The Role of Putative Fibrinogen Aalpha -, Bbeta -, and gamma A-chain Integrin Binding Sites in Endothelial Cell-mediated Clot Retraction*

(Received for publication, April 3, 1997, and in revised form, May 30, 1997)

Richard A. Smith Dagger §, M. W. Mosesson par , Michael M. Rooney **, Susan T. Lord **Dagger Dagger , A.U. Daniels § and T. Kent Gartner Dagger

From the Dagger  University of Memphis, Department of Microbiology and Molecular Cell Sciences, Memphis, Tennessee 38152, par  University of Wisconsin Medical School, Milwaukee Clinical Campus, Department of Medicine, Sinai Samaritan Medical Center, Milwaukee, Wisconsin 53233, Departments of ** Chemistry and Dagger Dagger  Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, and § University of Tennessee-Campbell Clinic, Department of Orthopaedic Surgery, Memphis, Tennessee 38163

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

In this study, endothelial cell-mediated clot retraction was supported by fibrin generated from several purified fractions of plasma fibrinogen, purified proteolytic fragments of plasma fibrinogen, recombinant normal fibrinogen, and recombinant variant fibrinogen. These results were surprising because some of these fibrinogens lack domains that are known binding sites for the integrin receptors that support clot retraction. Specifically, fibrinogens lacking Aalpha -chain RGD residues at 572-574 or lacking the gamma -chain residues AGDV 408-411 supported endothelial cell-mediated clot retraction as well as intact fibrinogen. Thus, clot retraction mediated by endothelial cells is not dependent on either of these sites. A variety of monoclonal antibodies against the integrin alpha vbeta 3 partially inhibited the endothelial cell-mediated retraction of clots formed from plasma fibrinogen. As expected, an antibody to the platelet integrin alpha IIbbeta 3 did not inhibit endothelial cell-mediated clot retraction. These results indicate that this retraction is mediated at least in part by alpha vbeta 3. These results support the conclusion that (a) neither of the two fibrinogen cell binding sites described above is required to support clot retraction or that (b) either site alone or in conjunction with other fibrin(ogen) region(s) can support clot retraction. Thus, endothelial cell-mediated clot retraction appears to be dependent on fibrinogen cell binding sites other than those required to support adhesion of resting platelets to immobilized fibrinogen and platelet aggregation.


INTRODUCTION

This study was undertaken to evaluate the role in clot retraction mediated by endothelial cells of presumptive endothelial cell and platelet binding sites on fibrinogen. Platelets, fibroblasts, melanoma cells, and endothelial cells are known to support clot retraction (1-4). However, it is not known whether clot retraction mediated by endothelial cells is dependent on either the presumptive endothelial cell fibrinogen Aalpha -chain binding site, the 572-574 RGD residues (5), or the fibrinogen gamma A-chain carboxyl-terminal AGDV sequence as is resting platelet adhesion to immobilized fibrinogen (6, 7) and platelet aggregation (8, 9).

Clot retraction is dependent on fibrin binding to activated alpha IIbbeta 3 in platelets (10, 11) or to the homologous integrin alpha vbeta 3 (12, 13) in nucleated cells (3, 4). Katagiri et al. (3) used monoclonal antibodies and immunoelectron microscopy to show that clot retraction mediated by melanoma cells is dependent on fibrin binding to unstimulated alpha vbeta 3. In their study, clot retraction mediated by melanoma cells was blocked by RGD-containing peptides and anti-beta 3 as well as anti-alpha vbeta 3 mAbs1 but not by an alpha IIbbeta 3-specific inhibitor. The conclusion that alpha vbeta 3 can support clot retraction mediated by nucleated cells was confirmed by Chen et al. (4). Alemany et al. (14) provided evidence that a fibrinogen gamma A-chain binding region of the platelet integrin alpha IIbbeta 3 is on its beta 3 subunit and that ligand binding to this site is independent of platelet activation. Their results support the possibility that the alpha vbeta 3 integrin of endothelial cells, like the homologous alpha IIbbeta 3 on platelets, may be able to bind fibrinogen via the gamma A-chain carboxyl termini and that this hypothetical binding may play a role in endothelial-mediated clot retraction.

