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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biochemistry, University of
Pavia, Italy.
Decorin is a small leucine-rich proteoglycan able to interact with
several molecules of the subendothelial matrix, such as collagen and
fibronectin. In this work, we investigated the ability of purified
decorin to support adhesion of human platelets. We found that
gel-filtered platelets were actually able to interact with immobilized
decorin. Platelet adhesion to decorin was time dependent, required the
presence of Mg2+ ions, and was totally mediated by the
protein core of the proteoglycan. Platelet stimulation with either
adenosine diphosphate (ADP) or a thrombin receptor-activating
peptide significantly increased interaction of these cells with the
proteoglycan. Upon adhesion to immobilized decorin a number of platelet
proteins were found to become tyrosine-phosphorylated. By
immunoprecipitation experiments with specific antibodies, the tyrosine
phosphorylation of the tyrosine kinase Syk and the phospholipase C Decorin is the prototype member of a growing
family of structurally related extracellular matrix proteoglycans,
known as small leucine-rich proteoglycans.1 It is composed
by a 40-kDa protein core and one chondroitin/dermatan sulfate side
chain linked to a serine residue at the N-terminal region of the
protein. The protein core contains 10 leucine-rich repeats of 24 amino
acids forming a typical Decorin plays a key role in the regulation of extracellular matrix
assembly by binding to several components such as
collagen,2-5 thrombospondin,6 and
fibronectin.7,8 Interaction of decorin with collagen has
been shown to affect fibril formation by causing an initial delay in
the lateral assembly and a reduction of the average fibril
diameter.2,9 These effects may explain the phenotype of
decorin-null mice characterized by abnormal skin fragility and loosely
packed collagen fibers.10
In addition to its ability to modulate the assembly of the
extracellular matrix, decorin also displays a number of
biologic effects. For instance, this proteoglycan binds to
growth factors, such as transforming growth factor Upon a vessel wall injury, molecules of the subendothelial matrix are
exposed to circulating blood cells. As an early event in the hemostatic
processes, platelets rapidly adhere to several subendothelial
components through specific membrane receptors. This event is followed
by rapid platelet activation that leads to the formation of cell
aggregates, representing a real hemostatic plug. Among the different
components of the subendothelial matrix, several glycoproteins have
been shown to mediate platelet adhesion and activation. For instance,
platelet interaction with collagen and von Willebrand factor has been
deeply investigated and certainly plays a major role in the recruitment
of these cells at the site of vessel wall injury.21-23
Similarly, adhesion to other glycoproteins of the subendothelial
matrix, such as thrombospondin,24
fibronectin,25 and laminin,26 has been
reported. By contrast, no information is available on the possible
interaction of human platelets with the proteoglycans, which are
relevant components of the subendothelial matrix, and, in addition to
adhesive glycoproteins, may play a role in primary hemostasis. In the
light of the increasing interest arising from the dual role of the
small proteoglycan decorin as regulator of matrix assembly and cell
function, we investigated its possible involvement in the hemostatic
processes by analyzing its ability to support platelet adhesion and
activation. In this work we demonstrate that human platelets
efficiently adhere to decorin under static conditions. This interaction
is mediated by integrin Materials
Decorin purification
Platelet preparation Blood was collected from healthy volunteers using citric acid-citrate-dextrose as anticoagulant and centrifuged at 120g for 10 minutes at room temperature to obtain the platelet-rich plasma (PRP). Platelets were then recovered by centrifugation of the PRP at 300g for 10 minutes and resuspended in a small volume (0.5-1 mL) of autologous plasma. Platelets were then isolated by gel-filtration on a 10-mL column of Sepharose CL-2B and eluted with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (10 mM HEPES, 137 mM NaCl, 2.9 mM KCl, 12 mM NaHCO3, pH 7.4). Platelet count was adjusted to 1 × 109 cells/mL with the same buffer and then diluted for the adhesion assay.Platelet adhesion assay Platelet adhesion to decorin was studied in 60-mm polystyrene dishes coated for 16 hours