©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Involvement of Integrin in Mediating Fibrin Gel Retraction (*)

(Received for publication, September 1, 1994)

Yasuhiro Katagiri (§) Takashi Hiroyama Noriko Akamatsu Hidenori Suzuki Hiroh Yamazaki Kenjiro Tanoue

From the Department of Cardiovascular Research, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Platelet integrin alphabeta(3) (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 beta(3) integrin family, alpha(v)beta(3), 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 alphabeta(3) complex. This retraction was inhibited by RGD-containing peptide, monoclonal anti-beta(3), and anti-alpha(v)beta(3) antibodies. Immunoelectron microscopic studies revealed a direct interaction between beta(3) 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 alpha(v)beta(1), but no alpha(v)beta(3), lacks fibrin gel retractile activity. Upon transfection of beta(3) DNA into 293 cells, the beta(3) subunit formed a complex with an endogenous alpha(v) subunit. The beta(3)-bearing transfectants were found to retract fibrin gels, which was specifically inhibited by anti-beta(3) antibody. In addition, a point mutation at Asp in the beta(3) ligand binding domain abolished the clot retractile activity of 293 transfectants, indicating the requirement of alpha(v)beta(3) ligand-binding activity. Our findings suggest that alpha(v)beta(3) is involved in mediating the interaction between the three-dimensional fibrin network and nucleated cells and in promoting ``post-receptor occupancy'' events.


INTRODUCTION

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, alphabeta(3) (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 alphabeta(3), do not show the dramatic reduction in clot volume(1, 2, 3) . Monoclonal antibodies against alphabeta(3) (A(2)A(9), 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 alpha 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 alpha- and beta-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 alpha(v)beta(3) integrin is involved in clot retraction. Moreover, our data indicate that ligand binding activity is required for clot retraction.


MATERIALS AND METHODS

Monoclonal Antibodies and Reagents

Monoclonal antibodies TM83 and TM60 are directed against beta(3) and human platelet glycoprotein Ib (GPIb), respectively(25, 26) . Monoclonal antibody T74 was generated in our laboratory and is directed against beta(3). Both TM83 and T74 can inhibit platelet aggregation induced by ADP and collagen. Monoclonal anti-human integrin alpha(v) (VNR147), anti-human alpha(v)beta(3) (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 alpha (98DF6) antibody was kindly donated by Dr. J. Ylänne (University of Helsinki, Finland)(29) .

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).

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 beta(3) was kindly provided by Dr. M. H. Ginsberg (The Scripps Research Institute). The cDNA was cloned into the XbaI sites of pEF-BOS (pBOSbeta(3)).

A beta(3) mutation (beta(3) Asp Ala) was introduced by PCR (^1)using the overlap extension method(32) . PCR products were generated using pfu polymerase (Stratagene). The beta(3) sequence from nucleotide -60 to 465 with the mutation was generated by PCR using beta(3) cDNA as a template, T7 promoter primer as an upstream primer, and a downstream primer consisting of the beta(3) sequence from 465 to 444 with the T at 454 mutated to G. Another beta(3) 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 beta(3) sequence from 788 to 771. The secondary PCR product was digested with NspV and KpnI and ligated into NspV/KpnI-digested pBOSbeta(3). The insert orientation and sequence of pBOSbeta(3)(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.

pBOSbeta(3) or pBOSbeta(3)(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 beta(3) or beta(3)(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(2), 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(2) at 37 °C for 5 min in a siliconized glass tube (1 times 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 times 10^5 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(2), 1 mM MgCl(2), 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^3 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 beta(3) 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-beta(3) 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.


RESULTS

A Human Melanoma Cell Line C32TG Induces Clot Retraction in the Absence of alphabeta(3) Complex

alphabeta(3) 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 times 10^6/ml cells. In this study, we used 2 times 10^6/ml C32TG cells unless stated otherwise. In addition, flow cytometric analysis revealed that C32TG cells contain neither the alpha subunit nor the alphabeta(3) complex on the cell surface, but do possess the beta(3) subunit (Fig. 1c).


