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(Received for publication, September 1, 1994) From the
Platelet integrin Vascular injury is widely recognized as a trigger for platelet
adhesion, spreading, and aggregation, as well as for activation of the
intrinsic coagulation cascade, resulting in the rapid conversion of
prothrombin to thrombin near the site of injury. The presence of a
local high thrombin concentration will quickly lead to the formation of
an insoluble gel composed of fibrin fibers, which reinforces the
platelet plug, halting blood loss. Once the fibrin network is in place,
the platelets begin to pull actively on the network strands and this
process leads to a dramatic reduction in clot volume. Clot retraction
is assumed to play a role in approximating the edges of a tissue defect
and concentrating the clot precisely in the injured area. The
retraction of a clot may be important for allowing the recanalization
of a partially or totally obstructed vessel. Many previous reports
have demonstrated that platelet integrin,
The
interaction of adhesive proteins with receptors in the integrin family
are associated with several significant biological events including
development, immune recognition, inflammation, hemostasis, and wound
repair(8, 9, 10, 11, 12, 13, 14) .
Integrins are expressed on the cell surface as heterodimers of Nucleated cells, such as fibroblasts
and tumor cells, have been reported to interact with three-dimensional
fibrin substrate and to induce the retraction of fibrin
clots(22, 23, 24) . Such retraction may be an
important feature of tissue reorganization. However, in contrast to
platelets, little is known about receptors on the cell surface or about
the mechanism of the process. In this report, we present biochemical
and ultrastructural evidences that
GRGDSP and GRGESP were purchased from Iwaki
(Chiba, Japan). GPIIb-IIIa-specific antagonist, Ro43-5054(30) ,
was generously provided by Nippon Rosche Research Center (Kamakura,
Japan).
A
pBOS
Figure 1:
Fibrin gel
retraction induced by platelets and C32TG cells. a, fibrin
gels containing washed platelets (2
Figure 2:
Effects of antibodies, peptides, and
Ro43-5054 on fibrin gel retraction induced by platelets and C32TG
cells. Cells were preincubated with buffer, appropriate antibodies,
peptides, or Ro43-5054 at room temperature for 15 min. Fibrin gels
containing treated cells (5
C32TG cells were able to induce clot
retraction in an RGD- and
Figure 3:
Clot
retraction induced by C32TG and 293 cells. a, analysis of
Figure 4:
Analysis of
Figure 5:
Clot retraction induced by 293
transfectants bearing
Figure 6:
Alignment of fibrin strands in fibrin gels
retracted by C32TG cells. a, fluorescence micrograph of
retracted fibrin gels. A clot containing 2
To
define the ultrastructure of fibrin clots, fibrin gels containing C32TG
cells were fixed at different stages of clot retraction and examined by
transmission electron microscopy. Fig. 7, a and b, shows transmission electron micrographs of fibrin clots
containing C32TG cells fixed at 5 min and at 45 min, respectively. At 5
min, the cells were frequently interconnected by long fibrin strands,
suggesting selective interaction. At 45 min, the cells showed much
closer contact with fibrin strands, and most of the cell surface was
coated with fibrin, a result that appears to be consistent with the
observation by phase contrast microscopy (Fig. 6). In addition,
it should be noted that the diameter of the fibrin fibers increased as
the clot underwent retraction, indicating altered fibrin structure.
Figure 7:
Transmission electron micrographs of
fibrin clots containing C32TG cells. Fibrin clots were fixed at 5 min (a) and 45 min (b) after the addition of thrombin.
Details are found under ``Materials and Methods.'' C32TG
cells were seen associated with fibrin fibers (arrows). At 45
min, the cells showed much closer contact with fibrin strands, and most
of the cell surface was coated with fibrin. The diameter of the fibrin
fibers increased. Bar represents 5
µm.
