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Blood, Vol. 109, Issue 9, 3745-3748, May 1, 2007
Endorepellin, the C-terminal angiostatic module of perlecan, enhances collagen-platelet responses via the  2ß1-integrin receptor
Blood Bix et al.
109: 3745
Supplemental materials for: Bix et al, Vol 109, Issue 9, 3745-3748
Endorepellin, platelets, and materials Human recombinant ER was purified from 293-EBNA cells as published.1 Reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. Platelet-rich plasma (PRP), obtained from medication-free healthy donors under IRB approval, was prepared as previously published.2 Briefly, blood was collected and anticoagulated with sodium citrate (0.38% final concentration). The blood was then centrifuged at 150g to obtain PRP and adjusted to a final concentration of 2 × 108 platelets/mL. Washed platelets were also prepared as previously published.3 Briefly, blood was anticoagulated in 39 mM citric acid, 75 mM trisodium citrate, 135 mM glucose (ACD), and centrifuged at 150g. The isolated PRP was then supplemented with 1/10 volume ACD and PGEsrc and indomethacin to prevent nonspecific platelet activation/aggregation.3 The 1300g pellet was washed twice by centrifugation, and resuspended in 12 mM sodium bicarbonate, 0.36 mM sodium phosphate,138 mM sodium chloride, 5.5 mM glucose, 2.9 mM potassium chloride, ± 1.1 mM magnesium chloride, pH 7.3 (Tyrode buffer). Platelet adhesion, activation, and aggregation assays Microtiter Immulon plates (Thermo Electron, Milford, MA) were coated overnight at 4°C with acid-soluble collagen I (BD Biosciences, Bedford, MA, 100 µg/mL), endorepellin (20 µg/mL), triple-helical peptide GFOGER (10 µg/mL),4 or 1% bovine serum albumin (BSA). The selected concentrations of collagen and endorepellin support optimal adhesion of other cell types.5 In some experiments, liquid-phase endorepellin (20-80 µg/mL) was preincubated for 30 minutes with platelets in the presence or absence of the following functional blocking antibodies to:  2 1 (MAB 1998Z, Chemicon, Temecula, CA), CD31 (Chemicon MAB2148), GPVI (Santa Cruz Biotechnology, Santa Cruz, CA), or nonfunction blocking 21 12F1 (BD Biosciences), IIb3 (Santa Cruz Biotechnology), or with GFOGER or PP1 (10 µM, Sigma-Aldrich). Platelet adhesion was measured by crystal violet colorimetric analysis using a Perkin Elmer (Boston, MA) plate reader at OD560 nm. Platelet activation was measured by immunoblotting using antibodies against P-Tyr (PY20, BD Biosciences), phosphorylated focal adhesion kinase (FAK-Y397, BD Biosciences), src-homology phosphatase-1 (SHP-1, Santa Cruz Biotechnology), cortactin (BD Biosciences), syk (Cell Signaling Technology, Beverly, MA) and -actin (Sigma-Aldrich). Src activation was measured by immunoblotting with anti-src pTyr418 (Calbiochem, San Diego, CA). The intensity of the bands in the linear range was quantified with NIH-ImageJ software. Platelet aggregation was determined with an aggregometer (Sienco, Wheat Ridge, CO), which was used to study responses to fibrillar collagen (Helena Laboratories, Beaumont, TX), acid-soluble collagen (Biodata, Horsham PA), ER, 21 function blocking antibody, 12F1 21 nonfunction blocking antibody, carbamyl platelet-activating factor (C-PAF, Biomol, Plymouth Meeting, PA) or ADP.
Files in this Data Supplement:
- Figure S1. PP1 inhibits phosphorylation of platelet src kinase (JPG, 48.6 KB)
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(A) Platelets in suspension were incubated with or without the src kinase inhibitor PP1, 10 µM for 10 minutes. The platelets were then pelleted, lysed with RIPA buffer, and src activation (P-src) was analyzed by Western immunoblotting with a src-specific pTyr-418 antibody (Calbiochem), which recognizes the activation loop of pp60src. Significantly less (75% reduced, P<.005) src pTyr418 could be detected after PP1 treatment. -Actin loading control is also shown. (B) Washed platelets (n=4) with or without preincubation with PP1 (10 µM) were added to wells coated with collagen, endorepellin or BSA for 1 hour. Platelet adhesion was determined by measuring crystal violet (OD at 560 nm). PP1 significantly inhibited platelet adhesion to collagen (P<.01), significantly enhanced platelet adhesion to endorepellin (P <.001), and had no effect on platelet adhesion to BSA.
- Figure S2. Endorepellin enhances collagen activation of src (JPG, 76.7 KB)
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(A) Platelets adherent to 1% BSA, collagen (100 µg/mL) with or without liquid-phase endorepellin (20 µg/mL) or collagen (100 µg/mL) plus liquid-phase endorepellin (20 µg/mL) plus PP1 (10 µM) were lysed with RIPA buffer, and src activation (P-src) was analyzed by Western immunoblotting of src pTyr-418. The detected bands migrated at 60 kDa. -Actin loading control is also shown. (B) OD quantification (ImageJ software, mean OD ± SE, n=3) of src pTyr418 signal demonstrates that the addition of liquid phase endorepellin (20 µg/mL) to collagen-adherent platelets significantly (P<.005) increased the pTyr418 signal, and that PP1 (10 µM) blocked this enhancement such that src pTyr418 was not significantly different (P>.05) from that obtained with collagen alone.
- Figure S3. Endorepellin enhances the rate of collagen-mediated FAK phoshorylation (JPG, 56.8 KB)
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Tyrode solution containing 2 × 108/mL platelets in suspension was treated with fibrillar collagen (10 µg/mL) with or without a 10-minute preincubation with endorepellin (ER, 20 µg/mL) for 0 to 90 seconds at 37°C with constant stirring. The platelets were then pelleted, and platelet lysate obtained with RIPA was analyzed for phosphorylated FAK (P-FAK) by Western immunoblotting. The resulting bands were normalized to -actin loading control and the relative OD for each is shown ± SE. After 0-seconds incubation, all collagen plus ER relative OD values were significantly higher than corresponding collagen values (P<.05 after 10 seconds and P<.001 after 30, 60, and 90 seconds).
REFERENCES
1. Mongiat M, Sweeney S, San Antonio JD, Fu J, Iozzo RV. Endorepellin, a novel inhibitor of angiogenesis derived from the C terminus of perlecan. J Biol Chem. 2003;278:4238-4249.
2. Bernardo A, Bergeron AL, Sun CW et al. Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation. J Thromb Haemost. 2004;2:660-669.
3. Santoro SA, Zutter MM, Wu JE et al. Analysis of collagen receptors. Methods Enzymol. 1994;245:147-183.
4. Sweeney SM, DiLullo G, Slater SJ et al. Angiogenesis in collagen I requires a2b1 ligation of a GFP*GER sequence and possible p38 MAPK activation and focal adhesion disassembly. J Biol Chem. 2003;278:30516-30524.
5. Bix G, Fu J, Gonzalez E et al. Endorepellin causes endothelial cell disassembly of actin cytoskeleton and focal adhesions through the a2b1 integrin. J Cell Biol. 2004;166:97-109.
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