Figure 1: Fibrin gel retraction induced by platelets and C32TG cells. a, fibrin gels containing washed platelets (2 times 10^8 cells/ml) and C32TG cells (2 times 10^6 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 alpha and beta(3) subunit expression on platelets and C32TG cells by flow cytometry. Cells were stained with anti-alpha (98DF6), anti-beta(3) (T74), or anti-alphabeta(3) complex (P2) antibodies. Dashed lines indicate the profile stained with murine control IgG.



Effects of Anti-beta(3) Integrin Antibodies and Peptides on Clot Retraction

To investigate the roles of beta(3) integrin, we examined the inhibitory effects of antibodies and peptides. Platelet-mediated clot retraction was inhibited by 40 µg/ml T74, a monoclonal anti-beta(3) antibody, and 0.5 mM GRGDSP peptide (Fig. 2). Another antibody against the beta(3) subunit, TM83, had a similar inhibitory effect (data not shown). A specific alphabeta(3) 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 alphabeta(3) 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.


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 times 10^8/ml for platelets and 2 times 10^6/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 beta(3) subunit-dependent manner since both GRGDSP and T74 inhibited the reaction (Fig. 2). Since alpha(v)beta(3) is another member of the beta(3) integrin family, we examined the effect of an anti-alpha(v)beta(3) complex antibody, LM609, on the reaction. LM609 has been reported to be a good inhibitor of alpha(v)beta(3)-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 alphabeta(3), whereas C32TG cell-induced clot retraction is due to alpha(v)beta(3). The fact that the alphabeta(3)-selective antagonist Ro43-5054 had no inhibitory effect on C32TG cell-induced clot retraction indicates the importance of alpha(v)beta(3) in the C32TG cell-mediated reaction.

Transfection of beta(3) Subunit Accelerates Clot Retraction

To further confirm the importance of alpha(v)beta(3) integrin, a human embryonal kidney cell line, 293, was used. The 293 cell line has been reported to have alpha(v)beta(1) complex but no beta(3) 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 beta(3) subunit (293beta(3)). Upon transfection of pBOSbeta(3) DNA into 293 cells, significant amounts of beta(3) subunit were expressed on the surface of the transfectants, 293beta(3), based on flow cytometric analysis (Fig. 4a). Furthermore, we found that the beta(3) subunit expressed was associated with the endogenous alpha(v) subunit since both anti-beta(3) and anti-alpha(v) antibodies immunoprecipitated the alpha(v)beta(3) complex from 293beta(3) cell lysates, while anti-alpha(v) antibody precipitated the alpha(v)beta(1) complex from 293 cell lysates (Fig. 4b). This alpha(v)beta(3) 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 beta(3) subunit accelerated clot retraction, a process which was specifically inhibited by the monoclonal anti-beta(3) antibody. This indicates that alpha(v)beta(3) is essential for clot retraction induced by cells not containing alphabeta(3) complexes.


Figure 3: Clot retraction induced by C32TG and 293 cells. a, analysis of alpha(v) and beta(3) subunit expression on C32TG and 293 cells by a FACScan. Cells were stained with anti-alpha(v) (VNR147), anti-beta(3) (T74), or anti-alpha(v)beta(3) 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 times 10^6 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 beta(3) and beta(3)(D119A) subunit expression in 293 transfectants by flow cytometry (a) and immunoprecipitation (b).a, analysis of alpha(v) and beta(3) 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-beta(3) (T74) and anti-alpha(v) (VNR147) antibodies. The immunoprecipitates were separated on a 7.5% SDS-polyacrylamide gel under nonreducing conditions, followed by autoradiography. beta(3) and alpha(v) denote the immunoprecipitates with T74 and VNR147, respectively. The positions of alpha(v), alpha, beta(1), and beta(3) are indicated by arrows.




Figure 5: Clot retraction induced by 293 transfectants bearing beta(3) and beta(3)(D119A) subunits. C32TG, 293, 293beta(3), and 293beta(3)(D119A) cells were preincubated with buffer or T74 (40 µg/ml) at room temperature for 15 min. Fibrin gels containing treated cells (2 times 10^6/ml) were maintained at 37 °C, and clot retraction was measured at 90 min.



