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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Hematology Unit, Hadassah University Hospital,
Mount Scopus, Jerusalem, Israel; the Flow Cytometry Unit, Hematology
Laboratory and Institute of Hematology, Soroka University Medical
Center, Beer-Sheva, Israel; the Max-Planck-Institut für
Biochemie, Martinsried, Germany; and the Department of Organic
Chemistry, Weizmann Institute of Science, Rehovot, Israel.
Serum amyloid A (SAA) is an acute phase reactant, and its level in
the blood is elevated to 1000-fold in response of the body to trauma,
infection, inflammation, and neoplasia. SAA was reported to inhibit
platelet aggregation and to induce adhesion of leukocytes. This study
looked at adhesion of human platelets to SAA. Immobilized SAA supported
the adhesion of human washed platelets; level of adhesion to SAA was
comparable to fibronectin and lower than to fibrinogen. Adhesion to SAA
was further enhanced by Mn2+ and the physiological agonist,
thrombin. Platelet adhesion to SAA was completely abolished by anti-SAA
antibody. SAA-induced adhesion was inhibited by antibodies against the
integrin receptor Serum amyloid A (SAA) is composed of a family of
proteins, and its level in the blood is elevated to 1000-fold as part
of the response of the body to various injuries, including trauma, infection, inflammation, and neoplasia.1,2 The SAA
proteins are expressed primarily in the liver, but extrahepatic
expression was described, including in cells of human atherosclerotic
lesions, ie, smooth muscle cells, endothelial cells, and
monocytes/macrophages,3,4 and in many histologically
normal human tissues, predominantly by the epithelium.5
SAA contains binding sites for the extracellular matrix (ECM)
components laminin6 and heparin/heparan
sulfate7 as well as YIGSR-like and RGD-like adhesion
motifs (YIGSDKYFHARGNY, residues 29 to 42). The
latter is present at the turn of an assumed The major integrin receptor expressed on platelet membrane, Antibodies and peptides
Other materials
Preparation of platelets Platelets were purified from fresh blood obtained from healthy human donors (men and women, aged 27 to 51 years) who had not taken any medication for at least 2 weeks. Blood was drawn in 3.8% buffered citrate solution (7:1 vol/vol). Washed platelets were prepared as described24 and resuspended in Tyrode buffer (NaCl 140 mM, KCl 2.7 mM, NaHCO3 11.9 mM, NaH2PO4 0.36 mM, CaCl2 2 mM, MgCl2 1 mM, glucose 5.4 mM, bovine serum albumen [BSA] 1 mg/mL, pH 7.3). Platelets were studied microscopically to ensure the absence of platelet aggregates and minimal presence of contaminating cells. They were counted (COULTER STKS, Coulter Electronics, Hialeah, FL) and adjusted to 3 to 4 × 108 platelets/mL in Tyrode buffer. The white blood cell count was 1 to 2 cells per 1000 platelets.Adhesion of platelets Platelet adhesion was carried out as described.25 Wells of 96-well titer plates were precoated (overnight at 4°C) with 50 µL of 50 µg/mL solution of each of the adhesive proteins, BSA, SAA, fibronectin, or fibrinogen and blocked (1 hour at 37°C) with Tyrode buffer containing 1% BSA. Platelet suspension (100 µL containing 3 to 4 × 107 platelets) was added to the precoated wells, platelets were allowed to adhere (1 hour at 37°C), and wells were then washed with Tyrode buffer to remove unattached platelets. Adherent platelets were fixed and stained in 2% toluidine blue/4% paraformaldehyde (30 minutes at 37°C), and, after rinsing in tap water until the washing was clear, solubilized with 100µL 1% sodium dodecyl sulfate (SDS). Absorbance at 595 nm was read in a spectrophotometer (UVICON 930; Kontron Instruments, Zurich, Switzerland). This method of staining25 was adapted after initial experiments showed its comparability to other methods (staining platelet-derived protein26 and measuring platelet acid phosphatase activity27). The acid phosphatase assay was used in some experiments (Figure 1) to quantitate the number of adherent platelets. Briefly, after the adhesion and washing procedure, as described above, the substrate solution (0.1 M citrate buffer pH 5.4, containing 5 mM p-nitrophenyl phosphate and 0.1% Triton X-100; 150 µL per well) was added and incubated for 1 hour at room temperature. The reaction was stopped, and the color was developed by the addition of 100 µL 2N NaOH, and absorbance at 405 nm was measured with a microplate reader (DYNATECH MR 5000; Dynatech Laboratories).
