 |
INTRODUCTION |
Thrombospondin-1 (TSP1)1
is a component of the platelet
-granule that is released upon
platelet degranulation (1). It is a member of a family of proteins that
includes TSP2, TSP3, TSP4, and TSP5 (COMP) (reviewed in Ref. 2). TSP1
and TSP2 are both large trimeric glycoproteins composed of 150-kDa
subunits covalently linked by interchain disulfides (3). From the amino
to the carboxyl terminus, each TSP1 or TSP2 monomer contains a
heparin-binding domain, oligomerization (heptad) domain, a procollagen
module, three properdin (type I) modules, three epidermal growth factor (EGF)-like (type II) modules, a calcium binding domain, and a globular
carboxyl-terminal domain (C-globe) (2). TSP3, TSP4, and TSP5 are
pentameric proteins that are composed of subunits that do not contain
procollagen or properdin modules (2). The disulfide-rich, central stalk
region of TSP1, containing the interchain disulfides, the
oligomerization domain, the procollagen module, the properdin modules,
and the EGF modules, is protease-resistant (4). Therefore, assigning
function to a discrete module or set of modules within the central
stalk of TSP1 has been difficult. Functions have been assigned to the
domains of TSP1 using proteolytic fragments, monoclonal antibodies, and
synthetic peptides (2, 5). Functions also have been ascribed to
recombinant properdin modules expressed as bacterial fusion proteins
including activation of latent transforming growth factor
(6),
attachment of Bowes melanoma cells (7), inhibition of decorin
interaction with TSP1 (8), and inhibition of TSP binding to MDA-MB-231
breast cancer cells (9). No attempts have been made, however, to
express the individual modules of the stalk region in their native
disulfide-bonded state.
The heparin binding domain of TSP1 has been localized to the amino
terminus of TSP1 (10). The 70-kDa core fragment of platelet TSP1, which
includes the procollagen and properdin modules, does not bind to
heparin (10, 11), sulfatides, or heparan sulfate proteoglycans (12).
Peptides based on sequences from the properdin modules, however, do
interact with heparin and sulfatides (13, 14).
Platelet TSP1 interacts with purified fibrinogen/fibrin (15, 16),
fibronectin (17), plasminogen (18), and histidine-rich glycoprotein
(19). Such interactions may occur during blood coagulation when TSP1 is
released from platelet
-granules (20). Binding of TSP1 to
fibronectin (21), plasminogen (11), fibrinogen (11), and histidine-rich
glycoprotein (19) is through the central stalk region as assessed by
solid phase binding assays or affinity chromatography. TSP1 becomes
bound to fibrin both noncovalently and covalently via activated factor
XIII (FXIIIa)-mediated transglutamination during blood coagulation (16,
22). FXIII mediates formation of an
-
-glutamyl cross-link between
specific glutamine and lysine residues. Incorporation of TSP1 into the fibrin clot results in fibrin polymers that are finer and thinner (22,
23). The 70-kDa core fragment of TSP1 has the potential to contribute
both lysine and glutamine residues to the cross-linking (24).
Antibodies to the fibrinogen A
-chain inhibit the fibrinogen-TSP1 interaction (25). In solid phase assays, TSP1 bound to both the A
-
and B
-chains of fibrinogen (26) and discrete peptides based on
sequences in the A
- and B
-chains (27). Binding of TSP1 to
fibrinogen may account for the ability of TSP1 to alter platelet
aggregation (28). Because the fibrin clot has an important role in
wound healing, including promoting cell migration, cell adhesion, and
endothelial tube formation (20, 29), the understanding of proteins
incorporated into the clot may provide a better understanding of wound remodeling.
In order to explore specific interactions of procollagen and properdin
modules of TSP1, we expressed the modules as fusion proteins with the
gelatin-binding domain (GBD) of fibronectin in insect cells using
baculovirus. The expression system allowed the modules to be processed
by the secretory machinery and thus optimizes the chances that the
modules will adopt their native fold and disulfides. The proteins were
purified from conditioned media by gelatin-agarose affinity
chromatography, examined for heparin binding activity, and studied in
protein-protein interactions.
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EXPERIMENTAL PROCEDURES |
Expression and Purification of Recombinant Proteins--
Five
constructs of the procollagen and properdin modules of human TSP1
cDNA were expressed in the GELEX (GE1) expression vector based on
the amino-terminal third of fibronectin (30): the first properdin
module (P1); the third properdin module (P3); the first and second
properdin modules in series (P12); the first, second, and third
properdin modules in series (P123); and the procollagen domain with the
first, second, and third properdin modules in series (CP123) (Fig. 1).
The amplified cDNA inserts for P1, P12, and P123 all encoded for
the amino-terminal sequence beginning at SDSA (positions 357-360),
selected based on intron/exon boundaries (31). The P3 constructs began
at INGG (positions 473-476) based on the same reasoning. The CP123
construct began at LRRP (positions 294-298), a choice based on a
cleavage site in TSP1 that gives rise to the anti-angiogenic gp140
fragment of hamster TSP1 generated by baby hamster kidney cells (32).