at room temperature with 1 mL of 100 µg/mL decorin solution or decorin protein core in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA). Control plates were coated with 0.5% BSA in PBS for the same time. Dishes were then washed 3 times with 5 mL PBS and then blocked with 2 mL 5% BSA in PBS for 3 hours at room temperature. Decorin-coated and control dishes were finally washed again 3 times with 5 mL PBS. Gel-filtered platelets were diluted at a final concentration of 2 × 108 cells/mL with HEPES buffer containing 0.1% BSA, 5.5 mM glucose, and 2 mM MgCl2. Then, 0.5 mL platelet suspension (1 × 108 cells) were added to decorin-coated or control dishes and incubated for 90 minutes at room temperature. In some experiments, platelet samples were incubated with agonists or specific antibodies just before addition to decorin-coated or control dishes. Platelet stimulation was performed with 10 µM ADP or 10 µM thrombin receptor-activating peptide (TRAP). At the end of the incubation, nonadherent platelets were gently removed and either discharged or collected in separate tubes for further analysis. Dishes were washed 3 times with 5 mL PBS, and the adherent platelets were lysed and scraped into 0.1 mL 2% sodium dodecyl sulfate (SDS) in HEPES buffer at 90°C. Platelet adhesion was quantified using a colorimetric assay based on the consideration that the number of adherent cells is proportional to the amount of cell-derived proteins as previously described.30,31 Lysed, adherent cells were transferred to a test tube, and the protein content was determined by the bicinchoninic acid assay. To correlate the amount of measured proteins with the number of adherent cells, parallel samples containing 1 × 108 platelets from the same cell suspension used for the adhesion experiments (corresponding to the number of cells added to each dish) were prepared, lysed with 2% SDS, and subjected to protein determination. Results are generally expressed as percentage of adherent cells referred to the total added platelets.Immunoprecipitation For immunoprecipitation experiments, adherent platelets were lysed with 0.25 mL ice-cold immunoprecipitation buffer (10 mM Tris/HCl pH 7.4, 158 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EGTA (ethyleneglycoltetraacetic acid), 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mM Na3VO4). Platelet lysates were placed on ice for 15 minutes. Nonadherent platelets, collected after the adhesion assay, were lysed by adding an equal volume of ice-cold immunoprecipitation buffer 2X and placed on ice. Similarly, an aliquot of total gel-filtered platelets in HEPES buffer was also lysed with immunoprecipitation buffer. Upon incubation on ice, the samples were centrifuged at 13 000 rpm for 10 minutes at 4°C to remove insoluble materials, and the protein concentration in the supernatant was determined by the bicinchoninic acid assay. Aliquots of each sample, containing equal amount of proteins, were precleared for 1 hour at 4°C with 100 µL protein A-Sepharose (50 mg/mL stock solution). The cleared lysates were incubated with 1 µg anti-Syk or anti-PLC 2 antisera for 2 hours at 4°C. Immunocomplexes
were then recovered by incubation with 100 µL protein A-Sepharose for
45 minutes. After centrifugation, immunoprecipitates were washed 3 times with immunoprecipitation buffer and finally resuspended with 25 µL SDS-sample buffer (25 mM Tris, 192 mM glycine, 2% SDS, 0.5% DTT,
10% glycerol, 0.01% bromophenol blue, pH 8.3) and heated at 95°C
for 3 minutes.
Immunoblotting Analysis of protein tyrosine phosphorylation was performed on both immunoprecipitates and whole cell lysates. In the latter case, aliquots containing the same amount of proteins from total platelets, nonadherent or adherent platelets to decorin of BSA-coated dishes, were added to an equal volume of SDS-sample buffer and heated at 95°C for 3 minutes. Immunoprecipitates, as well as samples of whole platelet lysates, were subjected to SDS-PAGE on 7.5% acrylamide gels. Proteins were transferred to nitrocellulose and tested with antibodies against phosphotyrosine, Syk, or PLC2, as previously
described.32
Purification of integrin  2 1 was purified from
platelet membranes by affinity chromatography on collagen-Sepharose 4B,
essentially as described by Kern et al.33 Platelet
membranes were prepared from transfusion units obtained from the local
blood bank, as previously described,34 and solubilized in
extraction buffer (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM
MnCl2, 2 mM MgCl2, 100 mM n-octyl
-D-glucopyranoside, 10 µg/mL leupeptin, 10 µg/mL aprotinin).