The distinction between C32TG cell-fibrin binding and nonspecific
trapping of cells in the fibrin network has also been confirmed by
immunoelectron microscopy with the monoclonal anti-
Figure 8:
Distribution of
In this report, we present several lines of evidence that
demonstrate that Fibrinogen clotting and platelet activation are
mediated by thrombin and are required for clot retraction. However,
clotting can be dissociated from clot retraction by the use of
reptilase (EC 3.4.21.29), the Bothrops atrox enzyme that clots
fibrinogen by releasing fibrinopeptide A, but does not activate
platelets(46) . Hantgan et al.(46) reported
that clot retraction requires platelet activation and that platelets in
the clots formed with reptilase do not retract unless the platelets are
first stimulated (e.g. by ADP). We examined whether C32TG
cells can retract clots formed with reptilase (purchased from Solco,
Basel, Switzerland). While unstimulated platelets retracted the
reptilase-formed clots poorly, C32TG cells had retractile
activity. A mechanism for platelet-mediated clot retraction has
been proposed(2, 49) . Fibrin strands conform to the
platelet surface as platelet pseudopods extend outward along fibrin
bundles. Platelets constrict through the contraction of microfilaments.
As the platelets constrict, fibrin strands are pulled into alignment,
tension develops, and forces are transmitted to the clot surface. When
the forces are sufficient to overcome the attachment of the clot to its
containment vessels, the clot pulls away and an irreversible reduction
in gel volume occurs. Frelinger et al.(21) reported
that antibodies specific for a ligand-occupied form of
It is becoming increasingly apparent that integrins
transmit signals into cells as a result of ligand binding.
Integrin-ligand interactions can result in reorganization of the
cytoskeleton, formation of focal contacts, altered cellular pH,
Ca
Volume 270,
Number 4,
Issue of January 27, 1995 pp. 1785-1790
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Integrin in Mediating Fibrin Gel
Retraction (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(GPIIb-IIIa) plays important roles in platelet-mediated clot
retraction. However, little is known about the mechanisms of clot
retraction mediated by nucleated cells. In this report, we demonstrate
that another member of the integrin family,
, is involved in clot retraction
mediated by nucleated cells. Retraction of fibrin clots was observed
using a human melanoma cell line, C32TG, which contains no
complex. This retraction was
inhibited by RGD-containing peptide, monoclonal anti-,
and anti- antibodies. Immunoelectron
microscopic studies revealed a direct interaction between integrin and fibrin fibers at an early stage of clot retraction.
We found that another human embryonal cell line, 293, which is known to
express 
, but no
, lacks fibrin gel retractile
activity. Upon transfection of DNA into 293 cells,
the subunit formed a complex with an endogenous
subunit. The -bearing transfectants
were found to retract fibrin gels, which was specifically inhibited by
anti- antibody. In addition, a point mutation at
Asp in the
ligand binding domain
abolished the clot retractile activity of 293 transfectants, indicating
the requirement of ligand-binding
activity. Our findings suggest that is involved in mediating the interaction between the
three-dimensional fibrin network and nucleated cells and in promoting
``post-receptor occupancy'' events.
(platelet membrane glycoprotein
GPIIb-IIIa), plays an important role in platelet-mediated clot
retraction. Clots made from the platelet-rich plasma of patients with
Glanzmann's thrombasthenia, who lack or are deficient in platelet
integrin , do not show the dramatic
reduction in clot volume(1, 2, 3) .
Monoclonal antibodies against (A
A
, 7E3, AP2, and LJ-CP8), which inhibit
platelet aggregation, also prevent clot
retraction(4, 5) . Moreover, RGD-containing peptides
inhibit clot retraction(4, 6) . Interestingly,
antibodies against a peptide located in the fibrinogen binding region
of also inhibit clot retraction(7) .
-
and -subunits, and each cell has a specific repertoire of
receptors that define its adhesive capabilities. Moreover, integrins
have been implicated in ``post-receptor occupancy'' events
such as gene induction, collagen gel contraction, lymphocyte
co-stimulation, and changes in intercellular levels of Ca and
pH(15, 16, 17, 18, 19, 20) .
