© 1996 American Heart Association, Inc.
Articles |
Heparin-Binding Domain of Fibrin Mediates Its Binding to Endothelial Cells
the Departments of Medicine (Hematology Unit: T.M.O., C.W.F., L.A.S., L.A.B., V.J.M., P.J.S.-H.), Pathology and Laboratory Medicine (L.A.S., V.J.M., P.J.S.-H.), and Microbiology and Immunology (P.J.S.-H.), University of Rochester School of Medicine and Dentistry, Rochester, NY.
Correspondence to Patricia J. Simpson-Haidaris, PhD, Hematology Unit–Department of Medicine, PO Box 610, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave, Rochester, NY 14642. E-mail phaida@gigli.medicine.rochester.edu.
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Abstract |
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Spreading of human umbilical vein endothelial cells (ECs) on fibrin requires thrombin cleavage of fibrinopeptide B (FPB) and subsequent exposure of the new ß15-42 N-terminus. To further understand the interactions between ECs and fibrin ß15-42 sequences, binding of fibrin(ogen) to EC monolayers was measured with polyclonal anti-fibrinogen (FBG) in parallel with monoclonal anti-FBG (18C6, ß1-21; J88B,
63-78) and anti-fibrin (T2G1, ß15-21) antibodies in an indirect enzyme-linked immunosorbent assay. To accomplish this, large, soluble fragments of fibrin were prepared by cyanogen bromide (CNBr) cleavage (fibrin-CNBr); CNBr-cleaved FBG (FBG-CNBr) served as the control ligand. N-terminal fibrin-CNBr bound to EC monolayers and cells in suspension in a dose-dependent and saturable manner. By contrast, FBG-CNBr bound only 50% as well to EC monolayers, with no significant binding of intact FBG, C-terminal FBG plasmic fragment D, or N-terminal plasmic fragment E, which lacks ß1-53. ECs bound the peptide ß15-42–bovine serum albumin (BSA) conjugate but neither a scrambled ß15-42 peptide conjugate nor conjugates of ß24-42, ß18-27, or ß18-31. Binding of fibrin-CNBr was inhibited 54% by the ß15-42–BSA conjugate and 17% by the Bß1-42-BSA conjugate but not by free ß15-42 peptide or RGDS-cell binding peptide. Binding of fibrin-CNBr was inhibited >95% by heparin in a concentration-dependent manner; the same concentrations of heparin inhibited binding of ß15-42–BSA by >75% but not the dose-dependent binding of fibronectin to ECs. These data suggest that in their native conformation, FBG Bß15-42 sequences are unavailable for binding to ECs and that thrombin-induced exposure of ß15-42 is required for binding by a heparin-dependent, RGD-independent mechanism at the new N-terminus of fibrin.
