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Blood, 1 May 2005, Vol. 105, No. 9, pp. 3561-3568. Prepublished online as a Blood First Edition Paper on January 6, 2005; DOI 10.1182/blood-2004-10-4089. This Article Abstract Full Text (PDF) All Versions of this Article: 2004-10-4089v1 105/9/3561    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Belkin, A. M. Articles by Medved, L. Search for Related Content PubMed PubMed Citation Articles by Belkin, A. M. Articles by Medved, L. Related Collections Cell Adhesion and Motility Signal Transduction Hemostasis, Thrombosis, and Vascular Biology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Transglutaminase-mediated oligomerization of the fibrin(ogen) C domains promotes integrin-dependent cell adhesion and signaling Alexey M. Belkin, Galina Tsurupa, Evgeny Zemskov, Yuri Veklich, John W. Weisel, and Leonid Medved From the Department of Biochemistry and Molecular Biology and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore; and the Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen C domains with factor XIIIa generates ordered C oligomers mimicking polymeric arrangement of the C domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the C domain–mediated interaction of fibrin with endothelial cells. Cell adhesion and chemical cross-linking experiments revealed that oligomerization of the C domains by both transglutaminases significantly increases their RGD (arginyl–glycyl–aspartate)–dependent interaction with endothelial V3 and to a lesser extent with V5 and 51 integrins. The oligomerization promotes integrin clustering, thereby increasing cell adhesion, spreading, formation of prominent peripheral focal contacts, and integrin-mediated activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways. The enhanced integrin clustering is likely caused by ordered juxtaposition of RGD-containing integrin-binding sites upon oligomerization of the C domains and increased affinity of these domains for integrins. Our findings provide new insights into the mechanism of the C domain–mediated interaction of endothelial cells with fibrin and imply its potential involvement in cell migration. They also suggest a new role for transglutaminases in regulation of integrin-mediated adhesion and signaling via covalent modification of integrin ligands.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The plasma protein fibrinogen plays a prominent role in hemostasis and a number of other physiological and pathological processes. Vascular injury initiates platelet aggregation and blood coagulation, resulting in conversion of soluble fibrinogen into insoluble fibrin and formation of fibrin-rich blood clot. The clot plugs damaged vessel walls, preventing the loss of blood, and serves subsequently as a provisional matrix for cell adhesion, migration, and proliferation during wound healing and neovascularization.1 Particularly, interaction of leukocytes and endothelial cells with the fibrin clot modulates the inflammatory response and stimulates angiogenesis.2,3 This multitude of fibrin(ogen) functions is based on its ability to interact with numerous adhesion receptors present on the surface of leukocytes, endothelial cells, fibroblasts, platelets, and other cell types. Among these receptors, integrins represent a large class of heterodimeric transmembrane adhesion receptors that participate in a wide range of cell-matrix interactions.4 Fibrinogen contains multiple recognition sites for integrins, some of which appear cryptic and become exposed upon its conversion into fibrin.5-10 Fibrinogen is a complex multidomain protein consisting of 2 identical subunits, each composed of 3 polypeptide chains, A,B, and .11,12 These chains are folded into a number of distinct domains grouped into several structural regions.13 The disulfide-linked NH2-terminal portions of all 6 chains form the central E region, while their COOH-terminal portions form 2 terminal D regions and 2 C domains.11,12,14,15 The C domains formed by the A chain residues 221 to 610 are located on the surface of the molecule and play an important role in modulation of various processes. They are involved in fibrin assembly,16,17 activation of factor XIII (FXIII),18 and modulation of fibrinolysis19,20 and cell adhesion via either bound fibronectin or their A572-574 arginyl–glycyl–aspartate (RGD) recognition motif.5,21,22 Previous studies showed that this motif is a predominant site for the interaction of fibrin(ogen) with endothelial cell integrin V3.5 Another endothelial cell integrin, 51, was also found to interact with fibrin (ogen) in an RGD-dependent manner via this motif.7,23 According to the current view, in fibrinogen the C