The carboxyl-terminal sequences of the constructs were as follows: P1,
QECD; P12, DACP; and P123, P3, and CP123, DCPI. TSP1 cDNA, a kind
gift from Dr. Vishva Dixit (33), was amplified using PCR with
appropriate primers to introduce a BstXI restriction site at
each end of the PCR-amplified DNA. The PCR products were digested with
BstXI and subcloned into GE1/pGEM4 producing module/GE1
constructs (30). The DNA sequence of the cloned constructs was verified
against errors introduced by polymerase chain reaction by dideoxy
sequencing of double-stranded DNA (Sequenase kit, U.S. Biochemical
Corp.). The module/GE1 constructs, P1/GE1, P12/GE1, P123/GE1, and
CP123/GE1, were cut from pGEM4 at the 5'-end with Acc65, blunt ended
with Klenow fragment, and modified with PstI linkers
(Promega, Madison, WI). The constructs were treated with
PstI and XbaI. The PstI to
XbaI properdin/GE1 sequence was isolated and subcloned into
the transfer vector pVL1392 (Invitrogen, San Diego, CA) for production
of recombinant baculovirus and expression in Sf9 insect cells.
P3/GE1 was cut from pGEM4 (Promega) using BamHI and directly
subcloned into pVL1392.
Cotransfection of recombinant DNA with Baculogold DNA (Invitrogen) was
done in Sf9 insect cells under serum-free conditions. Viruses
that had undergone homologous recombination were plaque-purified and
amplified to pass 2 or pass 3. For production of recombinant protein,
Hi5 or Sf9 insect cells were infected at a multiplicity of
infection between 5 and 10 in suspension or adherent cultures. Forty-eight to 72 h after infection, culture medium was spun down to remove cells and/or cellular debris, and phenylmethylsulfonyl fluoride was added to 2 mM. Protein was bound to
gelatin-agarose via the GBD; unbound material was removed by washing
with TBS (10 mM Tris, pH 7.4, 150 mM NaCl); and
recombinant protein was eluted with 3 M guanidine
hydrochloride in TBS, dialyzed against TBS, quick-frozen, and stored at
80 or
135 °C. Purity was assessed by SDS-polyacrylamide gel
electrophoresis (PAGE). Determination of protein concentration was
based upon the following calculated extinction coefficients (mg/ml/cm)
(34) and absorbance at 280 nm: CP123/GE1, 2.10; P123/GE1, 2.10;
P12/GE1, 2.03; P1/GE1, 1.96; P3/GE1, 1.98; GE1, 1.90; and P123, 2.55. The fusion proteins are designated with the suffix "GE1" in
contrast to proteins cleaved from the GBD that contain no suffix.
To remove the GBD, protein was bound to gelatin-agarose (Sigma) at 0.25 µg of protein/µl of gelatin-agarose by batch adsorption, poured
into a column, equilibrated with digestion buffer (50 mM Tris, pH 8.5, 150 mM NaCl, 2 mM
CaCl2), and treated with trypsin (0.2%, w/w) for 30 min
with occasional mixing at room temperature. The cleaved protein was
washed from the column in TBS and collected into tubes containing
soybean trypsin inhibitor bound to Sepharose (Pierce). The soybean
trypsin inhibitor-Sepharose was removed by applying the sample to a
chromatography column. The protein was found to be 85-90% pure by
SDS-PAGE with the primary contaminant being free GBD. For incorporation
of amines into the transglutaminase site(s), either
fluorescein-cadaverine (Molecular Probes, Inc., Eugene, OR) or
monodansylcadaverine (Sigma) was incubated with the recombinant protein
and blood coagulation FXIIIa under similar conditions to the
cross-linking experiments described below. The proteins were separated
by SDS-PAGE and examined for fluorescent bands with an ultraviolet
light source (35).
To compare trypsin sensitivity between untreated P123 and reduced,
denatured, and alkylated P123, P123 was denatured with 3 M
guanidine hydrochloride and reduced with 50 mM
dithiothreitol for 30 min at 37 °C. Then the protein was treated
with iodoacetic acid (100 mM) for 2.5 h at room
temperature and dialyzed overnight against TBS. Samples were treated
with 0.6% (w/w) L-1-tosylamido-2-phenylethyl chloromethyl
ketone-treated trypsin (Sigma) for 30 min at the indicated temperature.
The proteins were separated by SDS-PAGE, transferred to nitrocellulose,
and probed with rabbit anti-TSP1 antibodies (36).
Heparin Binding Assay--
NaCl was removed by dialysis of
fusion proteins into 20 mM Tris with subsequent addition to
the indicated NaCl concentration. Alternatively, P123 cleaved from GBD
was dialyzed against TBS. Heparin binding was determined by batch
adsorption of proteins for 2 h at 4, 22, or 37 °C to
heparin-agarose (Sigma). After batch adsorption, the unbound protein
was removed, the heparin-agarose was washed three times, and bound
protein was eluted with 20 mM Tris containing 300 mM NaCl or sample buffer (3% SDS, 9 M urea). Bound and unbound protein were analyzed by SDS-PAGE.