Key Words: fibrinogen • proteoglycans • RGDS peptide • cell adhesion • thrombin
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Introduction |
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Since vascular ECs are constantly in contact with circulating FBG, it is likely that specific epitopes exposed on conversion of FBG to fibrin are involved in the processes of wound healing and revascularization. Indeed, EC spreading1 and proliferation2 on fibrin matrices require exposure of the ß15-42 region at the neo–N-terminus of the fibrin molecule. The same structural requirement has been demonstrated for fibrin-induced release of von Willebrand factor from the Weibel-Palade body storage pool in ECs.3 Furthermore, ß15-42 is important in the formation of EC capillary tubes in response to fibrin.4
Several lines of evidence suggest that the interaction of ECs with the ß15-42 domain of fibrin is not likely mediated by integrin receptors. Although 
vß3 is a major receptor that mediates EC attachment to FBG, cell adhesion to desAB fibrin (fibrin lacking FPA and FPB) is partially resistant to inhibition by RGD-containing peptides and an MoAb against integrin receptor vß3 (7E3).5 6 These results suggest that in addition to vß3-mediated EC attachment, integrin-independent binding of ECs occurs in response to fibrin. In addition, MoAb 7E3 does not inhibit capillary tube formation on fibrin, which suggests that the EC vitronectin-FBG receptor alone is not responsible for this process.4 A 130-kD GP purified from EC membranes by ß15-42 affinity chromatography represents a putative cell surface receptor for ß15-42; however, unlike most integrin receptor-ligand interactions, its binding to ß15-42 is neither inhibited by RGDS cell binding domain peptides nor requires divalent cations.7
HBDs of adhesive GPs involved in mediating cell-cell and cell-matrix interactions include FN,8 9 10 laminin,11 12 vitronectin,13 14 von Willebrand factor,15 and thrombospondin.16 17 Our recent study demonstrates that exposure of the ß15-42 region by thrombin cleavage enhances fibrin-heparin binding and reveals that this site also constitutes an HBD.18 Therefore, we postulated that the newly exposed HBD of fibrin plays a role in mediating fibrin-EC interactions. In this study, the structural requirements that support binding of CNBr-solubilized fibrin(ogen) fragments to ECs were examined. We found that exposure of ß15-42 by thrombin cleavage enhanced binding of fibrin-CNBr to EC monolayers by a heparin-dependent mechanism.
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Methods |
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Materials
Human FBG was purchased from Kabi Vitrum and FN from Sigma Chemical Co. FBG plasmic fragments D and E were obtained from Crystal Chemicals. Thrombin (human plasma, 3554 NIH U/mg) was from Calbiochem. HRP- and alkaline phosphatase–conjugated secondary antibodies were from Jackson Scientific and streptavidin-HRP or streptavidin-FITC from GIBCO BRL. N-Hydroxysuccinimide ester–biotin, heparin sodium salt (porcine intestinal mucosa, grade II; 162 USP U/mg), CNBr, and formic acid were from Sigma. Fetal bovine serum was from Intergen, EC growth factor (crude extract) from Collaborative Research, and McCoy's 5A media from ICN Flow. The cell binding domain tetrapeptide RGDS was purchased from BACHEM. Synthetic peptides corresponding to regions of the FBG Bß-chain N-terminus were either purchased or custom synthesized on the basis of the primary structure of Bß1-42: QGVNDNEEGFFSARGHRPLDKKREEAPSLRPAPPPISGGGYR. Peptide ß24-42 was purchased from BACHEM; peptide ß15-42 and scrambled ß15-42 (ß15-42S, primary structure of DRGAPAHRPPRGPISGRSYPEKEKLLPG) were synthesized by the Cornell Biotechnology Facility (Ithaca, NY). Fibrin ß-chain peptides ß18-27 and ß18-31 were synthesized by Louisiana State University Medical Center (New Orleans, La). The purity of the peptides ranged from 95% to 100%, and mass spectroscopy data indicated that the peptides were of the expected sizes. Antibodies used in this study are described in Table 1
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CNBr Cleavage of FBG and Fibrin
The rate of thrombin cleavage of FPB is enhanced during fibrin polymerization.21 Therefore, fibrin was generated under conditions of low thrombin concentration (0.25 TAME U/mL) without exogenous factor XIII to reduce cross-linking and fibrin polymerization.22 Large, soluble fragments were generated by partial CNBr cleavage of fibrin essentially as described.22 SDS-PAGE, Western blotting, and ELISA were performed as described.23 The Sephadex G100 fractions that were positive by ELISA (Table 2) and Western blot (not shown) with the fibrin-specific MoAb T2G1 were pooled and designated fibrin-CNBr. FBG-CNBr fragments were prepared for comparison with fibrin-CNBr fragments with regard to their binding to ECs.
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Peptide Modification
BSA was biotinylated as described.24 Synthetic peptides corresponding to FBG Bß-chain sequences were covalently linked to BSA or biotinylated BSA by one-step coupling with glutaraldehyde.25 Molar ratios of peptide to BSA were determined by amino acid analysis performed by the Cornell Biotechnology Facility and were determined to be 20 to 40 molecules of peptide per molecule of BSA.