Type I collagen was coupled to cyanogen bromide-activated Sepharose
4B, according to the manufacturer's instructions. The efficiency of
the coupling was evaluated by hydroxyproline assay. A 5 mL column of
collagen-Sepharose 4B was packed and equilibrated with extraction
buffer at a flow rate of 30 mL per hour. Solubilized membrane proteins
were loaded at a flow rate of 10 mL per hour. After extensive washes of
the column, bound proteins were eluted with Tris/HCl 50 mM, pH 7.4, 150 mM NaCl, 25 mM n-octyl -D-glucopyranoside, 25 mM EDTA, and monitored continuously by spectrophotometry at 280 nm. Positive fractions were
pooled and extensively dialysed against Tris/HCl 50 mM, pH 7.4, 150 mM
NaCl, 25 mM n-octyl -D-glucopyranoside, 2 mM MgCl2, 1 mM
MnCl2. The amount of proteins was determined by the
bicinchoninic acid assay, and the purity of integrin
21 was confirmed by electrophoretical analysis followed by silver staining of the gels.
Solid-phase binding assay Wells of a microtiter plate were coated with 50 µL decorin (400 µg/mL in PBS) for 16 hours at 4°C. Plates were washed 3 times with 100 µL washing solution (150 mM NaCl and 0.1% Tween 20) to remove unbound proteins. Additional binding sites were saturated for 2 hours with 100 µL 1% BSA in PBS. Increasing amounts of purified integrin21 (0.1, 0.5, 1, and 2 µg) in
a final volume of 50 µL Tris/HCl 50 mM, pH 7.4, 150 mM NaCl, 25 mM
n-octyl -D-glucopyranoside, 2 mM MgCl2, 1 mM
MnCl2 were added, and incubation was performed for 2 hours
at room temperature. Wells were then washed 4 times with washing
solution containing 2 mM MgCl2 and incubated with 50 µL
of the anti-integrin 2 antibody 1936 (1:1000 dilution) in PBS containing 1% BSA, 0.05% Tween 20, and 2 mM MgCl2
for 2 hours at room temperature, followed by incubation with a
peroxidase-conjugated secondary antibody for an additional hour. Bound
proteins were detected by a colorimetric reaction using
o-phenylenediamine dihydrochloride as substrate. Control wells were
prepared by omitting either coating with decorin or incubation with
purified integrin 21.
Integrin -mannopyranoside in Tris/HCl 50 mM, pH 7.4, 135 mM NaCl, 1 mM
CaCl2, 2 mM MgCl2, 1 mM MnCl2, 10 mM n-octyl -D-glucopyranoside. Fractions containing eluted
glycoproteins were pooled, and 1 mL samples were pretreated with 200 µL streptavidin-Sepharose (20% slurry) for 1 hour at 4°C. The
cleared samples were incubated either with 100 µg biotinylated decorin or an equal volume of buffer for 2 hours at room temperature, and then streptavidin-Sepharose was added. After further incubation for
1 hour, the beads were collected by centrifugation and washed 3 times,
and the associated proteins solubilized with 25 µL SDS-sample buffer.
The presence of integrin 21 among the
glycoproteins bound by biotinylated decorin was analyzed by
immunoblotting with the anti-integrin 2 antibody 1936.
Characterization of purified decorin The decorin preparations used in this study were first characterized by electrophoretic analysis under denaturating conditions, before and after digestion with chondroitinase ABC: a single band of about 100 kDa and 40 kDa, respectively, was observed. Purified decorin was then subjected to N-terminal amino acid sequencing. In all the preparations, a unique and correct sequence was determined, confirming the absence of any contaminating proteins. The structural conformation of purified decorin was analyzed by circular dichroism spectroscopy: spectra at 20°C were very similar to those reported in the literature for recombinant decorin.In addition, we specifically tested the decorin preparations for contaminating collagen. Amino acid analysis of hydrolyzed samples did not reveal the presence of hydroxyproline, and the amount of proline was consistent with that expected for purified decorin. Moreover, the presence of collagen was also tested by dot blot experiments using a specific antiserum against collagen type I. Even when 10 µg purified decorin was tested, no reactivity to the anticollagen antibody was observed. By spotting on nitrocellulose filters increasing amounts of purified collagen, we found that this immunological system was able to detect amounts of collagen as low as 10 ng, indicating that if undetectable traces of collagen were present in the decorin preparations, they accounted for < 0.1% (data not shown). Therefore, we conclude that decorin used in this study was highly purified and essentially free of contaminating collagen. Platelet adhesion to immobilized decorin To investigate the ability of human platelets to interact with decorin, polystyrene dishes were coated with either purified decorin or BSA as a control. Gel-filtered platelets were incubated with immobilized ligands for 90 minutes at room temperature in the presence of 2 mM MgCl2, and the extent of adhesion was estimated by a colorimetric assay as described in "Material and methods." Figure 1 shows that under these conditions about 24% of added platelets were able to adhere to decorin-coated dishes, while the nonspecific binding to immobilized BSA was only about 10%. Under the same conditions, platelet adhesion to collagen-coated dishes was about 50%, indicating that interaction of platelets with decorin, although significant, was less efficient. When polystyrene dishes were coated with recombinant decorin, the percentage of platelet adhesion was very similar to that observed using dishes coated with the proteoglycan purified in our laboratory (Figure 1). To investigate whether decorin interaction with platelets was mediated by the proteoglycan protein core or by the glycosaminoglycan side chain, purified decorin was digested with chondroitinase ABC, and the isolated protein core was used to coat polystyrene dishes. As shown in Figure 1, a significant platelet adhesion to the protein core was observed. The level of adhesion to the decorin protein core was about 37%, and, therefore, subsequent experiments were performed using purified decorin protein core.