Cross-linking: Properdin-Protein Interaction in
Solution--
FXIII cross-linking was performed to examine the
interaction in solution of the P123 construct with proteins of
interest. Recombinant FXIII (generous gift of Paul Bishop,
Zymogenetics, Seattle, WA) was preactivated with thrombin (generous
gift of John Fenton II, New York State Department of Health, Albany,
NY) for 30 min at 37 °C, and thrombin was inactivated with hirudin (Sigma). The amino-terminal 70-kDa fragment of fibronectin (37), fibrinogen (38), histidine-rich glycoprotein (39), plasminogen (40),
and Hi2 DSK cyanogen bromide fragment of fibrinogen (41) were purified
as described previously. The proteins (0.2 µM) were incubated in solution with P123 (0.1 µM) at 37 °C in
the presence of FXIIIa (1 µg/ml) in the absence of Ca2+
in a total volume of 30 µl to allow binding in the absence of cross-linking. After 1 h, CaCl2 (3 mM) was
added for 10 min for cross-linking, and the reaction was stopped with
sample buffer containing EDTA (6 mM). Alternatively, in the
case using fibrinogen, the FXIIIa was inhibited with EDTA, and the
complex was treated with thrombin to cleave fibrinopeptide A and
fibrinopeptide B from the A
- and B
- chains of fibrinogen, respectively.
To analyze complex formation, the proteins were separated by SDS-PAGE,
transferred to nitrocellulose, and immunoblotted. The recombinant TSP1
modules were probed with anti-human TSP1 (36). The GBD of the fusion
proteins and the tails from GE1 that remain on the amino and carboxyl
terminus of the inserted protein after cleavage with trypsin (Fig. 1)
were probed with rabbit anti-GE1 (expressed without any insert)
antibodies. These antibodies were specific for rat fibronectin and did
not recognize human fibronectin in immunoblots. The 27-kDa fragment of
fibronectin was probed with rabbit anti-27 kDa (37). An ovalbumin
conjugate of a synthetic peptide corresponding to human fibrinopeptide
A (FPA, A
-(1-16)) was used for preparation of antibody FPA 19/7
(IgG1
). In contrast to a fibrinogen peptide A-specific
antibody identified previously (42), antibody FPA 19/7 not only reacts
with native and synthetic A
-(1-16) and synthetic A
-(7-16) but
also with intact fibrinogen (Western blotting and enzyme-linked
immunosorbent assay, data not shown). In fact, the reactivity of this
antibody with fibrinogen is significantly greater than with any of the
free peptides (IC50(fibrinogen) = 100 pmol/ml;
IC50(A
-(1-16)/A
-(7-16)) = 2000 pmol/ml). This antibody does not react with any chain of fibrin or FXIIIa cross-linked fibrin. Three other anti-A
monoclonal antibodies were used: 1D4 to
residues 349-406, 1C2-2 to residues 529-539, and T103 to residues 308-318 of fibrinogen. The epitope for T103 was defined by synthetic peptides similar to what was described previously for the epitopes of
ID4 and IC2-2 (43). Negative controls, i.e. cross-linking in the presence or absence of P123, were included in each experiment to
be certain there was no antibody cross-reactivity to other proteins.
Cross-linking of P123 or the fusion proteins to self was assessed in
each experiment and was negative under all conditions used.
Chemiluminescence reagents (NEN Life Science Products) were used to
detect horseradish peroxidase-conjugated goat secondary antibodies
(Cappel/Organon Teknika Corp., West Chester, PA) bound to the rabbit or
mouse primary antibodies.
Procollagen and Properdin: Fibrin Interaction during Clot
Formation--
To study the incorporation of the 27-kDa fragment of
fibronectin (purified as described (44)) and procollagen and properdin modules of TSP1 into fibrin clots, the various proteins (0.1 µM) were incubated with purified fibrinogen (1.47 µM), recombinant FXIII (3 µg/ml), CaCl2 (3 mM), and thrombin (1 unit/ml) in 10 mM Tris
with 150 or 300 mM NaCl in a total volume of 20 or 40 µl.
The clots were formed for 60 min as described (45). Lysis of fibrin
clots was done at 37 °C using 5-10 µg/ml concentration of
urokinase-activated plasminogen (40). To analyze P123 cross-linking to
fibrin in a more complex milieu, platelet poor plasma was prepared, and
the globulin proteins were precipitated with 40% saturated ammonium
sulfate. The precipitate was resuspended and dialyzed against TBS
containing 0.1 mM EDTA. After the addition of P123, the
plasma globulin fraction was clotted with thrombin and
CaCl2 for 60 min at 37 °C. Purified fibrinogen
supplemented with FXIII was used as a control. For plasma clots and the
respective control, the clot was separated from the supernatant but not
washed. The clot was reduced, denatured, and loaded onto the gel. For
fibrin clots, the entire clot and supernatant were denatured and in
some cases reduced, with the total volume loaded onto the gel. Proteins were separated by SDS-PAGE and analyzed by Western blot as described above. Alternatively, for 125I-TSP cross-linking
experiments, fibrin clots were formed with purified fibrinogen (20 µg/ml) and 125I-TSP (3.8 µg/ml) in 30 µl for 1 h
at 37 °C as described previously (16). P123 was tested at several
concentrations (10-54 µg/ml). The negative controls, the 70-kDa
fragment of fibronectin and GE1, were tested at equimolar concentration
of 2.4 µM (equivalent to 54 µg/ml P123). The entire
clot and supernatant were reduced and denatured, the total volume was
loaded onto the gel, and the proteins were separated on 5% SDS-PAGE,
in some cases in the absence of a stacker. Amounts of cross-linked
125I-TSP were quantitated by PhosphorImager analysis
(Molecular Dynamics, Inc., Sunnyvale, CA). P123/GE1 was iodinated as
described previously for the 70-kDa fragment of fibronectin (37), and
binding of 125I-P123/GE1 to fibrinogen (1-3 µg/ml)
coated onto plastic (Falcon Probind 96-well plate, Becton Dickinson,
Franklin Lakes, NJ) was determined in duplicate as described for
collagen V (46).