EC Culture
ECs were isolated from human umbilical cord veins by pronase perfusion as previously described26 ; seeded on 0.2% (wt/vol) gelatin-coated, 96-well microtiter tissue-culture plates; and used at confluence, which was typically reached after 4 to 5 days. Only P2 and P3 passage cells were used for the experiments.
EC-Ligand Binding ELISA
Confluent EC monolayers in microtiter wells were washed with Hank's balanced salt solution and fixed with 1% (vol/vol) glutaraldehyde in PBS for 10 minutes at room temperature. Fixation was required to prevent cell detachment from the culture wells during incubation and washing, as previously described.27 After fixation, ECs were washed five times with PBS and nonspecific binding sites were blocked with 5% (wt/vol) BSA in PBS followed by four washes with PBS. Each ligand (0 to 400 µg/mL) was applied to three wells and incubated for 1 hour at room temperature; unbound ligand was removed by five washes with PBS. The amount of bound ligand was determined by incubating the wells with the ligand-specific MoAb or polyclonal antibodies (10 µg/mL), washing, and subsequent colorimetric development after incubation with the appropriate secondary species-specific HRP antiserum. In the case of peptide-BSA conjugates, the peptide ligand was detected through a streptavidin-HRP reaction with the biotinylated carrier BSA.
Competitive Inhibition Assays
The binding of 50 µg/mL of fibrin(ogen)-CNBr fragments to EC monolayers was competed with increasing concentrations of peptide-BSA conjugates (0 to 500 µg/mL, based on BSA concentration). Heparin concentrations from 0 to 250 µg/mL were used to competitively inhibit the binding of 12.5 µg/mL of fibrin-CNBr fragments, ß15-42–BSA, or FN. The specificity of ß15-42–BSA binding to ECs was also tested by competitive inhibition of 25 µg/mL ß15-42–BSA with intact FBG, FBG-CNBr, or FN, ranging from 0 to 50 µg/mL.
Flow Cytometry Analysis
To analyze the binding of peptide-BSA conjugates, FBG-CNBr, FBG, or fibrin-CNBr to ECs, flow cytometry was performed on an EPICS C cytofluorometer (Coulter).25 ECs were washed in Hanks' balanced salt solution and detached by incubation with trypsin-EDTA for 1 to 2 minutes at 37°C; trypsin action was blocked by adding 20% (vol/vol) fetal bovine serum–McCoy's medium. The cells were washed in PBS/0.02% NaN3 (wt/vol) at 4°C to block metabolic activity. All further incubations were performed on ice in PBS/5% fetal bovine serum. ECs (106) were incubated with 50 µg/mL ligand or peptide conjugates for 30 minutes. Binding of primary antibodies for fibrin(ogen) fragments was detected with FITC-tagged goat anti-mouse F(ab')2; binding of peptide-BSA conjugates was detected with FITC-streptavidin.25 Nonspecific binding was determined with an irrelevant isotype-specific antibody or FITC-streptavidin alone.
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Results |
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Characterization of CNBr Fragments of FBG and Fibrin
To maintain the conformational integrity of fibrin N-terminal domains yet allow solubilization, partial CNBr cleavage was performed (Fig 1
). FBG-CNBr fragments were positive by ELISA with the FBG-specific MoAb 18C6, indicating that FPB was still intact (Table 2
). Fibrin-CNBr retained 
30% activity with 18C6, indicating that thrombin cleavage of FPB was incomplete (Table 2). The MoAb binding site A-RGDS-572-575 was preserved in the CNBr fragments of FBG and fibrin, whereas the platelet recognition domain 400-411 was lost after CNBr cleavage, fractionation, and dialysis (Table 2). ELISA (Table 2) and Western blot (not shown) of the plasmic E fragments indicated that they were mostly E3 (lacking Bß1-53); plasmic D fragments were a mixture of D1, D2, and D3 (lacking C-terminal residues; Fig 1). The CNBr-cleaved FBG and fibrin fragments ranged in molecular weight from 180 to 300 kD (Fig 2).