Platelet-mediated retraction of fibrin gels is one of the post-ligand
binding events(21) .
integrin is involved in clot retraction. Moreover, our data
indicate that ligand binding activity is required for clot retraction.
Monoclonal Antibodies and Reagents
Monoclonal
antibodies TM83 and TM60 are directed against and
human platelet glycoprotein Ib (GPIb),
respectively(25, 26) . Monoclonal antibody T74 was
generated in our laboratory and is directed against .
Both TM83 and T74 can inhibit platelet aggregation induced by ADP and
collagen. Monoclonal anti-human integrin (VNR147),
anti-human (LM609)(27) , and
anti-human platelet glycoprotein GPIIb-IIIa (P2) (28) antibodies were purchased from Life Technologies, Inc.,
CHEMICON, and Immunotech, respectively. Monoclonal anti-human
(98DF6) antibody was kindly donated by Dr. J.
Ylänne (University of Helsinki,
Finland)(29) .
DNA Construction
Restriction enzymes were
purchased from Takara (Kyoto, Japan). pRc/CMV was from Invitrogen.
pEF-BOS, a mammalian expression vector containing the promoter of the
human polypeptide chain elongation factor 1a chromosomal gene, was a
generous gift from Dr. S. Nagata (Osaka Bioscience Institute,
Japan)(31) . The cDNA of was kindly provided
by Dr. M. H. Ginsberg (The Scripps Research Institute). The cDNA was
cloned into the XbaI sites of pEF-BOS (pBOS). mutation ( Asp
Ala) was introduced by PCR (
)using the overlap
extension method(32) . PCR products were generated using pfu polymerase (Stratagene). The sequence
from nucleotide -60 to 465 with the mutation was generated by PCR
using cDNA as a template, T7 promoter primer as an
upstream primer, and a downstream primer consisting of the sequence from 465 to 444 with the T at 454 mutated to G. Another
sequence from 444 to 788 was generated by PCR using
an upstream primer consisting of the sequence from 444 to 465 with the
A at 454 mutated to C, and a downstream primer consisting of the
sequence from 788 to 771. These two PCR products were mixed, denatured,
and reannealed, and then an overlap extension reaction was performed
with Sequenase Version 2.0 (U. S. Biochemical Corp.). Secondary PCR was
done using the overlap extension reaction product as a template, T7
promoter primer as an upstream primer, and a downstream primer
consisting of the sequence from 788 to 771. The
secondary PCR product was digested with NspV and KpnI
and ligated into NspV/KpnI-digested
pBOS. The insert orientation and sequence of
pBOS(D119A) were verified by DNA sequencing. All
plasmid DNA was purified by two successive centrifugations through
cesium chloride before transfection.Cell Culture and Transfection
A human melanoma
cell line, C32TG, and a human embryonal kidney cell line, 293, were
obtained from the Japanese Cancer Research Resources Bank. C32TG cells
were maintained in Eagle's minimum essential medium supplemented
with 10% fetal bovine serum, glutamine, and kanamycin. 293 cells were
maintained in Dulbecco's minimum essential medium supplemented as
above. or pBOS(D119A) was
co-transfected with a neomycin resistant gene pRC/CMV into 293 cells
using a BTX electroporator (BTX)(29) . 293 cell lines
expressing high levels of or (D119A)
were selected using G418 (geneticin, Life Technologies, Inc.) and
subcloned.Clot Retraction Assay
Washed platelets were
prepared as described previously(33) . Cultured cells were
harvested with trypsin-EDTA (Life Technologies, Inc.) at room
temperature for 5 min. The cells were first washed with culture medium,
then 3 times with Tyrode's/Hepes buffer (150 mM NaCl,
2.5 mM KCl, 2 mM MgCl, 5 mM Hepes, pH 7.35) containing 1 mg/ml glucose and 3.5% BSA. Washed
platelets or nucleated cells were incubated with 10 mM tranexamic acid (Daiichi Seiyaku, Japan), 250 µg/ml fibrinogen
(Kabi Diagnostica, Stockholm, Sweden), and 2 mM CaCl at 37 °C for 5 min in a siliconized glass tube (1 
4.4
cm, AHS Japan Inc.). One unit of thrombin (Sigma) was added, after
which fibrin gels began to form almost immediately, and the tubes were
kept at 37 °C. Total volume of the reaction mixture was 1 ml.