 |
RESULTS |
Expression and Stability of the Procollagen and Properdin Fusion
Proteins--
To explore the structure/function of the procollagen and
properdin modules of TSP1, the modules (Fig.
1A) were expressed in insect
cells using the GE1 expression vector that encodes the pre-pro
secretion signal, the transglutaminase site(s), the protease-sensitive site, and GBD (Fig. 1B) (30). The modules of the GBD must be in their native conformation to bind to gelatin (47). This property allows the GBD to serve as a quality control for proper folding of the
fusion proteins during processing in the rough endoplasmic reticulum
and Golgi apparatus. After trypsin cleavage, the expressed module(s)
retain(s) from the expression vector the transglutaminase cross-linking
sites in the amino and carboxyl-terminal tails from the expression
vector. The primary transglutaminase acceptor site (Gln4)
is amino-terminal to the modules (Fig. 1B), while a
potential, additional site, Gln13, is carboxyl-terminal to
the expressed modules (35, 48).

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Fig. 1.
The domain structure of the subunits of TSP1
and TSP1 modules expressed in GE1. A, the polypeptide
backbone of TSP1 is shown schematically from the NH2
terminus (left) to the COOH terminus (right). The
structural domains and modules are labeled and discussed in the
Introduction. The recombinantly expressed modules are indicated as
follows: CP123, procollagen with first, second, and third properdin;
P123, first, second, and third properdin; P12, first and second
properdin; P1, first properdin; P3, third properdin. B,
model of the three tandem properdin repeats of TSP1 expressed in GE1
(P123/GE1). The tandem properdin modules (P123) are shown with the
signal sequence derived from fibronectin (gray
line), the transglutaminase cross-linking sites (×), the
protease-sensitive region (dotted line), and the
GBD composed of fibronectin type I (rectangles) and
fibronectin type II (ovals) modules. After cleavage with a
processing protease (upward arrow) and trypsin
(downward arrow), the properdin modules (P123)
are flanked with GE1 tails containing cross-linking sites.
Recombinantly expressed GE1 contains the transglutaminase cross-linking
sites (×), the protease-sensitive region (dotted
line), and the GBD but no insert (dashed
line).
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The proteins were expressed at concentrations of 2.5-20 µg/ml,
secreted into the culture medium, and purified by gelatin-agarose affinity chromatography (Fig.
2A). CP123/GE1, P123/GE1, and
P12/GE1 were approximately 68, 61, and 55 kDa, respectively, as
estimated from SDS-PAGE. P1/GE1 and P3/GE1 were similar in size,
approximately 48 kDa. The estimated sizes by SDS-PAGE were consistent
with the calculated masses. All proteins migrated more rapidly in the
absence of reducing agent (data not shown), indicating the presence of intramolecular disulfide bonds, and disulfide-linked oligomers were not
detected (data not shown). All proteins incorporated monodansylcadaverine or fluorescein-cadaverine when reacted with FXIIIa
(data not shown), indicating that the transglutaminase site(s) were
functional. There were no apparent differences in amine incorporation
among constructs. The protein preparations shown are representative in
that all contained GBDs of approximately 41 kDa, presumably due to
cleavage between the expressed modules and the GBD fusion proteins
during expression or purification. The stalk region of TSP1 is
resistant to protease digestion (4, 11); therefore, we tested P123 for
trypsin sensitivity. P123, cleaved from the GBD, resisted trypsin
digestion (Fig. 2B) at 25 but not 37 °C after a 30-min
incubation with 0.6% (w/w) trypsin. P123 that was reduced, denatured,
and alkylated with iodoacetic acid shifted in molecular weight due to
modification of the Cys residues with iodoacetic acid and was sensitive
to trypsin digestion at 25 or 37 °C (Fig. 2B).

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Fig. 2.
Expression of procollagen and properdin
modules of human TSP1. A, fusion proteins of the
indicated modules in GE1 were expressed by insect cells using the
baculovirus expression system. All five proteins were secreted into the
culture medium and purified by gelatin-agarose affinity chromatography.
The contaminating 41-kDa band in each protein is the GBD. Equal amounts
of proteins were stained with Coomassie Brilliant Blue. B,
purified P123 (cleaved from GBD) was untreated ( ) or reduced,
denatured, and alkylated (RDA) as indicated. The protein (30 µg/ml) was treated at 25 °C (25) or 37 °C (37) for 30 min with
trypsin (0.6%, w/w) or left untreated ( ) as indicated. The protein
was resolved on 12% SDS-PAGE under reducing conditions, and protein
was detected by anti-TSP1 antibodies on Western blot. The molecular
mass markers (kDa) are as indicated.