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Fibrin Fragment Binding to ECs Is Concentration Dependent and Saturable
The ability of fibrin-CNBr to bind to intact EC monolayers was studied with the EC-ligand ELISA. Binding of all immunoreactive fragments of fibrin-CNBr, FBG-CNBr, and intact FBG (100 µg/mL) to EC monolayers was determined with a polyclonal anti-FBG (Fig 3A). Fibrin-CNBr showed the highest level of binding compared with the 50% lower binding of FBG-CNBr; intact FBG showed nearly undetectable binding (Fig 3A). FBG fragment E3, which lacks ß1-53, and fragments D1, D2, and D3 derived from the C-terminal portion of FBG also showed no binding (not shown). The binding of fibrin-CNBr fragments was detected with the fibrin ß15-21–specific MoAb T2G1, and binding of FBG-CNBr fragments was detected with the FBG Bß1-21–specific MoAb 18C6 (Fig 3A). The results showed that fibrin and FBG N-terminal–containing fragments simultaneously bound to the apical surface of ECs and to their respective MoAbs. Thus, these MoAbs could be used to detect fibrin- and FBG-CNBr fragment binding, respectively. Fibrin-CNBr binding to ECs occurred in a dose-dependent manner, with saturation at 100 µg/mL when T2G1 was used for detection (not shown). When a panspecific -chain MoAb (J88B) was used to compare the binding of fibrin-CNBr with that of FBG-CNBr and FBG, only fibrin-CNBr showed significant binding in a dose-response manner (Fig 3B). FBG-CNBr binding was 60% to 70% lower, whereas FBG binding was negligible.
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To test whether peptide ß15-42 bound ECs in suspension, the cells were lifted by brief trypsinization and flow cytometric analysis was performed. Between 60% and 75% of ECs showed positive binding of ß15-42–BSA-biotin (100 µg/mL) when compared with the binding of the scrambled peptide at the same concentration (Fig 4A). Neither biotinylated BSA carrier protein nor fibrin-specific peptide ß24-42–BSA-biotin conjugate bound to ECs (not shown). The binding of fibrin-CNBr, FBG-CNBr, and FBG to ECs in suspension was also performed. Whereas FBG did not show appreciable binding to ECs, both FBG-CNBr and fibrin-CNBr showed significant binding (Fig 4B). However, the distribution in the FL1 shift of cells that bound FBG-CNBr was observed as a "shoulder" at the leading edge, with two peaks at 190 and 215 FL1, whereas ECs that bound fibrin-CNBr showed a single peak at 215 FL1 (Fig 4B). These results suggest that fibrin-CNBr contains a more homogeneous population of binding sites for ECs than does FBG-CNBr, which is consistent with our earlier results showing that CNBr cleavage of FBG in the absence of thrombin cleavage partially exposes the HBD.18 Taken together, the results suggest that fibrin-CNBr binding to ECs occurs at the cell surface and not to the extracellular matrix and that the ß15-42 domain contributes to the specificity of N-terminal fibrin(ogen) fragment binding to ECs.