domains interact intramolecularly with each other and with the central E region while in fibrin they switch to intermolecular interactions to form polymers,24 which are covalently cross-linked by activated plasma transglutaminase factor XIIIa (FXIIIa).25 Tissue transglutaminase (tTG) also cross-links the C domains in fibrin, although the cross-linking pattern seems to be different.26,27 Because fibrinogen is rather inert in the circulation while fibrin is highly reactive, the activity of the C domains appears to be connected with their polymerization and cross-linking. This was confirmed in our recent study in which we found that the recombinant C domains are able to form stable oligomers upon cross-linking with FXIIIa and that the adhesion of endothelial cells to the C domains increased upon their oligomerization.28 The underlying mechanism for such effects remains unclear. Our study also demonstrated that FXIIIa–cross-linked recombinant C domain oligomers have an ordered structure and may adequately mimic the structure and properties of the C domains in cross-linked fibrin.28 These oligomers, as well as the tTG–cross-linked C oligomers, were used in the present study as models to clarify the mechanism(s) of the C domain–mediated interaction of fibrin with endothelial cells. The experiments revealed that oligomerization and cross-linking of the C domains by both transglutaminases significantly promote adhesion of endothelial cells via their integrin receptors, facilitate focal adhesion assembly via integrin clustering, and amplify integrin-mediated signaling.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Proteins, antibodies, and recombinant fragments Bovine -thrombin and guinea pig liver tTG were from Sigma (St Louis, MO). Bovine serum albumin (BSA) and human FXIII were from Calbiochem (La Jolla, CA). The recombinant human fibrinogen C domain including the A chain residues 221 to 610 was prepared as described earlier.29 A membrane-impermeable thiol-cleavable cross-linker 3,3'-dithiobis[sulfosuccinimidyl propionate] (DTSSP) was from Pierce (Rock-ford, IL). Purified integrins V3, V5, and 51; anti–5 integrin polyclonal antibodies; and monoclonal antibodies (mAbs) P3G8 (anti-V), LM609 and 23C6 (anti-V3), P1F6 (anti-V5), P1D6 (anti-51), BHA2.1 (anti-21), NKI-GoH3 (anti-61), JB1A (anti-1), and 25E11 (anti-3) were obtained from Chemicon (Temecula, CA). Antiphosphotyrosine polyclonal antibodies were from BD Biosciences (San Diego, CA). Polyclonal antibodies to focal adhesion kinase (FAK) and phosphospecific antibodies to pTyr residues of FAK were from BioSource (Camarillo, CA). Polyclonal antibodies against extracellular signal-regulated kinase 1/2 (ERK1/2) and dually phosphorylated ERK1/2 were from Cell Signaling Technology (Beverly, MA). Monoclonal antibody 1D4 against an epitope located in the A349-406 region of the C domain30 was a gift from Dr B. Kudryk (New York Blood Center). Cell culture Human umbilical vein endothelial cells (HUVECs) and Clonetics endothelial cell growth medium EBM-2 supplemented with EGM-2 SingleQuots were obtained from BioWhittaker (Walkersville, MD). Cross-linking of the C domains with factor XIIIa or tTG and purification of cross-linked oligomers Cross-linking of the recombinant C domain with FXIIIa and preparation of soluble cross-linked C oligomers were performed as described earlier.19,28 Cross-linking of the C domain with tTG was performed similarly to that with FXIIIa. The reaction mixture containing the C domain at 1 mg/mL and tTG at 50 µg/mL in Tris (tris(hydroxymethyl)aminomethane)–buffered saline (TBS) with 10 mM Ca2+ was incubated for 2 hours at room temperature, centrifuged to remove nonsoluble material, and then fractionated by size-exclusion chromatography on a Superdex 200 HR column to prepare soluble cross-linked C oligomers. Solid-phase binding assays Solid-phase binding was performed in microtiter plates using enzyme-linked immunosorbent assay (ELISA). Microtiter plate wells (Fisher Scientific, Hampton, NH) were coated overnight with 100 µL per well monomeric C domains (C monomers) or C oligomers cross-linked by either factor XIIIa (C(FXIII) oligomers) or tissue transglutaminase (C(tTG) oligomers), all at 20 µg/mL in 0.1 M Na2CO3, pH 9.5 (coating buffer). The wells were then blocked with 1% BSA in TBS. The amounts of C monomers and oligomers adsorbed to microtiter wells were the same as revealed by ELISA with mAb 1D4. After washing with TBS containing 0.05% Triton X-100, 1 mM MgCl2, and 1 mM MnCl2, the V3 integrin in the same buffer was added to the wells at 20 µg/mL and incubated for 1 hour. Bound V3 was measured by reaction with mAb P3G8 and peroxidase-conjugated antimouse polyclonal