Fibrinogen platelet binding sites have been identified using a variety of experimental systems. Farrell et al. (8) used recombinant forms of fibrinogen to show that platelet aggregation appears to be dependent on residues within the sequence 408-411 of the fibrinogen gamma A-chain. This was shown more directly by Rooney et al. (9). These observations confirmed and extended earlier results obtained using aggregated genetically modified gamma -chains (15). Under static conditions, the adhesion of both resting and stimulated platelets to immobilized fibrinogen appears to be dependent on the fibrinogen gamma A-chain carboxyl-terminal platelet binding sites (6, 7, 16). The activation-independent adhesion of platelets to fibrinogen also appears to be dependent on the fibrinogen gamma A-chain carboxyl-terminal platelet binding sites under flow conditions (17). Rooney et al. (9), using a recombinant form of fibrinogen (without the gamma A-chain terminal sequence AGDV on either gamma A-chain), tested gamma A-chain involvement in platelet aggregation and clot retraction. The recombinant fibrinogen did not support platelet aggregation in response to ADP but did support clot retraction. Therefore, the ligand sites on fibrinogen that support platelet aggregation may be different than the sites on fibrin that support clot retraction. These latter studies did not exclude the possibility that normal fibrinogen secreted from the platelet alpha  granules may have provided functional fibrinogen gamma A-chains to support the clot retraction. However, a recent study by Holmback et al. (18) confirms the conclusion of Rooney et al. (9) by demonstrating that mice which have only fibrinogen lacking the QAGDV sequence of both gamma -chains can support clot retraction even though the altered fibrinogen cannot support platelet aggregation. These results raise the interesting possibility that non-gamma -chain platelet binding sites on fibrinogen can support platelet-mediated clot retraction (9, 18).

Others have reported the blocking of clot retraction by certain RGD-containing peptides (presumably by binding to the receptors that mediate retraction). Unfortunately this inhibition did not identify the receptor binding site(s) on the ligand (10, 11). Likewise, the role of the gamma A-chain carboxyl-terminal platelet binding regions is not clear. Despite the fact that the gamma A-chain carboxyl-terminal peptide mimetics LGGAKQAGDV (L10) and HHLGGAKQAGDV (H12) have been shown to inhibit platelet-mediated clot retraction (10), the dependence of clot retraction on the corresponding platelet binding sites on fibrinogen has not been shown. In fact, as described above, recent evidence demonstrates that clot retraction mediated by human and mouse platelets is not dependent on the AGDV sequence of the fibrinogen gamma A-chain (9, 18). Thus, care must be taken in interpreting the results of peptide inhibition studies (19).

In the experiments described here, human umbilical vein endothelial cells (HUVECs) were tested in clot retraction assays because, unlike platelets, they do not secrete fibrinogen in response to treatment with thrombin. Thus, various forms of exogenous fibrinogen in conjunction with HUVECs would be useful to try to identify the cell binding site(s) on fibrinogen that is required to support clot retraction. Peak 1 fibrinogen (gamma Agamma A), fibrinogen fraction I-9 (a fibrinogen fragment that lacks about 100 carboxyl-terminal residues from each Aalpha -chain including the 572-574 RGD sequence and that is bivalent with respect to the gamma A-chain binding sites (20)), peak 2 fibrinogen fraction I-9 (a fragment of peak 2 fibrinogen that has one platelet reactive gamma A-chain and one nonplatelet reactive gamma '-chain and the same Aalpha -chain composition as fibrinogen fraction I-9), recombinant normal human fibrinogen (9), recombinant fibrinogen gamma 407, which lacks residues 408-411 in both gamma A-chains (9), and the mAb 4A5 (a mAb specific for the carboxyl-terminal region of the gamma A-chain) (21) were used to evaluate the role of presumptive fibrinogen cell binding sites in clot retraction mediated by HUVECs.


EXPERIMENTAL PROCEDURES

Cell Culture

HUVECs and endothelial cell growth medium (containing 10 ng/ml human recombinant epidermal growth factor, 1 µg/ml hydrocortisone, 50 µg/ml gentamicin, 50 ng/ml amphotericin B, 3 mg/ml bovine brain extract, 2% fetal bovine serum) were purchased from Clonetics Corp. (San Diego, CA). The cells were grown to 80-95% confluence, and then a 0.025% trypsin, 0.01% EDTA solution (Clonetics) was used to release the cells from the surface of the flask, after which trypsin neutralization solution (Clonetics) was added. The cell suspension was centrifuged at 220 × g for 5 min, the supernatant liquid was decanted, and the cells were suspended in fresh endothelial cell growth medium. The suspended cells were allowed to recover from trypsinization by incubation for 30 min at 37 °C before use in the clot retraction assay.