Tranexamic acid was added as an inhibitor to prevent the fibrinolytic
activity displayed by many types of cultured cells and which could
interfere with clot retractile activity. The addition of tranexamic
acid up to 100 mM to this system did not change the results of
clot retraction induced by washed platelets. In inhibition experiments,
the cells were preincubated with antibodies, GRGDSP peptide, or
Ro43-5054 for 15 min at room temperature before the addition of
fibrinogen and tranexamic acid. Clot retraction was monitored by taking
photographs every 15 min. The outlines of whole reaction mixture and
clot were traced from the photographs onto sheets of tracing paper. The
sheets of paper were then digitized into a computer, and the contours
of the whole reaction mixture and clot were obtained by thresholding.
The areas inside the contours were then calculated. These processes
were performed by an image-processing system which included a Nexus
6810 color image processor and a Nexus 68322 A/D converter (products of
Kashiwagi Research Corporation, Japan) and a color TV camera. The whole
system was under the control of a workstation system, ss 7 model 22, of
Unisys Corp. (Blue Bell, PA). Clot retraction was expressed as the
percentage of the area excluded by the clot in the area of the whole
reaction mixture.Flow Cytometric Analysis
The surface expression of
integrins was analyzed by flow cytometry with specific antibodies.
Primary antibodies were incubated on ice for 20 min with 2
10
cultured cells in Tyrode's Hepes buffer (pH 7.4)
containing 0.35% BSA and 1 mg/ml dextrose. The cells were washed with
Tyrode's Hepes buffer and then incubated on ice for 20 min with
fluorescein-labeled goat anti-mouse IgG antibody (Tago). Cells were
pelleted, resuspended in Tyrode's-Hepes buffer, and analyzed on a
FACScan (Becton Dickinson). For platelets, 2 µl of platelet-rich
plasma in 50 µl of Tyrode's Hepes buffer was incubated with
primary antibodies at room temperature for 30 min. Twenty minutes after
the addition of fluorescein-labeled goat anti-mouse IgG antibody,
samples were diluted with the same buffer and analyzed on a FACScan.Immunoprecipitation
Cells were surface-labeled by
the IODOGEN method according to the manufacturer's instructions
(Pierce) and solubilized in lysis buffer (10 mM Hepes, 0.15 M NaCl, 1% Triton X-100, 1 mM CaCl, 1
mM MgCl, 2 mM phenylmethylsulfonyl
fluoride, 0.1 mM leupeptin, 5 mMN-ethylmaleimide, 100 kallikrein-inhibiting units/ml
aprotinin, pH 7.3). Cell extracts were immunoprecipitated with T74 or
VNR147 as described previously(34) . Briefly, cell extracts
were incubated with mouse nonimmune serum and Protein G-Sepharose
(Pharmacia) for 1 h at 4 °C. After centrifugation, the aliquots of
the supernatant were incubated with monoclonal antibodies for 2 h at 4
°C and then with Protein G-Sepharose for 30 min at 4 °C. The
Sepharose beads were washed extensively with lysis buffer. The
immunoprecipitates were subjected to SDS-polyacrylamide gel
electrophoresis, followed by autoradiography.Immunohistochemistry
The distribution of
fibrin(ogen) in frozen sections of fibrin clots containing C32TG cells
was determined by an indirect immunofluorescence method. Serial clot
sections (5 µm thick) were prepared with a cryostat microtome and
thaw-mounted on glass slides that had been cleaned with ethanol. The
mounted sections were dried in the air for 2 h and fixed with acetone
at 4 °C for 5 min. Sections were washed twice with PBS at 4 °C
for 5 min and then incubated with 5% BSA in PBS for 15 min at room
temperature. After washing twice with PBS, the sections were incubated
with rabbit anti-human fibrinogen antibody (Cappel Laboratories) at
room temperature for 60 min. After washing four times with cold PBS,