antibodies. A TMB Microwell peroxide substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added to the wells, and the amount of bound ligand was measured spectrophotometrically at 450 nm. Transmission electron microscopy studies Samples for electron microscopy were prepared by spraying the C monomer or C oligomers, both at 40 µg/mL in a volatile buffer (50 mM ammonium formate, pH 7.4, or 0.125% acetic acid, pH 3.5) and 25% to 30% glycerol, onto freshly cleaved mica and rotary shadowing with tungsten in a vacuum evaporator as previously described.31 Specimens were examined in a Philips 400 electron microscope (Philips Electronic Instruments, Hillsboro, OR) at 80 kV and x 60 000 magnification. Cell adhesion assays Twenty-four–well tissue culture plastic plates (Midwest Scientific, St. Louis, MO) were coated with C monomers, C(FXIII) oligomers, or C(tTG) oligomers, all at 20 µg/mL in TBS, for 16 hours at 4°C, and then blocked with 10 mg/mL BSA. The amounts of C monomers and oligomers adsorbed to tissue culture plastic wells were the same as revealed by ELISA with mAb 1D4. For adhesion experiments, HUVECs were labeled overnight with 20 µCi (0.74 MBq)/mL Tran35S-Label (MP Biomedicals, Irvine, CA). After detachment by trypsinization and blocking excess of trypsin with 0.5 mg/mL soybean trypsin inhibitor, 5 x 104 35S-labeled HUVECs were plated into each well in serum-free Dulbecco modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA) containing 5 mg/mL BSA and allowed to adhere for 20 minutes at 37°C. In some experiments, cells were plated in the presence of 250 µg/mL GRGDSP or GRGESP peptides (American Peptide, Sunnyvale, CA). To study the role of individual integrins in HUVEC adhesion to C monomers and oligomers, the 35S-labeled cells were preincubated for 30 minutes on ice with 20 µg/mL function-blocking anti-V3 mAb LM609, anti-V5 mAb P1F6, and anti-51 mAb P1D6 before plating on the substrates in the presence of the antibodies. Adherent cells were washed 3 times with phosphate-buffered saline (PBS) and lysed in 1% sodium dodecyl sulfate (SDS). The bound radioactivity was counted in a Beckman LS 3801 scintillation counter (Beckman Coulter, Fullerton, CA) and converted into the number of adherent cells by referring to the levels of 35S incorporation per 103 cells. Cell spreading assays and quantitation of cell area on substrates A total of 2 x 104 unlabeled HUVECs were plated in serum-free DMEM with 5 mg/mL BSA at 37°C for indicated periods of time on glass coverslips coated with 20 µg/mL C monomers, C(FXIII) oligomers, or C(tTG) oligomers. At different time points of spreading, cells were fixed with 3.7% paraformaldehyde, stained with Coomassie blue, destained, and photographed. The outlines and cell areas of 100 randomly chosen nonadjacent cells were analyzed using Image-Pro Plus microscopy software (Media Cybernetics, Baltimore, MD) calibrated with an Applied Micro Stage micrometer (EF Precision Group, Willow Grove, PA). Immunofluorescence Glass coverslips were coated with 20 µg/mL C monomers, C(FXIII) oligomers, or C(tTG) oligomers and then blocked with 10 mg/mL BSA. Serum-starved HUVECs were trypsinized and then plated in DMEM with 10 mg/mL albumin on the C domain monomers or oligomers. After 2 hours the cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. The cells were double stained with 20 µg/mL mAb 23C6 against V3 and 10 µg/mL rabbit polyclonal antiphosphotyrosine antibodies, followed by rhodamine-conjugated anti–mouse and fluorescein-labeled anti–rabbit immunoglobulin G (IgG). Cells were photographed using a Nikon Eclipse E800 microscope (Nikon, Melville, NY) with a 60x/1.4 objective and Spot RT digital camera. Images were acquired with Advance Spot software (Diagnostic Instruments). Quantitation of ligand-bound integrins by reversible chemical cross-linking to substrates A total of 107 HUVECs were plated in serum-free DMEM with 10 mg/mL BSA on T150 tissue culture flasks coated with 20 µg/mL C monomers, C(FXIII) oligomers, or C(tTG) oligomers. Two hours later cells were washed with PBS and chemically cross-linked to substrates with 2 mM DTSSP in PBS for 30 minutes at 4°C. To stop the cross-linking, the cells were incubated with TBS for 10 minutes at 4°C. Then they were extracted 4 times for 20 minutes with 25 mL 0.1% SDS in H2O containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride [PMSF], 1 mM benzamidine, 10 µg/mL leupeptin, 10 µg/mL aprotinin). Recovery of the cross-linked material (de–cross-linking) was performed on an orbital shaker at 40°C for 1 hour with 12 mL TBS containing 100 mM dithiothreitol (DTT), 0.1% SDS, and 5 µg/mL BSA. To account for a difference in the number of adherent cells on the