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Heparin Binding Properties of Procollagen-Properdin/GE1 Fusion
Proteins--
Peptides based on the properdin modules of human TSP1
(13) and a reduced and alkylated fragment that includes the first properdin module (46) bind to heparin. To test whether properdin modules of TSP1 expressed in insect cells also bind to heparin, we
incubated the GE1 fusion proteins at 4 °C with heparin-agarose at
three different salt concentrations: no added NaCl, 50 mM
NaCl, or 150 mM NaCl. CP123/GE1, P123/GE1, and P12/GE1 all
bound to heparin in the absence of added NaCl but did not bind heparin in the presence of 150 mM NaCl (Fig.
3, A-C). In the presence of
50 mM NaCl, a small fraction of CP123/GE1, P123/GE1, or
P12/GE1 bound to heparin-agarose. P1/GE1 did not bind to
heparin-agarose in the absence of added NaCl, 50 mM NaCl,
or 150 mM NaCl (Fig. 3D). The majority of the
P3/GE1 did not bind heparin-agarose in the absence of added NaCl, and
none bound at 50 and 150 mM NaCl (Fig. 3E). The
41-kDa GBD, contaminating all proteins, was found in the unbound
fraction. Similarly, GE1 without any insertion did not bind to
heparin-agarose (data not shown). P123 (cleaved from GBD) did not bind
to heparin-agarose in the presence of 150 mM NaCl at 4, 22, or 37 °C (data not shown). To summarize, CP123, P123, and P12
modules bound to heparin at 4 °C but only at nonphysiologic, low
salt concentrations, and P1 and P3 did not bind.

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Fig. 3.
Binding of the procollagen and properdin
modules of human TSP1 to heparin-agarose. CP123/GE1
(A), P123/GE1 (B), P12/GE1 (C), P1/GE1
(D), and P3/GE1 (E) were tested for their ability
to bind heparin-agarose at 4 °C in 20 mM Tris in the
presence of no added NaCl (0 mM), 50 mM NaCl
(50 mM), or 150 mM NaCl (150 mM).
The starting material (S) and proteins in the bound
(B) and unbound (U) fractions are shown by
SDS-PAGE. Proteins were detected by Coomassie Brilliant Blue staining.
The molecular mass markers (kDa) are as indicated.
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Cross-linking of P123 to TSP1-binding Proteins--
TSP1 has been
reported to bind numerous proteins, including fibrinogen, fibrin,
plasminogen, histidine-rich glycoprotein, and the amino-terminal 70-kDa
portion of fibronectin (11, 16, 49). The transglutaminase cross-linking
sites remaining in P123 after cleavage from the GBD (Fig.
1B) were utilized to analyze protein-protein interactions in
solution. This strategy was employed previously to demonstrate that the
EGF modules of blood coagulation factor IX interact specifically with
zymogen but not with activated factor X (35). The primary
transglutaminase acceptor site, Gln4, is contained within a
flexible region of fibronectin and thus should be available for
cross-linking to a variety of adjacent lysines (50). Proteins were
allowed to interact in solution with P123 and then cross-linked using
FXIIIa. Covalent complexes were analyzed by Western blotting with
anti-TSP1. No cross-linking of P123 to itself was detected (Fig.
4), as was the case under all
experimental conditions. P123 cross-linked to fibrinogen but not to
plasminogen, albumin, histidine-rich glycoprotein, or the 70-kDa
amino-terminal fragment of fibronectin (Fig. 4).
125I-P123/GE1 bound fibrinogen in a solid phase assay as
well (data not shown).

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Fig. 4.
Cross-linking of P123 to TSP1 binding
proteins. Cross-linking conditions were as described under
"Experimental Procedures." The P123 was incubated alone
(C) or with fibrinogen (Fbg), plasminogen
(Plg), bovine serum albumin (BSA), histidine-rich
glycoprotein (HRGP), or the amino-terminal 70-kDa fragment
of fibronectin (Fn). After cross-linking, proteins were
separated by 8% SDS-PAGE under reducing conditions, and complexes
containing P123 were detected by anti-TSP1 antibodies on Western blot.
P123 formed covalent complexes with fibrinogen (arrow) but
not with other proteins. Non-cross-linked P123 (P123) and
molecular mass markers (kDa) are as indicated.
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The covalent complexes of P123 (23 kDa when not cross-linked) and
fibrinogen had estimated sizes of 93 kDa (major band) and 83 kDa (minor
band) under reducing conditions (Fig. 4) and were larger than
fibrinogen in the absence of reducing agent (>330 kDa) (data not
shown). The 93- and 83-kDa bands under reducing conditions were
recognized by antibodies to both TSP1 and the fibrinogen fibrinopeptide
A (Fig. 5). When the complex was treated with thrombin after cross-linking to remove fibrinopeptide A, the 93- and 83-kDa bands, as recognized by antibodies to TSP, shifted in
molecular weight (Fig. 5A), and recognition by the monoclonal antibody to fibrinopeptide A (FPA 19/7) was lost (Fig. 5B). These data indicate that P123 cross-linked to the
A
-chain of fibrinogen, which has a size of 70 kDa. The 83-kDa band
presumably represents the 23-kDa protein cross-linked to the
approximately 60-kDa remnant of the A
-chain seen in Fig.