To test the effect of various agents, platelets were preincubated (15 minutes at 37°C) with the indicated agent before application onto the precoated wells. All experiments were performed in triplicates and repeated at least 3 times. Adhesion of melanoma cells Adhesion of the transfected human melanoma cells was carried out as described earlier with modifications. Cells were detached by minimal trypsinization (Trypsin 0.25%/EDTA 0.05%, 1 to 2 minutes), washed, and resuspended in serum-free Dulbecco modified Eagle medium supplemented with 1 mg/mL BSA. Cell suspension (100 µL containing 1 × 105 cells) was added to wells of 96-well titer plates precoated with each of the adhesive proteins, BSA, SAA, and fibronectin and was allowed to adhere for 1 hour at 37°C. Nonadherent cells were rinsed off with phosphate-buffered saline (PBS) containing CaCl2 2 mM, MgCl2 1 mM (warmed to 37°C), and adherent cells were fixed and stained in 2% toluidine blue/4% paraformaldehyde (30 minutes at 37°C). Wells were then rinsed in tap water. Stained cells were solubilized in 100µL 1% SDS, and absorbance of the solution was measured at 595 nm in microplate reader. Experiments were performed in triplicates and repeated at least 3 times. When indicated, cells were preincubated (15 minutes at 37°C) with 2 mM MnCl2 and/or anti- IIb 3 antibody (c7E3Fab),
and then applied onto the precoated titer plate wells.
Western blot analysis Recombinant human SAA and C5a (control) proteins were loaded onto 15% SDS-polyacrylamide gel and run at reduced conditions. After electrophoretic separation, the proteins were transferred to nitrocellulose membrane. The membrane was probed with anti-SAA monoclonal antibody (clone mc29), treated with horseradish peroxidase-conjugated goat anti-mouse IgG, and developed with Chemiluminescence Luminol Reagent (Santa Cruz Biotechnology, Santa Cruz, CA).Flow cytometry analysis Transfected human melanoma cells were stained for flow cytometry by using standard indirect procedure. Briefly, cells were detached by minimal trypsinization, washed, and adjusted to 1.5 × 106 cells/50 µL PBS containing CaCl2 2 mM, MgCl2 1 mM, and MnCl2 2 mM. Fifty-microliter cell suspensions were incubated (60 minutes at 4°C) with anti-IIb/CD41 (clone 5B12), anti-V3 (clone LM609), or
control isotype-matched mouse IgG. Cells were then washed and stained
with FITC-conjugated goat anti-mouse IgG. The samples were then
washed, fixed in 1% formaldehyde, and analyzed in EPICS XL-MCL flow
cytometer with System II (Beckman Coulter, Miami, FL) and EXPO32
software programs (Applied Cytometry Systems, Sheffield, United
Kingdom). In experiments detecting the binding of soluble SAA, 50-µL
cell suspensions were incubated (60 minutes at 37°C) with SAA or
control BSA (each at 200 µg/mL). Suspensions were then washed and
incubated with a mixture of anti-SAA antibodies (clones mc29 and mcl)
or control mouse IgG, and the assay proceeded as above.
Statistical analysis Unless otherwise indicated, data are presented as mean ± SEM from at least 3 independent experiments. Statistical significance was calculated by using the paired t test.