substrates, all 3 fractions were normalized to represent 2 x 106 adherent cells. The recovered proteins were concentrated in Amicon Ultra-4 concentrators (Millipore, Bedford, MA) and then precipitated with ice-cold acetone. Integrins in the recovered protein fractions were analyzed by 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with antibodies against selected integrin subunits; 10 ng purified V3, 51, and V5 integrins were used as external standards on the blots. Blots were developed with SuperSignal West Pico Substrate (Pierce). The developed blots were subjected to densitometry using NIH Image 1.63f software. A calibration with different integrin loadings showed a linear increase in signal within the range of 0.5 to 25 ng. The amounts of cellular V3, 51, and V5 integrins chemically cross-linked to the 3 substrates were compared with purified integrin standards by densitometry and expressed relative to those in HUVECs adherent on C monomers. Using this method, a total number of integrin receptors per cell was estimated as follows: (2.1 ± 0.2) x 105; (2.3 ± 0.2) x 105; and (1.6 ± 0.2) x 105 for V3, 51, and V5, respectively. Analysis of adhesion-mediated phosphorylation of FAK and ERK1/2 Analysis of adhesion-mediated FAK and ERK1/2 phosphorylation was performed as described previously.32-34 A total of 106 serum-starved HUVECs in serum-free DMEM with 10 mg/mL BSA were kept in suspension or plated on tissue culture plates coated with 20 µg/mL C monomers or C(FXIII) oligomers. Two hours later the adherent cells were washed with PBS and lysed in ice-cold buffer (20 mM Tris; pH 7.4, 100 mM NaCl, 1 mM EDTA [ethylenediaminetetraacetic acid], 1 mM EGTA [ethyleneglycoltetraacetic acid], 20 mM Na4P2O7, 1 mM NaF, 1% Triton X-100, 0.1% SDS) containing 2 mM Na3VO4 and protease inhibitors (1 mM PMSF, 1 mM benzamidine, 10 µg/mL leupeptin, 10 µg/mL aprotinin). Protein concentrations were determined with BCA protein Assay Kit (Pierce). A total of 200 µg of total cell extracts was subjected to immunoprecipitation with anti-FAK antibodies. The resulting immune complexes and total cell lysates were separated on 8% SDS-PAGE gels and analyzed by immunoblotting with antibodies to FAK, phosphotyrosine, and phosphospecific antibodies against selected pTyr residues of FAK. Blots were developed with SuperSignal West Pico Substrate and scanned by densitometer with NIH Image 1.63f software. The extent of FAK phosphorylation was normalized to the amounts of total FAK in each sample and expressed relative to that in HUVECs kept in suspension. Alternatively, the total cell extracts were also separated on 15% SDS-PAGE gels and examined by immunoblotting with antibodies to ERK1/2 and dually phosphorylated (activated) ERK1/2. ERK1/2 bands visualized by ECL chemiluminescence with West Pico Substrate were scanned and digitized by NIH Image 1.63f software. The levels of ERK1 and ERK2 phosphorylation were normalized to the amounts of total ERK1 and ERK2 in each sample and expressed relative to those in HUVECs in suspension.    Results Top Abstract Introduction Materials and methods Results Discussion References   Characterization of C oligomers generated by cross-linking with transglutaminases We demonstrated recently that treatment of the recombinant C domains with factor XIIIa results in cross-linked C oligomers, C(FXIII), which are soluble, stable, and have an ordered structure.28 In the current study, we prepared and characterized C oligomers cross-linked with guinea pig liver tissue transglutaminase, C(tTG). SDS-PAGE analysis revealed that, similarly to cross-linking with factor XIIIa, treatment of the C domain with tTG led to the formation of covalently cross-linked material with the mobility expected for dimers, trimers, and higher order oligomers (Figure 1A). The fraction containing soluble oligomers with molecular mass higher than 200 kDa was separated by size-exclusion chromatography and analyzed by electron microscopy after preparation by the method of rotary shadowing. The analysis revealed that these oligomers were similar to those prepared with factor XIIIa (Figure 1B-F). In both cases, the oligomers consisted of branched or bundled strands built of 4 nm globular structures. These oligomers seemed to be regular in structure, with strands that were about 8 nm or 2 monomers in width, but they were often branched and quite complex. Some very large polymers were observed in these preparations. Overall, cross-linking of the C domains with both transglutaminases produced C oligomers with similar regular structure. Both types of oligomers, C(FXIII) and C(tTG), were further used in experiments with endothelial cells. View larger version (71K): [in this