5B.

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Fig. 5.
P123 cross-links to the fibrinogen
A -chain. P123 was cross-linked to fibrinogen (lane
1), or P123 was cross-linked to fibrinogen and then treated
with thrombin (lane 2). The complexes were
separated on 8% SDS-PAGE under reducing conditions and detected by
Western blotting. The cross-linked complex (arrow) was
detected with polyclonal anti-thrombospondin ( -TSP)
(A) or with monoclonal antibody FPA 19/7 directed to
fibrinopeptide A at the amino terminus of fibrinogen A -chain
(B). Non-cross-linked P123 (P123) and molecular
mass markers (kDa) are as indicated.
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P123 Incorporation into a Plasma Clot--
We also analyzed
cross-linking of P123 to fibrin after clot formation of purified
fibrinogen. For these studies, FXIII was added as the zymogen, and
thrombin was added as the final reagent to initiate clotting that
proceeded for 60 min. Using a constant concentration of P123/GE1 (55 µg/ml) and increasing concentrations of fibrinogen (30 µg/ml to 1 mg/ml), there was a dose-dependent increase in
incorporation of P123/GE1 into fibrin clots (data not shown). To test
the specificity of the interaction of P123 with fibrin, we incubated
P123 with the fibrinogen-rich globulin fraction of platelet-poor plasma
containing FXIII. The P123 was detected in the clot of the plasma
globular fraction on Western blot by antibody to the GE1 fusion protein
as a single band with a molecular mass of 93 kDa after reduction (Fig.
6). Although the bands were distorted due
to high concentrations of 60-100-kDa proteins in the plasma globulin
fraction of the unwashed clot, the molecular weight of the cross-link
product was the same as the cross-link product found in a clot formed
with purified fibrinogen and FXIII. The 93-kDa band was recognized by a
monoclonal antibody to fibrin
-chain (1D4) (data not shown). Higher
-chain multimers also were recognized by 1D4 (data not shown). With
longer exposure times, P123 could be detected in higher multimers as
well (data not shown; see Fig. 8). Inclusion of unlabeled P123 (54 µg/ml) resulted in a decrement in cross-linking of
125I-TSP (3.8 µg/ml) to fibrin (fibrinogen, 20 µg/ml)
with 60 ± 10% decrease (mean ± S.D. of three experiments)
that was dose-dependent with a 25% decrease at 20 µg/ml
and 40% decrease with 30 µg/ml. Neither the 70-kDa fragment of
fibronectin nor GE1 caused a decrease in cross-linking of
125I-TSP into the fibrin clot (<10% decrease).

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Fig. 6.
Incorporation of P123 into plasma or fibrin
clots. The plasma globulin fraction (Plasma) or
purified fibrinogen (Fbg) was clotted in the presence of
P123 as described under "Experimental Procedures." The clots were
solubilized in reducing gel sample buffer and separated using 8%
SDS-PAGE, and complexes were detected by Western blotting. The P123
complex (arrow, approximately 93 kDa) was detected by
antibodies (anti-GE1) to the fusion protein tails that do not recognize
human proteins. Molecular mass markers (kDa) are as indicated.
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Plasmin Digest of Fibrin Clots Containing P123--
Fibrin clots
containing P123 were digested with plasmin to identify the segment(s)
of the
-chain that cross-link(s) to P123. Under nonreducing
conditions, the cross-link material containing P123 as detected by
anti-TSP1 was in a >200-kDa band (Fig.
7A). After 40 or 80 min of
plasmin digestion (5 µg/ml) at 37 °C, P123 was detected in a band
at approximately 180 kDa, the expected size of the D-dimer fragment of
cross-linked fibrin (51). Higher molecular weight bands persisted,
reflecting the plasmin resistance of cross-linked fibrin (52). Protein
staining of the digests revealed that about 85% of the fibrin was
converted to D-dimer by 80 min (data not shown). In reduced samples,
P123 was present in a band of 70 kDa at 20 and 40 min and 56 kDa at 80 min (Fig. 7B). The 180- and 56-kDa bands were not recognized
by monoclonal antibodies 1C2-2, 1D4, and T103, which recognize
residues 529-539, 349-406, and 308-318 of the A
-chain,
respectively (43). P123 protein did not cross-link to the monomeric
Hi2DSK fragment of fibrin encompassing residues 241-476 of the
-chain (data not shown). As a control, the amino-terminal 27-kDa
fragment of fibronectin was cross-linked to fibrin (38) and plasmin
treated. After 20, 40, or 80 min of plasmin digestion, the majority of
the 27-kDa fragment was contained in cross-linked material migrating
between 35 and 60 kDa under nonreducing conditions (data not
shown).

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Fig. 7.