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Fibrin Binding to ECs Involves Residues ß15-42
To more precisely determine the EC binding domain on fibrin-CNBr fragments, partially overlapping peptides from the ß15-42 region of fibrin were synthesized and conjugated to biotinylated BSA carrier. Whereas ß15-42–BSA showed saturable binding, there was no binding of ß18-31, ß18-27, or ß24-42 conjugates (Fig 5A). The role of fibrin–ß15-42 compared with that of FBG–Bß1-42 sequences in mediating fibrin-CNBr and FBG-CNBr binding to ECs was demonstrated by competitive inhibition with increasing concentrations of nonbiotinylated ß15-42–BSA or Bß1-42–BSA conjugate. Nonbiotinylated ß15-42–BSA conjugate (25 to 400 µg/mL) bound to ECs did not react with T2G1 (not shown); in contrast, fibrin-CNBr bound to ECs could be detected with T2G1. Maximum inhibition (54%) of fibrin-CNBr binding to ECs occurred at concentrations of nonbiotinylated ß15-42–BSA conjugate 
250 µg/mL (4.1 µmol/L, based on BSA concentration; Fig 5B), whereas the same sequences in FBG–Bß1-42 were much less effective in inhibiting binding of fibrin-CNBr to EC monolayers. The total binding of FBG-CNBr fragments to EC monolayers was significantly less than that of fibrin-CNBr (Fig 3). Moreover, ß15-42–BSA was a stronger competitive inhibitor of FBG-CNBr binding than was Bß1-42–BSA conjugate (Fig 5B). The free ß15-42 peptide in concentrations up to 430 µmol/L showed no binding to ECs, either directly as biotinylated peptide or indirectly with the specific MoAb T2G1 (not shown), suggesting that multivalency of the ß15-42 ligand may be required for binding to ECs. No inhibition of fibrin-CNBr binding to ECs occurred when free ß15-42 peptide, carrier BSA alone, or ß24-42–BSA conjugate (not shown) was used. Together these results suggest that the sequences important in mediating binding of fibrin to EC monolayers are exposed after thrombin cleavage and are localized to the ß15-42 N-terminus.
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Fibrin Fragment Binding to ECs Is Divalent Cation and RGD Independent
To investigate the possible involvement of divalent cations in mediating fibrin binding to ECs, the influence of Ca2+, Mg2+, and Mn2+ on the binding of ß15-42–BSA-biotin and fibrin-CNBr fragments was tested. Binding of ß15-42–BSA (not shown) and of fibrin-CNBr fragments to ECs was significantly lower when Ca2+, Mg2+ (Fig 6), and Mn2+ (not shown) were present. These results suggest that binding of fibrin-CNBr and ß15-42–BSA to ECs is not mediated through a divalent cation–dependent mechanism like those identified with integrin receptor binding of ligand.32 33 34 When increasing concentrations of the cell binding domain peptide RGDS were used to inhibit N-terminal fibrin-CNBr fragment binding to EC monolayers, no inhibition of binding occurred (not shown). Moreover, binding of ß15-42–BSA-biotin (Fig 7) and fibrin-CNBr (not shown) was not competitively inhibited by the RGD-containing ligands FBG, FN, and FBG-CNBr.
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Fibrin Fragment Binding to ECs Is Heparin Dependent and ß15-42 Specific
To test the possibility that fibrin-CNBr binding to ECs is mediated through a heparin-dependent mechanism, increasing concentrations of heparin were preincubated with fibrin-CNBr, FN, or ß15-42–BSA before exposure to EC monolayers. Binding of fibrin-CNBr was inhibited >83% at a concentration of 7.5 µg/mL and nearly completely inhibited (>95%) at concentrations of heparin >62.5 µg/mL. Under the same conditions, binding of the ß15-42–BSA peptide conjugate was inhibited by almost 80% (Fig 8B). This inhibition is consistent with heparin's involvement in mediating the binding of fibrin fragments to ECs, most likely through the recently identified HBD composed of ß15-42 sequences at the fibrin ß-chain neo–N-terminus.18 Furthermore, heparin failed to inhibit binding of FN to ECs (Fig 8B), even though concentration-dependent binding of FN to ECs was demonstrated (Fig 8A).