window] [in a new window]   Figure 1.. Preparation and characterization of C(FXIII) and C(tTG) oligomers. (A) SDS-PAGE analysis of the recombinant C domain fragment and its oligomers. The C domain (lane 1) was covalently cross-linked with factor XIIIa (lane 2) or tTG (lane 4), and the resulting material was subjected to size-exclusion chromatography to separate high molecular mass fractions containing C(FXIII) oligomers (lane 3) or C(tTG) oligomers (lane 5); the right outer lane contains protein markers of the indicated molecular mass. (B-D) Electron microscopy of the rotary shadowed samples of the C domain fragment (B) and its C(FXIII) and C(tTG) oligomers (C-D, respectively). Bar indicates 100 nm. (E-F) Higher magnification and contrast images of small portions of panels C and D, respectively, showing details of the branching polymers. Bar indicates 100 nm.   Oligomerization of the C domains promotes RGD-dependent adhesion of endothelial cells via V3, V5, and 51 integrins First, we compared adhesion of human umbilical vein endothelial cells (HUVECs) to monomeric recombinant C domains and C(tTG) or C(FXIII) oligomers in short-term static cell adhesion assays (Figure 2A). In both cases, HUVEC adhesion to C oligomers was about 3-fold higher than that to C monomers. This indicates that oligomerization of C domains increases their adhesive capacity for endothelial cells and that the cell-binding properties of tTG- and FXIIA–cross-linked C oligomers are similar. The adhesion to all species was abolished by treatment of the cells with GRGDSP peptide, while it remained unaffected by the control scrambled GRGESP peptide, in agreement with the previous findings that this interaction is RGD dependent. View larger version (22K): [in this window] [in a new window]   Figure 2.. Oligomerization of the C domains stimulates RGD-dependent adhesion of endothelial cells via V3, V5, and 51 integrins. Quantitative adhesion assays were performed with 5 x 104 HUVECs plated in serum-free DMEM for 20 minutes at 37°C on plastic wells coated with 20 µg/mL C monomers, C(FXIII) oligomers, or C(tTG) oligomers. (A) Effects of oligomerization and RGD-containing peptide on cell adhesion to the C domain species (, C monomers; , C(FXIII); , C(tTG)). Cells were plated without treatment or in the presence of 250 µg/mL GRGDSP or control GRGESP peptides. The number of untreated adherent cells on C monomers was taken as 100%. (B) The role of individual integrins in adhesion to C monomers and oligomers. Cells were plated without treatment or in the presence of 20 µg/mL blocking antibodies to 51 (mAb P1D6; ), V5 (mAb P1F6; ), or V3 (mAb LM609; ). The numbers of untreated cells () adherent to C monomers or C(FXIII) oligomers were taken as 100%. The results shown in both panels are the means ± standard deviations of 3 independent experiments performed in duplicates.   HUVECs express at least 3 integrins, V3, V5, and 51, which interact with their ligands in an RGD-dependent manner. Therefore, we examined the roles of these integrins in adhesion of HUVECs to C monomers and C(FXIII) oligomers using function-blocking antibodies (Figure 2B). Treatment of the cells with mAbs P1D6 and P1F6 against, respectively, the 51 and V5 integrins, moderately decreased (by about 15% to 30%) adhesion of HUVECs to C monomers and C(FXIII) oligomers. In contrast, the use of anti-V3 mAb LM609 sharply reduced (by about 75% to 85%) adhesion of HUVECs to both the monomeric and oligomeric C domains. In control experiments with function-blocking mAbs, we found no involvement of RGD-independent 21 and 61 integrins expressed on HUVECs in adhesion of these cells to the C domain species (data not shown). Thus, formation of covalently cross-linked C domain oligomers by either FXIIA or tTG strongly stimulates RGD-dependent adhesion primarily via the V3 integrin and to a lesser extent via the 51 and V5 integrins. Oligomerization of the C domains enhances endothelial cell spreading and facilitates focal adhesion assembly To clarify the relationship between the oligomerization and the increased adhesion, we first tested binding of purified V3 integrin to immobilized C monomers and C(FXIII) or C(tTG) oligomers by ELISA (Figure 3). While no difference in the amounts of C monomers and oligomers immobilized on microtiter plastic wells was detected with anti–C domain mAb 1D4 (data not shown), a moderate increase (1.5-fold to 1.7-fold) in binding of V3 to both types of C oligomers was observed. Although such increased binding could be a result of an increased affinity of RGD-containing binding sites in the oligomers, it cannot itself account for the dramatic increase in the adhesive capacity of the oligomers. Therefore, we focused on morphological changes and localization of integrins in HUVECs adherent to the C monomers and oligomers. View larger version (13K): [in this window] [in a new window]   Figure 3.. Binding of purified V3 integrin to immobilized C monomers and C(FXIII) or C(tTG) oligomers. Plastic wells were coated with 20 µg/mL C monomers (), C(FXIII) oligomers (), or C(tTG) oligomers (). Binding of purified V3 integrin (20 µg/mL) was measured by ELISA with anti-3 mAb 25E11 followed by secondary goat anti–mouse IgG coupled with peroxidase. Binding to C monomers was taken as 100%. Results are the means ± standard deviations of 2 independent experiments performed in triplicates.   