(Received for publication, September 1, 1994)
From the
Platelet integrin (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.
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,
(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) .
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 -
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) .
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 integrin is involved in clot retraction. Moreover, our data
indicate that ligand binding activity is required for clot retraction.
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 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.
pBOS 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.
Figure 1:
Fibrin gel
retraction induced by platelets and C32TG cells. a, fibrin
gels containing washed platelets (2 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.
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 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.
C32TG cells were able to induce clot
retraction in an RGD- and 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.
Figure 3:
Clot
retraction induced by C32TG and 293 cells. a, analysis of
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.
Figure 4:
Analysis of 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.
Figure 5:
Clot retraction induced by 293
transfectants bearing 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.
Figure 6:
Alignment of fibrin strands in fibrin gels
retracted by C32TG cells. a, fluorescence micrograph of
retracted fibrin gels. A clot containing 2 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.
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- 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.
Figure 8:
Distribution of 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.
In this report, we present several lines of evidence that
demonstrate that 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. (
)
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. 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.
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
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.
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 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.