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Discussion |
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Cellular responses of adhesion, spreading, or migration to adhesive GPs are mediated by multiple recognition domains of individual molecules.33 35 Many adhesive GPs involved in cell-cell and cell-matrix interactions, including fibrin(ogen), have both HBDs8 9 10 11 12 13 14 15 16 17 18 and RGD cell binding domains.32 33 34 36 37 38 39 Therefore, fibrin solubilized by CNBr cleavage was used to explore the structural requirements of binding to cultured EC monolayers and to study the potential involvement of both RGDS- and heparin-mediated interactions in this binding. Because the coiled-coil region imparts structural rigidity to fibrin(ogen) molecules,40 we postulated that limited cleavage within this region would aid in retention of the conformational integrity of the cryptic ß15-42 HBD.18 Therefore, partial CNBr cleavage of methionine residues was performed to produce large-molecular-weight but soluble fragments of fibrin (Fig 2
). ELISA and Western blot analysis of CNBr fragments of FBG and fibrin confirmed the presence of binding sites or epitopes for A-RGDS-572-575, FBG Bß1-21, or fibrin ß15-21, which lies within the ß-chain HBD ß15-42, and the absence of platelet recognition domain 400-411 (Fig 1). Residual activity of fibrin-CNBr with 18C6 indicated incomplete cleavage of FPB by thrombin, as expected under the conditions that were used for fibrin formation.
During fibrin polymerization, not only are new peptide domains exposed by the proteolytic action of thrombin but also other domains are likely to be brought into close proximity.41 Therefore, multiple polymerized binding sites may contribute to specific cellular interactions with fibrin. Saturable binding of fibrin(ogen) fragments to EC monolayers occurred and was enhanced in thrombin-cleaved compared with uncleaved preparations, suggesting ß15-42 involvement (Fig 3). Fibrin-CNBr bound to EC monolayer (Fig 3) and ECs in suspension (Fig 4), suggesting that the binding was EC specific and not dependent on the extracellular matrix. Binding of intact FBG was not detected and, therefore, binding of FBG-CNBr was likely due to partial exposure of the FBG Bß15-42 domain after CNBr cleavage.18 40 Using synthetic peptides conjugated with BSA, we determined that ß15-42 but not ß18-31, ß18-27, or ß24-42 bound saturably to ECs (Fig 5A). ß15-42–BSA conjugate partially inhibited (54%) binding of fibrin-CNBr, whereas Bß1-42–BSA conjugate was essentially ineffective (Fig 5B). Furthermore, even high concentrations of free ß15-42 peptide failed to bind to ECs or competitively inhibit binding of fibrin-CNBr. This incomplete inhibition, although typical when peptides are used as substitutes for native ligands,42 43 could indicate that ß15-42 represents an incomplete binding domain. By contrast, BSA conjugation may be required to secure the peptide in an appropriate or more stable conformation or binding site density.44 Alternatively, insufficient conformational constraints on bound peptide or cross-linking of the peptide to BSA via a (portion of) residue(s) that is important in mediating binding may account for the partial inhibition. However, the primary structure of ß15-42 rather than a charge interaction likely accounts for binding specificity, since a scrambled ß15-42 peptide with the same net +3 charge did not bind to ECs (Figs 4 and 5). The observed binding of fibrin to ECs did not occur in a typical RGDS-dependent fashion, since it was independent of divalent cations32 33 34 and not inhibited by soluble RGDS or intact RGD-containing ligands FBG and FN (Fig 7), although FN bound to ECs in a concentration-dependent manner (Fig 8A). These findings differ from integrin-related mechanisms that mediate EC adhesion to FBG after adsorption to plastic surfaces, which is mediated via RGDS sequences and ligand-induced activation of integrin receptor vß3.6 32 42 43 45 46 47 48 The lack of association between FBG and ECs contradicts these other studies, in which specific binding of FBG to ECs was demonstrated; however, the lack of FBG binding to ECs in our study most likely reflects differences in the methods used to study the heparin-dependent binding of fibrin(ogen) to ECs.