We next evaluated whether oligomerization of the C domains affects cell spreading and focal adhesion formation. Ninety minutes after plating on substrates in serum-free medium, HUVECs appeared significantly more spread on the C(FXIII) and C(tTG) oligomers than on the C monomers (Figure 4A-C). Quantification of the average spreading areas showed a time-dependent increase for all 3 substrates; however, HUVECs displayed a greater increase of the average spreading area when adherent to the C(FXIII) and C(tTG) oligomers (Figure 4D). View larger version (107K): [in this window] [in a new window]   Figure 4.. Oligomerization of the C domains facilitates endothelial cell spreading. A total of 2 x 104 HUVECs in serum-free DMEM were plated at 37°C for indicated periods of time on plastic wells coated with 20 µg/mL C monomers (A), C(FXIII) oligomers (B), or C(tTG) oligomers (C). At different time points of spreading, cells were fixed with 3.7% paraformaldehyde, stained with Coomassie blue, destained, and photographed. Shown are representative photographs of cells 90 minutes after plating on the substrates. Bar = 50 µm. (D) Time-dependent increase in cell spreading on C monomers () and oligomers (, C(FXIII); , C(tTG)). The average areas were determined for 120 sparsely plated cells on each substrate. Results are the means ± standard deviations of 2 independent experiments performed in triplicate.   To assess the state of adhesion complexes in HUVECs adherent to the C domain monomers and oligomers, immunostaining for the V3 integrin and phosphotyrosine was performed and 120 sparsely plated cells on each substrate were examined. In HUVECs plated on C monomers, V3 and phosphotyrosine were uniformly distributed over the cell surface with occasional punctate staining along the cell periphery (Figure 5A). Analysis at higher magnification revealed few peripheral focal complexes but no mature focal adhesions in the case of the monomeric substrate (Figure 5B). In contrast, a distinctive accumulation of V3 and phosphotyrosine along the cell edges was visible in HUVECs adherent to C(FXIII) and C(tTG) oligomers (Figure 5C,E). At higher magnification, well-developed peripheral focal adhesions exceeding 2 µm in length were visualized in at least 80% of HUVECs on either C(FXIII) or C(tTG) oligomers (Figure 5D,F), whereas less than 5% of cells on C monomers displayed prominent focal contacts (Figure 5B). These results indicate that oligomerization of the C domains promotes clustering of cell surface integrins as judged by a significant increase in the size of adhesion complexes. View larger version (71K): [in this window] [in a new window]   Figure 5.. Endothelial cells assemble prominent focal adhesions on C(FXIII) and C(tTG) oligomers but not on C monomers. HUVECs in serum-free DMEM were plated on 20 µg/mL C monomers (A-B), C(FXIII) oligomers (C-D), or C(tTG) oligomers (E-F) for 2 hours. Paraformaldehyde-fixed, Triton X-100–permeabilized cells were double stained with anti-3 integrin mAb 25E11 and polyclonal antiphosphotyrosine antibodies, followed by rhodamine-labeled anti–mouse and fluorescein-conjugated anti–rabbit IgG. A clear peripheral staining for V3 integrin and phosphotyrosine was observed at lower magnification in HUVECs on C oligomers (C,E) but not on monomeric C domains (A). At higher magnification, well-developed focal contacts containing V3 integrin and phosphotyrosine were detected in HUVECs on C oligomers (D,F), whereas no distinct focal adhesions were formed on C monomers (B). Bars indicate 20 µm. Arrowheads mark colocalization of V3 integrin and phosphotyrosine in the peripheral focal contacts of HUVECs on C oligomers.   