Plasmin digests of fibrin clots containing
P123. Fibrin clots were formed with P123 for 60 min and then
treated with plasmin (5 µg/ml) at 37 °C. At the indicated times
(0, 20, 40, and 80 min) the clots were solubilized with nonreducing
(A) or reducing (B) sample buffer, separated by
8% SDS-PAGE, and detected by Western blotting with anti-TSP1
polyclonal antibodies. The arrow indicates the primary
cross-link product. Non-cross-linked P123 (P123) and
molecular mass markers (kDa) are as indicated.
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The plasmin digestion pattern of P123 cross-linked to fibrinogen was
different from the digestion of fibrin. After digestion with plasmin
(10 µg/ml) for 3 min, all P123-antigen was in a band of 23 kDa,
indistinguishable from the size of non-cross-linked P123, although
monomeric D-fragment remained intact as ascertained by protein staining
(data not shown).
Incorporation of Procollagen and Properdin Fusion Proteins into the
Fibrin Clot--
Additional studies were conducted to characterize the
interaction of the procollagen and each properdin module of TSP1 with fibrin clots. Fibrinogen was incubated with CP123/GE1, P123/GE1, P12/GE1, P1/GE1, P3/GE1, GE1, or P123 in the presence of FXIII, and
clotting was initiated with thrombin. The incorporation of the proteins
into the fibrin clot was analyzed by Western blot with antibodies to
GE1. CP123/GE1, P123/GE1, P12/GE1, and P3/GE1 were incorporated into
the fibrin clot and were present in a band of the expected size for
cross-linking to a single
-chain as well as larger complexes
consistent with
-chain multimers (Fig. 8). The unincorporated protein is present
on the blot and labeled "module/GE1." A larger proportion of the
added P123 or P123/GE1 was incorporated into the fibrin clot than
cross-linked with fibrinogen (compare Fig. 4 with Fig. 8). These data
are consistent with 30-50% of added TSP1 incorporated into a fibrin
clot (16). P1/GE1 and GE1 did not incorporate into the fibrin clot to
the same extent as other proteins, although in some experiments a small
amount of GE1 or P1/GE1 was cross-linked into the fibrin clot. The
small incorporation is consistent with previously published data
showing that a proportion of the GE1 is incorporated into the fibrin
clot (53). These data indicate that the second and third properdin modules, but not the first, mediate the interaction of the GE1 fusion
constructs with fibrin.

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Fig. 8.
Incorporation of the procollagen and
properdin modules of TSP1 into fibrin clots. Fibrin clots were
formed with purified fibrinogen alone (C) or in the presence
of CP123/GE1, P123/GE1, P12/GE1, P1/GE1, P3/GE1, GE1, or P123. The
clots were solubilized in reducing sample buffer, separated by 8%
SDS-PAGE, and detected by Western blotting with GE1 antibodies. The
cross-linked material is bracketed as fibrin/module
cross-links and non-cross-linked material is indicated as
module/GE1 or P123. Molecular masses (kDa) are as
indicated.
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DISCUSSION |
We expressed recombinant procollagen and properdin modules of TSP1
as chimeras with the GBD of fibronectin in insect cells. The GE1 system
has previously been used to probe the disulfide pattern of module I-12
of fibronectin (30), binding activities of the EGF modules of factor IX
(35), and binding activities of the modules I-1 to I-5 of
fibronectin (53). The present fusion proteins were folded and
disulfide-bonded correctly as assessed by gelatin binding, resistance
to trypsin, and shift in molecular weight upon reduction. Functional
studies indicate that the binding properties of the properdin modules
of TSP1 are more restricted than would be inferred from studies of
larger proteolytic fragments of TSP1 or of TSP1-based peptides.
Constructs containing all three properdin modules or the first and
second properdin modules bound to heparin at 4 °C but not in the
presence of physiological concentrations of NaCl (150 mM). P123 cleaved from GE1 did not bind heparin at physiologic salt concentration, indicating that the lack of binding is not due to the
presence of the GBD of the fusion protein. These findings are
consistent with reports that the thrombin or chymotryptic fragments of
TSP1 containing the procollagen and properdin modules do not bind
heparin, sulfatide, or proteoglycans under physiologic conditions (11,
12). The data contrast with results showing that peptides based on the
properdin modules of TSP1 bind to solid phase heparin at 4 °C in
physiologic salt (13, 14) similar to an endopeptidase-derived fragment
of bovine TSP1 that is reduced and alkylated and binds to solid phase
heparin at 22 °C (46). Therefore, the recombinant procollagen and
properdin modules of TSP1 expressed as disulfide-bonded proteins in
baculovirus act similarly to the native 70-kDa fragments of TSP1 rather
than a smaller reduced and alkylated fragment or short peptide.
Individual properdin modules P1 and P3 did not bind heparin even in the
absence of NaCl, while properdin modules in series, P12, P123, and
CP123, did bind. A likely explanation is that the KRFK sequence between P1 and P2, which causes enhanced heparin binding when introduced into
synthetic peptides (13), binds to heparin under conditions that favor
ionic interactions.
