzed using Slidebook 5.0 (Intelligent Imaging Innovations, Denver, CO).
Image analysis For each thrombus generated by laser injury, a rectangular mask was defined that included a portion of the vessel upstream of the site of injury. The maximum fluorescence intensity of the pixels contained in this mask was extracted for all frames (pre- and post-injury) for each thrombus. The mean value calculated from the maximal intensity values in the mask for each frame was determined and used as the background value. Finally, for each frame the integrated fluorescence intensity was calculated as per following equation: 5
Integrated fluorescence intensity = Sum Intensity of signal – (mean of the maximal background intensity X area of the signal). This calculation was performed for all frames in each thrombus and plotted versus time to provide the kinetics of thrombus formation. For multiple fluorescence channels, calculations of background were made independently for each channel. The data from 22-40 thrombi were used to determine the median value of the integrated fluorescence intensity to account for the variability of thrombus formation at any given set of experimental conditions.
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Supplementary Figure 1. Expression of ERp5 antigen in the developing thrombus in vivo. Anti-ERp5 antibody labeled with Alexa 488 (0.05 µg/g body weight) or preimmune IgG labeled with Alexa-488 (0.05 µg/g body weight) and antiCD42b antibody labeled with Dylight 649 (0.1 µg/g body weight) were infused into a mouse 5-10 min prior to arteriolar injury. Representative images of the fluorescent signal from platelets (red) and anti-ERp5 (green) or control IgG (green) over 180 sec after laser-induced vessel wall injury. Merge=yellow. Minimal signal was detected from control IgG.
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Supplementary Figure 1
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control IgG
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