Ligand-binding Activity of alpha(v)beta(3) Is Required for Clot Retraction

Loftus et al. reported that a point mutation in beta(3), Asp Ala, abolishes the beta(3) integrin function originally found in a Glanzmann's thrombasthenia patient(39) . We examined whether the ligand-binding activity of the alpha(v)beta(3) complex exogenously expressed on 293 transfectants is required for clot retraction. As shown in Fig. 4, a and b, a beta(3)(D119A) subunit formed a complex with an alpha(v) subunit. In contrast to the wild-type beta(3) subunit, the beta(3)(D119A)-bearing transfectants failed to retract fibrin gels (Fig. 5) even though 293beta(3)(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 beta(3) Integrin and Fibrin Strands

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.


Figure 6: Alignment of fibrin strands in fibrin gels retracted by C32TG cells. a, fluorescence micrograph of retracted fibrin gels. A clot containing 2 times 10^6 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-beta(3) 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 beta(3) 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 beta(3) integrin and fibrin fibers, at least during the early stages of clot retraction.


Figure 8: Distribution of beta(3) 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-beta(3) 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 beta(3) integrin were present on the surface membrane, some of them interacting with fibrin fibers (arrowheads). Bar represents 1 µm. M, mitochondria; N, nucleus.




DISCUSSION

In this report, we present several lines of evidence that demonstrate that alpha(v)beta(3) plays a crucial role in fibrin clot retraction mediated by cells not containing alphabeta(3) complexes. First, human melanoma C32TG cells, which have no alphabeta(3) 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-alpha(v)beta(3), anti-beta(3) antibodies, and RGD-containing peptide, whereas Ro43-5054, an antagonist specific for alphabeta(3), has no inhibitory effect. Third, human embryonal 293 cells, which have neither beta(3) subunits nor alpha(v)beta(3) complexes, show no clot retractile activity. Transfection of beta(3) subunits, however, restores the activity which is specifically inhibited by monoclonal anti-beta(3) antibody. Finally, a beta(3) 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 alpha(v)beta(3) ligand binding activity is required for clot retraction. Thus, we conclude that alpha(v)beta(3) is involved in the retraction of fibrin clots mediated by these cells. It is still possible that alpha(v) together with other beta subunit(s) can support clot retraction since the alpha(v) subunit is reported to associate with at least five different beta subunits(38, 40, 41, 42, 43, 44) . Platelets have been reported to have alpha(v)beta(3) on their cell surface(45) . However, clot retraction mediated by platelets appears to utilize alphabeta(3) rather than alpha(v)beta(3) since LM609 does not prevent the reaction. This may be because platelets have much more alphabeta(3) than alpha(v)beta(3). To compare the clot retractile activity of alpha(v)beta(3) with that of the alphabeta(3) complex, we also established 293 cell transfectants bearing the alphabeta(3)complex. However, it was hard to evaluate clot retraction mediated by alphabeta(3) complex alone in this system since the transfected beta(3) subunit formed a complex with an endogenous alpha(v) subunit as well as the transfected alpha subunit. (^2)

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.^2 This finding suggests that the activation of alpha(v)beta(3) is not necessary for clot retraction mediated by C32TG cells. Upon platelet stimulation, alphabeta(3) binds to soluble fibrinogen. Conformational changes in the extracellular domain of alphabeta(3) regulate its ligand binding affinity, and the cytoplasmic domains are directly involved in physiological affinity modulation(47, 48) . Since alpha(v)beta(3) and alphabeta(3) are structurally similar and bind to many of the same adhesive ligands, a chimeric alpha subunit that has the extracellular domain of alpha(v) and cytoplasmic domain of alpha 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 alphabeta(3) 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 beta(3) 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 alpha(v)beta(3) 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 alpha(v)beta(3)-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.^2 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 beta(3) cytoplasmic domain abolished alphabeta(3)-mediated clot retraction, indicating that the beta(3) cytoplasmic domain is required for the transmission of the contractile force to the fibrin matrix. A crucial role of integrin alpha 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, alpha(v)beta(3)-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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 81-3-3823-2101 (Ext.5144); Fax: 81-3-3823-2965.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; BSA, bovine serum albumin; PBS, phosphate-buffered saline.

(^2)
Y. Katagiri, T. Hiroyama, N. Akamatsu, H. Suzuki, H. Yamazaki, and K. Tanoue, unpublished data.


ACKNOWLEDGEMENTS

We express our gratitude to Dr. Mark H. Ginsberg for generously providing beta(3) cDNA and Dr. Jari Ylänne for the monoclonal antibody to alpha. 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.


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