Human washed platelets adhere to immobilized SAA Human washed platelets were prepared by standard procedure24 and tested for their adhesion to immobilized proteins by using the microtiter plate adhesion assay.25,27 BSA served as a negative control for background adhesion, whereas fibronectin and fibrinogen served as positive controls. The number of platelets that adhered to the coated wells was quantified by using the acid phosphatase assay27 in which 1 optical density (absorbance at 405 nm) represents approximately 2 × 106 platelets (Figure 1A). Significant adhesion of platelets to SAA was observed; the level of adhesion to SAA was comparable to fibronectin and lower than to fibrinogen (Figure 1B). The number of adherent platelets to all 3 adhesive substrates was proportional to the number of platelets added to the coated wells and was similar to the number reported by others.28 Adhesion to SAA was further enhanced by Mn2+ ions (1 mM) or the physiologic agonist thrombin (1 U/mL) (Figure 1C). Mn2+ at lower concentration was less effective in enhancing platelet adhesion to SAA (3.8-, 1.7-, and 0.8-fold enhancement for 1, 0.5, and 0.1 mM Mn2+, respectively [representative experiment]). Adhesion was not enhanced by treatment with 10 µM adenosine diphosphate (102% ± 8.2% compared with nontreated platelets [defined as 100%]). Platelets prepared by the Mustard method and resuspended in Tyrode buffer without apyrase become insensitive to adenosine diphosphate, as previously reported.24Specificity of platelet adhesion to SAA The specificity of platelet adhesion to SAA was studied using various approaches. In the first approach, SAA-coated wells preincubated with monoclonal anti-SAA antibody, mc29 (500 µg/mL for 1 hour) failed to support platelet adhesion. However, platelet adhesion to fibronectin-coated wells, similarly pretreated with mc29, was not inhibited (Figure 2A). The specificity of mc29 antibody19,20 was confirmed by Western blot analysis, demonstrating its recognition of SAA (more then 90% of the recombinant SAA consisted of a SAA monomer of expected size 12.5 kDa) but not of C5a, an unrelated protein of similar size (Figure 2B).
In the second approach, the peptide GRGDSP inhibited platelet
adhesion to SAA in a dose-dependent manner, whereas the peptide GRGESP
had no significant effect (Figure 3,
upper panel). Furthermore, these peptides had similar differential
effect on platelet adhesion to the control adhesion protein,
fibronectin (Figure 3, lower panel). Additional control peptides,
scrambled RGD (DGPSGR) and reverse RGD (PSDGRG), had no
significant inhibitory effect on platelet adhesion to SAA
(89% ± 7.2% and 96% ± 7.6% of control adhesion, respectively)
or to fibronectin (90.6% ± 9.6% and 115% ± 7.3% of control
adhesion, respectively).
In the third approach, antibody recognizing the platelet receptor
In the fourth approach, SAA-derived peptide 29 to 42 (YIGSDKYFHARGNY) inhibited platelet adhesion to SAA in a
dose-dependent manner, whereas SAA peptides 28 to 40 (NYIGSDKYFHARG) and 83 to 104 (NEWGRSGKDPNHFRPAGLPEKY) had
no significant effect (Figure 5, upper
panel). Furthermore, SAA-derived peptide 29 to 42 also inhibited
platelet adhesion to wells coated with the control adhesion protein,
fibronectin (Figure 5, lower panel). Additional control peptides,
scrambled 29 to 42 (NYAGRKFHYSGDYI), reverse 29 to 42 (YNGRAHFYKDSGIY),
and peptide enclosing RGN (AARGNAA), had no significant inhibitory
effect on platelet adhesion to SAA (88% ± 5%, 93.4% ± 6%, and
93.8% ± 7.3% of control adhesion, respectively) or to fibronectin
(82.7% ± 9.2%, 96.7% ± 3.2%, and 93% ± 3.8% of control
adhesion, respectively).
Adhesion and flow cytometry of human melanoma cells expressing platelet receptors Adhesion of 2 transfected human melanoma cell lines expressing either theIIb3 or V3 integrin receptor on their
surface23 was studied (Figure
6). The control protein, fibronectin,
supported the adhesion of both cell lines, and the adhesion was
somewhat enhanced by Mn2+. SAA supported the adhesion of
IIb3-expressing cells but not V3-expressing cells; the
adhesion of IIb3-expressing cells was dependent on
Mn2+ (Figure 6A). The adhesion of IIb3-expressing
cells to SAA (in the presence of Mn2+) was inhibited, in a
dose-dependent manner, by the anti-IIb3 antibody, c7E3Fab
(Figure 6B).