fluorescein-labeled goat anti-rabbit IgG antibody (Tago) was added, and
the sections were incubated for 30 min. After washing four times with
cold PBS, the sections were observed and photographed with a Zeiss
photomicroscope equipped with IIIRS fluorescence optics.Electron Microscopy
Fibrin clots containing C32TG
cells were fixed with 4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). The samples were dissected into
1-mm or smaller blocks, postfixed with 1% osmium tetroxide
in 0.1 M phosphate buffer for 60 min at 4 °C, dehydrated
with a graded ethanol series, and embedded in Epon as described
previously(34) . Ultrathin sections were prepared, stained with
uranyl acetate and lead citrate, and then examined with a JEM1200EX
transmission electron microscope (JEOL, Ltd., Tokyo, Japan) at an
accelerating voltage of 80 kV.Immunoelectron Microscopy
The distribution of
integrin in the frozen sections of fibrin clots
containing C32TG cells was determined by immunostaining using ultrathin
frozen sections. Fibrin gels containing C32TG cells were prepared as
described above, and clot retraction was terminated by the addition of
4% paraformaldehyde in 0.1 M phosphate buffer. The fixed clots
were cut into small pieces and rinsed three times with PBS. Ultrathin
frozen sections were obtained and mounted on nickel grids according to
the procedures described by Tokuyasu (35) with minor
modifications(36) . The sections on the nickel grids were
floated onto a droplet of PBS for rinsing and then transferred to a
droplet of 0.5% BSA in PBS for blocking. The sections were incubated
with monoclonal anti- antibody (T74) overnight at room
temperature. After rinsing five times with PBS, the sections were
incubated with goat anti-mouse IgG coupled to 15 nm colloidal gold
(Amersham) for 60 min at room temperature. The sections were rinsed
three times with PBS and five times with distilled water and then
adsorption-stained as described previously(36) . The stained
sections were examined with a JEM1200EX transmission electron
microscope (JEOL Ltd., Tokyo, Japan) at an accelerating voltage of 80
kV.
A Human Melanoma Cell Line C32TG Induces Clot
Retraction in the Absence of
Complex on platelets
has previously been reported to be essential for clot
retraction(3, 4, 5, 37) . To
investigate the mechanism of clot retraction induced by nucleated
cells, we used cultured tumor cell lines. Among the cell lines
screened, a human melanoma cell line, C32TG, showed the strongest
activity. Fig. 1a shows the clot retraction induced by
platelets and C32TG cells. The retraction of a fibrin clot mediated by
C32TG cells was cell number-dependent as shown in Fig. 1b.
Compared to platelets, the rate of clot retraction induced by C32TG
cells was slow, and there was little activity after 120 min of
incubation with fewer than 0.25 10
/ml cells. In
this study, we used 2 10/ml C32TG cells unless
stated otherwise. In addition, flow cytometric analysis revealed that
C32TG cells contain neither the subunit nor the
complex on the cell surface, but
do possess the subunit (Fig. 1c).
10
cells/ml)
and C32TG cells (2 10 cells/ml) were incubated at
37 °C, and clot retraction was observed by taking photographs.
Details are found under ``Materials and Methods.'' b, effect of cell concentration on C32TG-induced retraction of
fibrin gels. Fibrin gels containing C32TG cells at the concentrations
shown were incubated at 37 °C, and clot retraction was measured at
120 min. c, analysis of and
subunit expression on platelets and C32TG cells by flow
cytometry. Cells were stained with anti- (98DF6),
anti-
(T74), or anti- complex (P2) antibodies. Dashed lines indicate the
profile stained with murine control IgG.