In time course experiments, platelet interaction with decorin appeared to be quite rapid. After 30 minutes of incubation the percentage of cell adhesion was already about 28%, and then further increased to reach a maximum after 90 minutes (data not shown). The morphology of decorin-adherent platelets was analyzed by fluorescence microscopy upon staining with trypan blue. Platelets adhered to immobilized decorin as single cells, and no aggregates were detected. Adherent platelets appeared as round cells with several thin and long pseudopods, indicating that some degree of activation occurred (data not shown). Platelet adhesion to decorin was investigated in the presence of 2 mM
MgCl2. To analyze the role of this ion on platelet-decorin interaction, adhesion assays were performed in the absence of MgCl2 or in the presence of different divalent cations such
as Ca++ and Zn2+. Figure
2 shows that platelet adhesion to decorin
was actually Mg2+ dependent. In the presence of EDTA,
adhesion dramatically dropped to a percentage comparable to that
observed in BSA-coated dishes. Figure 2 also shows that
Mg2+ could not be substituted with Ca++, while
only a slight increase of platelet adhesion to decorin was observed in
the presence of Zn2+. Another divalent ion,
Mn2+, could not be tested in our experimental system
because it was found to strongly interfere with the colorimetric assay
used to quantify platelet adhesion.
It is known that the ability of platelets to interact with several
adhesive proteins is increased upon stimulation of these cells.23 Therefore, we investigated the effect of platelet
stimulation on interaction with decorin. Platelets were allowed to
adhere to decorin-coated dishes in the presence of 10 µM ADP or 10 µM TRAP, a selective thrombin receptor activating peptide.
Stimulation with either ADP or TRAP increased the ability of platelets
to interact with immobilized decorin (Figure
3). In particular, in ADP-stimulated
platelets the percentage of adhesion was approximately 70%. Because,
even in the absence of stirring, platelet activation may result in the
formation of small cell aggregates that can cause an overestimation of
the percentage of adherent cells, assays with stimulated platelets were
performed in the presence of 1 mM Gly-Arg-Gly-Asp-Ser (GRGDS)
peptide, a potent antagonist of fibrinogen binding to integrin
Platelet adhesion to decorin activates tyrosine kinases It is known that platelet interaction with some components of the subendothelial matrix such as collagen, von Willebrand factor, or fibrinogen leads to cell activation associated with stimulation of tyrosine kinases and tyrosine phosphorylation of several intracellular proteins.32,36,37 Therefore, we analyzed the level of protein tyrosine phosphorylation in decorin-adherent platelets. Gel-filtered platelets were incubated with BSA- or decorin-coated dishes for 90 minutes. Adherent as well as nonadherent platelets were lysed, and an equal amount of proteins was separated on a 7.5% acrylamide gel, transferred to nitrocellulose, and probed with antiphosphotyrosine antibodies. In samples from nonadherent platelets, a single tyrosine phosphorylated protein with an apparent molecular mass of about 60 kDa (that in some samples appeared as a doublet by unknown reasons) was evident (Figure 4). This band was also present in lysates from untreated resting platelets and probably represented the tyrosine kinase pp60src, which is highly expressed and constitutively tyrosine phosphorylated in these cells.38 By contrast, a number of additional tyrosine phosphorylated proteins were detected in platelets adherent to decorin- but not to BSA-coated dishes (Figure 4). The main bands appeared to have molecular masses of about 38, 50, 60, 70, 120, and 150 kDa. These results indicate that adhesion of platelets to decorin specifically induces the tyrosine phosphorylation of multiple substrates.