The surface expression of either
SAA is an acute phase plasma protein, and its level in the blood is elevated to 1000-fold in response of the body to various insults, including tissue injury and inflammation. SAA is an ECM-bound protein12,13 that induces migration and adhesion of human leukocytes,10-12 and its derived peptides inhibit adhesion of leukocytes to ECM proteins.16 Because SAA expression was found in atherosclerotic lesions, including endothelial cells at the luminal sites of the vessels, it was postulated that SAA may modulate platelet aggregation or adhesion at the endothelial cell surface, thereby controlling platelet activity at vascular injury sites.3 Indeed, SAA was reported to inhibit thrombin-induced platelet aggregation9; however, its role in platelet adhesion was not studied. Here we describe that immobilized SAA supports the adhesion of human platelets and its derived peptide inhibits platelet adhesion to the subendothelial ECM protein, fibronectin. These findings identify a novel adhesive ligand for platelets and implicate its role in platelet adhesion thereby in hemostasis and thrombosis. The specificity of platelet adhesion to SAA is supported by the following findings. First, the level of adhesion to SAA was comparable to other platelet adhesive proteins, ie, fibronectin and fibrinogen. Furthermore, the physiologic agonist thrombin, as well as the divalent ion Mn2+ (an artificial stimulus that mimics physiologic agonists), could enhance platelet adhesion to SAA. The enhancement of platelet adhesion by these 2 agents is well documented and may result from an increase in the binding affinity of the platelet receptor(s) toward SAA, as reported for other adhesive proteins.28,32-34 Second, anti-SAA antibody completely inhibited SAA-induced platelet adhesion and had no inhibitory effect on fibronectin-induced adhesion. This antibody (clone mc29) is likely to bind to a functionally relevant epitope of SAA, because it was prepared against the invariant segment of SAA containing the RGD-like motif.19,20 Third, the peptide GRGDSP but not GRGESP inhibited platelet adhesion to SAA and to fibronectin, suggesting that the adhesion to both proteins is RGD dependent. The differential inhibitory effect of these 2 peptides on platelet adhesion to fibronectin, as well as other adhesive proteins including vitronectin and fibrinogen, is well documented.35,36 Fourth, antibody recognizing the platelet receptor Fifth, the SAA-derived peptide 29 to 42, containing an RGD-like
motif (aspartic acid [D] in the typical RGD motif is substituted by
asparagine [N]), inhibited platelet adhesion to SAA and to fibronectin; the peptides 28 to 40, containing incomplete RGD, and 83 to 104, the C-terminus of the protein lacking adhesion motif, had no
effect. Although the aspartic acid is very important for interactions
with integrins, the asparagine, a noncharged amino acid, is isosteric
and capable of establishing efficient interactions, ie, by hydrogen
bonding. Furthermore, the presence of the RGD-like motif in SAA at the
turn of an assumed The role of In conclusion, our data indicate that human platelets
specifically adhere to SAA in an RGD- and Dr Yaacov Matzner tragically passed away in
a plane crash on Saturday, November 24, 2001, returning home from a
scientific meeting in Germany; his many contributions to hematology
will be remembered, and his presence in our community will be greatly missed.
We thank Dr Mark H. Ginsberg, The Scripps Research Institute, for the transfected human melanoma cells and Dr Barry S. Coller, Mount Sinai School of Medicine, for the 10E5 antibodies.
Submitted March 20, 2001; accepted October 12, 2001.
Yaacov Matzner died on November 24, 2001.
Supported by the Israel Cancer Research Fund, the Israel Science Foundation (grant 686/00-1), the Israel Cancer Association, the Israel Ministry of Absorption, and the Deutsche Forschungsgemeinschaft, Bonn, Germany (grant Li 247/12-3).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Simcha Urieli-Shoval, Hematology Unit, Hadassah University Hospital, Mount Scopus, Jerusalem 91240, Israel; e-mail: matzner{at}cc.huji.ac.il.
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Ligands activate integrin |
Top
Abstract
Introduction
) or GRGESP (
) at the indicated concentrations and then
added to BSA-, SAA- or fibronectin-coated wells. The results are
expressed as a mean ± 1 SEM for 3 experiments, each designed with
triplicates for each peptide concentration. In each experiment, 100%
adhesion (control) is the adhesion achieved in the absence of the
tested peptide after subtracting adhesion to BSA.
*P < .05, **P < .01 when comparing adhesion
with and without the tested peptide.
) at the indicated concentrations and
then added to BSA-, SAA-, or fibronectin-coated wells. For details see
legend to Figure 
Ed