Effects of Anti-
To investigate the roles of
Integrin Antibodies and
Peptides on Clot Retraction integrin, we examined the inhibitory effects of
antibodies and peptides. Platelet-mediated clot retraction was
inhibited by 40 µg/ml T74, a monoclonal anti- antibody, and 0.5 mM GRGDSP peptide (Fig. 2).
Another antibody against the subunit, TM83, had a
similar inhibitory effect (data not shown). A specific
antagonist, Ro43-5054, inhibited
platelet-mediated clot retraction at a concentration of 1
µM. Neither GRGESP peptide nor TM60, an antibody against
platelet GPIb, inhibited the reaction. Taken together, these results
indicate that plays a crucial role
in platelet-mediated clot retraction, a finding consistent with
previous reports(4, 6) . Although Cohen et al.(5) reported the enhancing effect of RGD-containing
peptide on clot retraction, this may be, at least in part, due to the
different assay method.
10/ml for platelets and
2 10/ml for C32TG cells) were incubated at 37
°C. Clot retraction was measured at 15 min for platelets and 90 min
for C32TG cells. Concentrations used were 40 µg/ml T74 and TM60 and
a 1:2000 dilution of LM609 ascites. As inhibitors, 500 µM GRGDSP and GRGESP and 1 µM Ro43-5054 were
used.
subunit-dependent manner
since both GRGDSP and T74 inhibited the reaction (Fig. 2). Since
is another member of the integrin family, we examined the effect of an
anti- complex antibody, LM609, on the
reaction. LM609 has been reported to be a good inhibitor of
-dependent cell adhesion and ligand
binding(27) . As shown in Fig. 2, LM609 inhibited clot
retraction mediated by C32TG cells. However, the antibody failed to
inhibit platelet-mediated clot retraction, indicating that
platelet-mediated clot retraction is mainly due to
, whereas C32TG cell-induced clot
retraction is due to . The fact that
the -selective antagonist Ro43-5054
had no inhibitory effect on C32TG cell-induced clot retraction
indicates the importance of in the
C32TG cell-mediated reaction.Transfection of
To further confirm the importance of
Subunit Accelerates
Clot Retraction integrin, a human embryonal kidney
cell line, 293, was used. The 293 cell line has been reported to have
complex but no subunit(38) . Our data from flow cytometric analysis
provided the same result (Fig. 3a). Importantly, 293
cells expressed little activity to retract fibrin gels (Fig. 3b). Next we generated 293 cell-stable
transfectants bearing the subunit
(293). Upon transfection of pBOS DNA
into 293 cells, significant amounts of subunit were
expressed on the surface of the transfectants, 293,
based on flow cytometric analysis (Fig. 4a).
Furthermore, we found that the subunit expressed was
associated with the endogenous subunit since both
anti- and anti- antibodies
immunoprecipitated the complex from
293 cell lysates, while anti- antibody precipitated the complex from 293 cell lysates (Fig. 4b). This
complex formation was also confirmed
by staining the cells with LM609, a complex-specific monoclonal
antibody (Fig. 4a). Fig. 5shows the clot
retractile activity of C32TG cells and 293 transfectants. The
transfection of the subunit accelerated clot
retraction, a process which was specifically inhibited by the
monoclonal anti- antibody. This indicates that
is essential for clot retraction
induced by cells not containing complexes.
and subunit expression on C32TG and
293 cells by a FACScan. Cells were stained with anti- (VNR147), anti- (T74), or
anti- complex (LM609) antibodies. Dashed lines indicate the profile stained with murine control
IgG. b, time course of fibrin gel retraction by C32TG and 293
cells. Fibrin gels containing 2 10 cells/ml C32TG
and 293 cells were incubated, and clot retraction was measured at the
times shown. Inset, photographs of retracted fibrin gels at
180 min.