The central stalk of TSP1 has been shown to interact with numerous
proteins (11). We were unable to detect solution phase interaction with
plasminogen, histidine-rich glycoprotein, or the amino-terminal region
of fibronectin by cross-linking of the module constructs to purified
proteins. Furthermore, no interactions with plasma globulins other than
clotting fibrin were detected in the complex milieu of a clot made from
plasma globulins. The results indicate that plasminogen, fibronectin,
and histidine-rich glycoprotein interact with TSP1 in regions outside
of the properdin modules of TSP1. These results are in accord with
solution phase studies that indicate that the fibronectin and
fibrinogen binding sites in TSP1 are distinct (49) but not with solid
phase binding assays in which the two proteins cross-compete for the
binding sites in TSP1 (17). A weakness of the GE1 strategy is that
binding partners may not have a lysine readily accessible for
transglutaminase cross-linking despite the flexibility of the
cross-linking site (50). Nonetheless, the FXIII cross-linking strategy
has been used successfully to identify binding partners for the amino
terminus of fibronectin (37) and for the EGF modules of coagulation
factor IX (35).
The results suggest that the incorporation of TSP1 modules into the
fibrin clot involves highly specific protein-protein interactions. Properdin modules cross-linked only to fibrin in a complex mix of
plasma globulins. Further, the properdin modules competed for cross-linking of full-length 125I-TSP to fibrin.
Cross-linking to fibrin was to the
-chain based on the size of the
complexes, the higher order multimers, and reaction with monoclonal
antibody to fibrin. Analysis of plasmin digests indicates that
cross-linking was to a portion of the
-chain in D-dimer rather than
to the COOH-terminal 60% of the
-chain. Finally, P1 did not
cross-link to fibrin, whereas P2 and P3 did.
The amino-terminal tail of GE1 (EAQQIVQPPSPW) is similar to
the EAQQIV fibronectin-based peptide (54) and
NQEQVSPLTLLK
2-antiplasmin-based peptide
(55) that have been used to probe for lysine cross-linking partners in
-chains. NQEQVSPLTLLK cross-links to at least 12 lysines
in the
-chain (55). The glutamine-containing tails derived from GE1
are necessary for P123 (derived from GE1) cross-linking because P123
expressed in a different vector with a histidine tag does not
cross-link into a fibrin
clot.2 This result indicates
that active glutamines within the type I modules of TSP1 do not mediate
cross-linking, although the central stalk region of TSP1 is known to
contain reactive glutamines (24), and also that a lysine in the
properdin modules does not cross-link to a reactive glutamine in
fibrin. P123 was found cross-linked to the D-dimer fragment of fibrin
after plasmin digest, whereas the amino-terminal 27-kDa fragment of
fibronectin, also with the EAQQIVQ sequence, was
cross-linked to smaller fragments presumably derived from the
C
domain (A
-(220-610)) of the fibrin
-chain. These results show
that the protein module associated with the cross-linking peptide
directs the site of cross-linking.
P123-fibrinogen cross-linked material digested with plasmin for 3 min
was indistinguishable from non-cross-linked P123 by SDS-PAGE. In
contrast, plasmin digests of P123 cross-linked into fibrin clots
indicated that P123 remains associated with D-dimer of fibrin (180-kDa
band) after 80 min of plasmin treatment. The D-dimer includes the
pieces of the
-chain (residues 120-180) and
-chain (residues
134-461) that contain the previously identified binding sites for TSP1
in the fibrinogen A
-chain residues, 113-126, and B
-chain
residues, 243-252 (27). The x-ray crystal structure of the D-fragment
shows that the
-chain doubles back upon itself, apposing the
A
-chain residues of 113-126 of the TSP1 binding site to three
potential cross-link sites, Lys208, Lys219,
and/or Lys224 (56). Lys208, Lys219,
and Lys224 all were found cross-linked to the
2-antiplasmin peptide, although none were a preferred
site (used only 2-5% of the time) (55). The results are consistent
with P123 binding to region(s) within the D-domain as directed by P123
and cross-linking to Lys208, Lys219, and
Lys224 within the D-domain via the flexible amino-terminal
tail derived from GE1. There are seven potential plasmin sites between
residues 197 and 253 of the A
-chain of fibrinogen (57). The
differences in the final products of the P123-fibrinogen and
P123-fibrin complexes are due to differential cleavage of these sites
by plasmin in fibrinogen compared with cross-linked fibrin.
The fibrin clot has a critical role in wound healing. Cells migrate
into the area of tissue injury through multiple cell surface receptors
that interact with proteins incorporated into the fibrin clot (1, 20).
Other adhesive glycoproteins incorporated into the fibrin clot, like
fibronectin, are highly susceptible to plasmin proteolysis (58). The
central stalk region of TSP1 is protease-resistant (11) and may provide
a scaffold for cell migration even as the clot begins to be dissolved
by plasmin. Therefore, properdin modules would remain bound to the clot
and may provide interaction with CD36 (59) or a breast cancer cell
receptor (60) even after cleavage of the RGD sequence in the
Ca2+-binding region (61). Furthermore, TSP1 alters the
structure of the fibrin clot making finer, thinner fibrils and reducing the opacity of the clot (22, 23). Recent studies show that fibrin clots
with a reduced opacity stimulate endothelial tube formation (29).
Therefore, TSP1 in the fibrin clot could also regulate endothelial cell
function independently of direct interactions with cell surface receptors.