In light of our recent study18 of a heparin-binding function of the ß15-42 region of fibrin, we sought to determine whether fibrin binding to ECs occurs at least partially by a heparin-dependent mechanism. Such a mechanism is likely, since heparin competitively inhibits binding of fibrin-CNBr to ECs (Fig 8B). Furthermore, only peptides with the intact ß15-42 HBD and no overlapping fragments bind to ECs (Fig 5A). Our data indicate that lower-affinity HBDs previously identified in FBG Bß- and -chain C-terminal fragments38 39 also occur in plasmin-generated D fragments18 and do not mediate fibrin(ogen) binding to EC monolayers. Moreover, FN binding to ECs was not inhibited by heparin (Fig 8B), suggesting that heparin-dependent binding of fibrin to ECs is not only sequence specific but also fibrin specific. These data suggest that only N-terminal ß15-42 sequences, newly exposed by thrombin release of FPB, mediate heparin-dependent fibrin binding to ECs. The HBD of fibrin may function in one of several ways in mediating its binding to ECs. Fibrin may interact directly with heparin-containing molecules on the EC surface, or the interaction of ß15-42 with heparin may alter the conformation of fibrin to facilitate interaction with cellular receptors. Heparin binds preferentially to surface-adsorbed fibrin than to fibrin(ogen) in solution,40 suggesting a role for direct interaction of heparin or heparan sulfate PGs in regulating fibrin-mediated adhesion. Together, these data are consistent with our finding that EC responses to fibrin depend on specific structural and conformational features that are induced by thrombin conversion of soluble FBG to an insoluble fibrin polymer.1 2 3
A heparin-dependent interaction may facilitate fibrin interaction with integrins or other as-yet-unidentified cellular receptors, as has been shown for other adhesive proteins. Heparin binding to basic regions of proteins has been shown to stabilize the conformation of the HBD.35 Furthermore, clearance of vitronectin from the extracellular matrix occurs only after -thrombin induces exposure of a cryptic HBD.50 Although cellular recognition of this conformationally altered vitronectin occurs via vß5 integrins,51 such recognition is PG dependent.50 Coordinate interaction of cell surface PGs and integrins has also been proposed for cell adhesion to FN52 53 and for integrin-mediated interactions of cells with thrombospondin.16 54 Moreover, heparan sulfate PGs55 enhance the affinity of basic fibroblast growth factor for its cognate receptor by concentrating and stabilizing the ligand in the proper conformation for receptor binding, uptake, and intracellular signaling.56 57 58
Intravascular fibrin formation, whether in response to injury or pathological thrombosis, results in intimate contact of fibrin with the vascular endothelium. Cellular interactions with and responses to this "neomatrix" may be critical in wound healing and revascularization. Adhesion, spreading, and migration likely involve recognition of and binding to unique structural features exposed after conversion of FBG to fibrin. In adhesive processes, integrins provide strong adhesion but only after they are activated by other stimuli.33 47 48 The RGD independence of fibrin-CNBr binding to ECs is consistent with the binding characteristics of the EC-specific, 130-kD surface GP, which binds to ß15-42 in a divalent cation –independent and integrin-independent fashion.7 Perhaps heparin-dependent binding of fibrin to ECs occurs as an activation-independent step in cell adhesion. Thus, interaction of fibrin with an EC-specific heparan sulfate PG must be considered, and the hypothesis that fibrin ß15-42 binding to ECs involves a cell-surface PG, possibly heparan sulfate, is suggested by the results of this study.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported in part by grants HL-50615 (to P.J.S.-H.), HL-02790 (to L.A.B.), and HL-30616 (to L.A.S., C.W.F., V.J.M., P.J.S.-H.) from the National Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, Md, and a grant from the University of Rochester Strong Children's Research Center (to P.J.S.-H.). The authors wish to thank Sarah Lawrence, Laura Triou, and Li Hua Rong for expert technical assistance.
Received February 2, 1996;
revision received August 6, 1996;
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