and
(D119A) subunit expression in 293 transfectants by
flow cytometry (a) and immunoprecipitation (b).a, analysis of and subunit expression on 293 transfectants was carried out by a
FACScan. Dashed lines indicate the profile stained with murine
control IgG. b, lysates of surface-labeled platelets, C32TG,
293, and 293 transfectants were immunoprecipitated with monoclonal
anti- (T74) and anti- (VNR147)
antibodies. The immunoprecipitates were separated on a 7.5%
SDS-polyacrylamide gel under nonreducing conditions, followed by
autoradiography. and denote the
immunoprecipitates with T74 and VNR147, respectively. The positions of
, ,
, and
are indicated by arrows.
and (D119A)
subunits. C32TG, 293, 293, and
293(D119A) cells were preincubated with buffer or T74
(40 µg/ml) at room temperature for 15 min. Fibrin gels containing
treated cells (2 10/ml) were maintained at 37
°C, and clot retraction was measured at 90
min.
Ligand-binding Activity of
Loftus et al. reported that a point mutation in Is Required for Clot Retraction, Asp
Ala, abolishes the
integrin function
originally found in a Glanzmann's thrombasthenia
patient(39) . We examined whether the ligand-binding activity
of the complex exogenously expressed
on 293 transfectants is required for clot retraction. As shown in Fig. 4, a and b, a (D119A)
subunit formed a complex with an subunit. In contrast
to the wild-type subunit, the
(D119A)-bearing transfectants failed to retract fibrin
gels (Fig. 5) even though 293(D119A) cells
expressed comparable levels of recombinant integrin (Fig. 4a). These results demonstrate the requirement of
ligand-binding activity for clot retraction.Direct Interaction between
To examine C32TG cell-fibrin interaction
directly, an immunofluorescence study was performed. As shown in Fig. 6, many long fibrin strands were oriented like cables in
the axis of tension. Concurrent observation of the same field by phase
contrast micrography demonstrated that all C32TG cells were in close
contact with fibrin strands. The pattern of the cells connected by
bridges of numerous roughly parallel fibrin strands was a common
feature, confirming the suggestion that C32TG cells play a role in
directing the molecular architecture of the fibrin strands. Integrin and
Fibrin Strands
10 C32TG
cells was allowed to undergo retraction for 45 min, and frozen sections
were stained with rabbit anti-human fibrinogen antibody and then
incubated with fluorescein-labeled goat anti-rabbit IgG antibody. C32TG
cells are indicated by arrows. b, phase contrast
micrograph of the same fields as in a. Bar represents
20 µm. Fibrin strands can be seen aligning in the direction of
tension.
antibody. Fibrin clots containing C32TG cells were fixed 1 min
after the addition of thrombin. Short and thin fibrin fibers appeared
even at 1 min, and some of them were associated with the surface of
C32TG cells at the end of fibers where gold labels for integrin were present (Fig. 8). At 5 min or later, it was
hard to examine direct interactions with this immunostaining technique
since the cells were surrounded by fibrin fibers (data not shown).
These immunoelectron microscopic data strongly suggest a direct
interaction between integrin and fibrin fibers, at
least during the early stages of clot retraction.
integrin
in C32TG cell-containing fibrin clots by immunostaining using ultrathin
frozen sections. Fibrin clots containing C32TG cells were fixed at 1
min, and ultrathin frozen sections were prepared. The sections were
incubated with monoclonal anti- antibody (T74)
followed by goat anti-mouse IgG antibody coupled to colloidal gold (15
nm). Short and thin fibrin fibers appeared at 1 min (arrows).
Gold labels for integrin were present on the surface
membrane, some of them interacting with fibrin fibers (arrowheads). Bar represents 1 µm. M,
mitochondria; N, nucleus.
plays a crucial
role in fibrin clot retraction mediated by cells not containing
complexes. First, human melanoma
C32TG cells, which have no complex
on their cell surface, retract fibrin gels in a time- and cell
number-dependent manner. Second, this retraction is inhibited by the
addition of monoclonal anti-,
anti- antibodies, and RGD-containing peptide, whereas
Ro43-5054, an antagonist specific for
, has no inhibitory effect. Third,
human embryonal 293 cells, which have neither subunits nor complexes, show
no clot retractile activity. Transfection of subunits, however, restores the activity which is specifically
inhibited by monoclonal anti- antibody. Finally, a
mutation (Asp
Ala) in the
ligand-binding domain originally found in a Glanzmann's
thrombasthenia patient abolishes the clot retractile activity of 293
cell transfectants, indicating that
ligand binding activity is required for clot retraction. Thus, we
conclude that is involved in the
retraction of fibrin clots mediated by these cells. It is still
possible that together with other subunit(s)
can support clot retraction since the subunit is
reported to associate with at least five different
subunits(38, 40, 41, 42, 43, 44) .