Oligomerization of the C domains increases the amounts of ligand-bound V3, V5, and 51 integrins Given the enhancement of cell adhesion and assembly of large integrin clusters, we expected an increase in ligand-bound integrins for HUVECs adherent on C oligomers. Integrin-ligand binding is a prerequisite for chemical cross-linking of integrins to their ligands, while the amounts of chemically cross-linked integrins are proportional to the number of integrin-ligand bonds and overall adhesion strength.34,35 A reversible cell-impermeable cross-linker DTSSP with a short (1.3 nm [13 Å]) spacer arm was used to compare the amounts of ligand-bound integrins on HUVECs adherent to the C-domain monomers and oligomers. In these experiments, a relatively small proportion of cellular pools of V3, V5, and 51 integrins (1.5% to 3%) was found to be cross-linked to immobilized C monomers, whereas the amounts of cross-linked integrins were increased in the cells attached to the C oligomers (Figure 6A). Quantitative immunoblotting for the 3, 1, and 5 integrin subunits and normalization for the number of adherent cells revealed a significant (about 2.3-fold to 3.0-fold) increase in the amounts of cross-linked integrins in HUVECs on the C(FXIII) and C(tTG) oligomers (Figure 6B). Hence, transglutaminase-mediated oligomerization of the C domains stimulates their ability to bind V3, V5, and 51 integrins. View larger version (40K): [in this window] [in a new window]   Figure 6.. Oligomerization of the C domains increases the amounts of V3, V5, and 51 integrins chemically cross-linked to substrate. (A) HUVECs in serum-free DMEM were plated for 2 hours on 20 µg/mL C monomers, C(FXIII) oligomers, or C(tTG) oligomers; 5 x 106 adherent cells were cross-linked to the substrates with 2 mM DTSSP in PBS for 30 minutes at 4°C and extracted 4 times for 20 minutes with 0.1% SDS. The cross-linked material was recovered by treating plates for 1 hour at 37°C with a buffer containing 100 mM DTT and 0.1% SDS and then concentrated and analyzed by SDS-PAGE and immunoblotting for the 3, 1, and 5 integrin subunits. Right lanes on the gels contained 10 ng purified V3, V5, and 51 integrins. Shown is a representative of 3 experiments. Numbers below the blots refer to the relative amounts of individual integrins, as determined by densitometry and normalized to their amounts cross-linked to C monomers. (B) Bands corresponding to cellular integrins in panelAwere compared with external standards of purified integrins and then converted to the numbers of ligand-bound integrin receptors per cell. indicates C monomers; , C(FXIII) oligomers; and , C(tTG) oligomers. Results are the means ± standard deviations of 3 independent experiments.   Oligomerization of the C domains amplifies integrin-mediated signaling to FAK and ERK Integrins serve as signal transduction receptors, which require both clustering and ligand occupancy for a full biologic response.4 Because transglutaminase-mediated oligomerization of the C domains both increases integrin binding and promotes clustering of these receptors, we set out to test the effects of such oligomerization on integrin-dependent signal transduction (Figure 7). Activation of FAK upon integrin engagement is a critical early step in integrin signaling that is involved in many aspects of cell behavior.36 Up to 6 tyrosine residues of FAK can be phosphorylated upon integrin-mediated cell-matrix adhesion.37 In the present study, antiphosphotyrosine and phosphospecific polyclonal antibodies to pTyr-FAK397, pTyr-FAK577, and pTyr-FAK861 were employed to evaluate the cellular response to adhesion on the C monomers and cross-linked C(FXIII) oligomers. Compared with HUVECs in suspension, adhesion to either C monomers or C(FXIII) oligomers raised the overall level of FAK phosphorylation, with a more robust increase observed on the oligomers (Figure 7A). Phosphorylation of Tyr397 in FAK, which reports the level of integrin tethering proportional to the number of integrin-ligand bonds,34 was markedly enhanced by adhesion to both substrates. Yet again, the increase appeared 2 times higher for HUVECs plated on the C(FXIII) oligomers compared with C monomers. Very little if any phosphorylation of Tyr577 residue of FAK was observed in nonadherent and adherent HUVECs. Phosphorylation of FAK residue Tyr861, which occurs in response to integrin clustering but does not require ligand binding by integrins,34 was induced about 2-fold by plating cells on the C monomers and almost 3-fold by adhesion to the C(FXIII) oligomers. No difference in adhesion-mediated signaling was observed between the C(tTG) and C(FXIII) oligomers (data not shown). Together, these results indicate that transglutaminase-mediated oligomerization of the C domains amplifies adhesion-dependent phosphorylation of FAK due to up-regulation of both integrin binding and clustering. View larger version (45K): [in this window] [in a new window]   Figure 7.. Oligomerization of the C domains amplifies adhesion-dependent activation of FAK and ERK. HUVECs were either kept in suspension or plated in serum-free DMEM for 2 hours on tissue culture plates coated with 20 µg/mL C monomers or C(FXIII) oligomers. (A) Phosphorylation of FAK tyrosines 397, 577, and 861 was examined by SDS-PAGE and immunoblotting with specific polyclonal antibodies (see "Materials and methods"). Overall tyrosine phosphorylation of FAK was tested by immunoprecipitation of FAK, followed by SDS-PAGE and immunoblotting with polyclonal antiphosphotyrosine antibodies. (B) Adhesion-dependent phosphorylation of ERK was analyzed by immunoblotting with antibodies against dually phosphorylated ERK1/2 and total ERK1/2. Panels A and B are representative of 3 independent experiments for FAK and ERK.   