We next tried to identify some of the proteins that became tyrosine
phosphorylated upon platelet adhesion to decorin. It is known that
platelet interaction with collagen or von Willebrand factor induces the
tyrosine phosphorylation of the kinase Syk, which in turn mediates the
phosphorylation of PLC
Platelet adhesion to decorin is mediated by integrin
IIb3, CD38, or CD31 did not affect
platelet adhesion to immobilized decorin (Figure
6). By contrast, 2 distinct antibodies
against integrin 21 (1998 and P1E6)
almost completely inhibited the adhesion of human platelets to decorin.
Because the P1E6 antibody was used as ascite, to confirm that its
ability to block platelet interaction with decorin was actually due to
the antibody specificity, aliquots of P1E6 were pretreated with protein
G-Sepharose. As shown in Figure 6, antibody depletion by protein
G-Sepharose specifically abolished the ability of P1E6 to inhibit
platelet adhesion to decorin.
To confirm the involvement of integrin
Finally, we analyzed the ability of purified integrin
In the present work we have demonstrated that the small proteoglycan decorin is able to promote platelet adhesion and activation. These results represent the first evidence for a direct interaction of a member of the small leucine-rich proteoglycan family of the subendothelial matrix with circulating platelets and indicate that, in addition to its role as a regulator of matrix assembly and cell growth, decorin may also be involved in hemostasis and thrombosis. Decorin is composed of a protein core and a glycosaminoglycan side chain. We found that interaction with platelets is mediated by the decorin protein core. We consistently found a higher percentage of platelet adhesion to the isolated decorin protein core than to the intact proteoglycan. It is known that the glycosaminoglycan side chain linked to the N-terminal region of decorin has a high molecular mass, even greater than that of the protein core. Therefore, it is possible that in the native proteoglycan, the glycosaminoglycan chain may mask some platelet binding sites on the protein core, thus reducing the efficiency of the interaction. We have also provided convincing evidence that integrin
In this work we have also found that platelet adhesion to decorin is
increased upon activation of these cells. This may reflect a positive
regulation of the receptor function of integrin
We have also demonstrated that adhesion to decorin leads to
platelet activation through the stimulation of protein tyrosine phosphorylation. However, we have found that addition of soluble decorin to a platelet suspension did not cause detectable aggregation (data not shown). We have found that the tyrosine kinase Syk and the
lipid metabolizing enzyme PLC In conclusion, this work demonstrated for the first time a novel role for the small proteoglycan decorin in platelet adhesion and activation. Decorin is a member of a related family of small leucine-rich proteoglycans expressed in the subendothelial matrix, including the highly homologous biglycan. Further studies will be required to verify whether other members of this family of proteoglycans might also be involved in the regulation of platelet function.
Submitted November 14, 2001; accepted April 19, 2002.
Supported by grants from Consiglio Nazionale delle Ricerche (CNR, target project biotechnology), CIB (Consorzio Interuniversitario Biotecnologie), and the University of Pavia (Progetto di Ateneo).
G.G. and A.B. contributed equally to this work.
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: Mauro Torti, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy; e-mail: mtorti{at}unipv.it.
1. Iozzo R. The biology of the small leucine-rich proteoglycan. J Biol Chem. 1999;27:18843-18846 |
Top
Abstract
Introduction
-aminocaproic acid, 10 mM EDTA (ethylenediaminetetraacetic
acid), 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 5.6, and
purified by preparative ultracentrifugation (100 000g) in a
CsCl gradient in the presence of buffered 4 M guanidinium
hydrochloride. The fraction with density 1.5 g · mL
1
was adsorbed on diethylaminoethanol (DEAE)-Sephacel and eluted with a linear 0-0.8 M NaCl gradient in the presence of 4 M urea. Decorin was desalted on PD-10 columns, freeze-dried, and stored at
80°C. The protein content of the decorin preparations was determined with the Bradford method. Decorin protein core was prepared by digestion of purified decorin with chondroitinase ABC (EC
4.2.2.4) as reported by Yamagata et al.