Platelets have been reported to have on their cell surface(45) . However, clot retraction
mediated by platelets appears to utilize rather than since LM609 does
not prevent the reaction. This may be because platelets have much more
than
. To compare the clot retractile
activity of with that of the
complex, we also established 293
cell transfectants bearing the
complex. However, it was hard to
evaluate clot retraction mediated by complex alone in this system since the transfected subunit formed a complex with an endogenous subunit as well as the transfected subunit. (
) This finding suggests that the activation of
is not necessary for clot retraction
mediated by C32TG cells. Upon platelet stimulation,
binds to soluble fibrinogen.
Conformational changes in the extracellular domain of
regulate its ligand binding
affinity, and the cytoplasmic domains are directly involved in
physiological affinity modulation(47, 48) . Since
and are structurally similar and bind to many of the same adhesive
ligands, a chimeric subunit that has the extracellular domain of
and cytoplasmic domain of may
explain the relevance of the cytoplasmic domain in fibrin gel
retraction.
inhibit platelet-mediated
retraction of fibrin clots, but not fibrinogen binding, suggesting that
occupancy of the receptor leads to ``post-occupancy'' events.
On the contrary, the exact mechanisms of fibrin gel retraction mediated
by nucleated cells remains obscure. In this report, we provide
ultrastructural evidences that fibrin fibers interact with integrin on C32TG cell surface during the early stages of clot
retraction and that the cells are interconnected with each other and
totally surrounded by thick fibrin fibers during the late stages,
resulting in the alignment of fibrin strands and a reduction in clot
volume. It should be noted that neither cell spreading nor pseudopods
were observed during the reaction, which is in contrast to
platelet-mediated clot retraction. Based on our observations, C32TG
cell-induced clot retraction is due to an altered fibrin network rather
than morphological changes in the cells themselves. Our biochemical
evidence reveals the involvement of in this reaction, and we assume that the retraction of fibrin
gels observed here is one of the ``post-receptor occupancy''
events. Thus, the extent of clot retraction is probably an
-mediated increase in the diameter of
fibrin fibers which may be a cellular event subsequent to the ligand
binding. fluxes, and phosphorylation
events(19, 20, 50, 51, 52) .
It is likely that clot retraction involves a direct or indirect linkage
of actin to fibrin because cytochalasin D treatment of C32TG cells
prevents the retraction.
Moreover, the development of an
effective force presupposes the binding of actin and fibrin bundles at
specific sites on the membrane. Ylänne et al.(29) reported that truncation of the cytoplasmic domain abolished
-mediated clot retraction,
indicating that the cytoplasmic domain is required
for the transmission of the contractile force to the fibrin matrix. A
crucial role of integrin subunit cytoplasmic domains has also
been identified in collagen gel contraction that is another
integrin-mediated contraction of extracellular matrix(17) .
While the molecular mechanism of clot retraction awaits further
studies, -mediated increase in the
diameter of fibrin strands could provide insights into the
``post-receptor occupancy'' events including
integrin-dependent reorganization of the cytoskeleton and the
three-dimensional fibrin network.
))
We express our gratitude to Dr. Mark H. Ginsberg for
generously providing cDNA and Dr. Jari
Ylänne for the monoclonal antibody to
. We are grateful to Reiko Minamikawa-Tachino for
her help in image analysis. We thank Noriko Saito, Kotomi Maeda, Kaori
Fujimaki, Yukari Hanaue, and Makoto Nabatame for their technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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