We also examined integrin-dependent activation of ERK in HUVECs plated on C monomers and oligomers (Figure 7B). Immunoblotting with phosphospecific antibodies revealed that adhesion to both substrates sharply increased phosphorylation of ERK1/2. However, a more robust increase in ERK1/2 phosphorylation was observed for cells plated on the C(FXIII) oligomers, particularly in the case of ERK1. Thus, adhesion-dependent phosphorylation of ERK1/2 is also enhanced by oligomerization of the C domain.    Discussion Top Abstract Introduction Materials and methods Results Discussion References   Interaction of fibrin(ogen) with endothelial cells occurs through a number of cell receptors. They include vascular endothelial (VE)–cadherin, intercellular adhesion molecule-1 (ICAM-1), and at least 2 integrins, V3 and 51, which interact with complementary binding sites located in different fibrin(ogen) domains. In particular, the N domains of the central E region interact with VE-cadherin,38 the chain sequences located in the coiled coil and C domains of the D regions interact with ICAM-1 and V3 integrin, respectively,39,40 and the C domains interact with V3 and 51.5,7,23,41 In this study, we have examined the mechanism of the C domain–mediated interaction of fibrinogen and fibrin with endothelial cells. Notably, in fibrinogen the C domains are monomeric/dimeric, while in fibrin they form ordered cross-linked polymers24 in which their RGD-containing and other binding sites are brought into close proximity. Because of this structural difference, we tested both the monomeric C domains and the transglutaminase–cross-linked C oligomers that mimic the arrangement and properties of these domains in fibrinogen and fibrin, respectively.28 Factor XIII (FXIII) and tissue transglutaminase (tTG) are the most abundant members of transglutaminase family. Proenzyme FXIII, for which fibrin is the major physiological substrate, is present in plasma and platelets, whereas tTG is localized in the extracellular matrix and on the surface of various cell types. tTG is particularly abundant on the basal surface of endothelial monolayer in blood vessels.42,43 Moreover, it is enzymatically active and is able to cross-link fibrinogen on the surface of endothelial cells in situ.44 Thus, when the endothelial lining is damaged and tTG becomes exposed, it may substantially contribute to the cross-linking of fibrin. Therefore, in this study we prepared C domain oligomers cross-linked with tTG, C(tTG), and compared them with FXIIIa–cross-linked C domain oligomers, C(FXIII). The experiments demonstrated that both transglutaminases produced C oligomers with similar properties. Both types of oligomers, C(FXIII) and C(tTG), had similar ordered structure and exhibited dramatically increased adhesion capacity for HUVECs in comparison with that of the monomeric C domains. Previous work showed that 2 integrins on endothelial cells, V3 and 51, mediate their interaction with fibrin(ogen) via the RGD sequence of the C domains.5,7,23,41,45 Cell adhesion experiments with isolated C domain monomers/oligomers and function-blocking monoclonal antibodies presented here confirmed those findings and revealed that another integrin, V5, is also involved in RGD-dependent adhesion of endothelial cells to fibrin via its C domains. The relative contribution of each integrin to the adhesion process was found to be different. While blocking anti-V5 and anti-51 mAbs inhibited adhesion by only 15% to 30%, the mAb against V3 reduced adhesion by about 75%. A similar pattern was obtained by chemical cross-linking of endothelial integrins to the C domain species. Altogether, these results indicate that HUVECs utilize all these 3 integrins for the interaction with the fibrin(ogen) C domains, with V3 playing a predominant role in this process. Because our estimates showed that HUVECs contain similar numbers of these integrin receptors, such a prominent role of V3 could be explained by its potentially higher affinity for the