Clot Retraction

The clot retraction assay was a modification of published methods (3, 4, 10). The cell suspension was centrifuged, the liquid was decanted, and the cells were washed three times with a Tyrodes Hepes solution (150 mM NaCl, 2.5 mM KCl, 2.0 mM MgCl2, 5.0 mM Hepes, pH adjusted to 7.35) containing 1 mg/ml glucose and 3.5% bovine serum albumin. The washed cells were added to the incubation solution (wash solution containing 2 mM CaCl, 3 µg/ml aprotinin, and 250 µg/ml fibrinogen or fragment) and allowed to incubate for 5 min at 37 °C. Cell numbers were estimated using a hemocytometer. ~2 × 105 cells were suspended in 0.5 ml of clotting medium (250 µg/ml fibrinogen or fragment in Tyrodes Hepes solution containing glucose, bovine serum albumin, CaCl2, and aprotinin) in a sylanized (using trimethylsylyl) aggregometer cuvette. mAbs that bind integrins were added to cells in incubation buffer before addition to clotting medium. mAbs that bind fibrinogens or their fragments were added to the clotting medium before the addition of the cells. Thrombin (Sigma) (1 unit/ml) was added to inititate fibrin formation. The retracting clot was photographed at several time intervals, and the photos were traced on a back-lit digitizing pad (Ortho-Graphics, Inc.) providing automated data entry into a computer. The longitudinal cross-sectional area not occupied by the clot was calculated and expressed as a percentage of the total area. The results are reported as percent clot retraction where 0% is no retraction and 100% would be complete retraction, which is undefined. Each clot retraction experiment was repeated at least once. Some experiments were repeated only once due to the large amount of antibodies and recombinant fibrinogen required. Although data are presented as quantitative, these studies are not meant to be a stringent quantitative analysis; however, the experiments provide unequivocal data as to the support or inhibition of HUVEC-mediated clot retraction by the forms of fibrinogen, antibodies, and peptides used in the experiments described above.

Antibodies

mAbs used were LM609 (50 µg/ml), which binds to the alpha vbeta 3 receptor (22); CLB-706 (50 µg/ml), which binds to alpha v (LM609 and CLB-706 were from Chemicon International, Inc.); 7E3 (50 µg/ml), which binds to alpha IIbbeta 3 (23) and alpha vbeta 3 (24) (courtesy of Dr. Barry Coller); A2A9 (50 µg/ml), which binds to alpha IIbbeta 3 (25) (courtesy of Dr. J.S. Bennett); AP3 (100 µg/ml), which binds the beta 3 subunit (26) (courtesy of Dr. Peter Newman); 4A5 (21) 50 µg/ml (courtesy of Dr. Gary Matsueda), which binds the fibrinogen gamma A-chain carboxyl terminus and prevents platelet adhesion; and JB1a (1:100 dilution from ascites fluid), an anti-beta 1 antibody (27). Mouse IgG (mIgG) (Sigma) (50 µg/ml) was used as control IgG.

Fibrinogens

The following fibrinogens were used in this study to identify the binding sites utilized by endothelial cells to support clot retraction: peak 1 fibrinogen (gamma Agamma A) (6), peak 1 fibrinogen fraction I-9 (20), peak 2 fibrinogen fraction I-9 (6), and fibrinogen 325, which lacks the first 42 amino acids (amino terminus) of the Bbeta -chains (28, 29). The fibrinogens used and the important binding sites present or absent on each form are listed in Table I. Peak 1 (gamma Agamma A) and peak 2 (gamma Agamma ') fibrinogens were prepared as described by Mosesson and Finlayson (30) from fraction I-2 fibrinogen (31) containing >80% intact Aalpha -chains (20) or from fraction I-9 fibrinogen (20). Fraction I-9 fibrinogen is devoid of intact Aalpha -chains and contains instead Aalpha -chain derivatives of the size of Bbeta -chains (54 kDa) or smaller (20) that lack carboxyl-terminal segments. Its composition was verified by SDS-polyacrylamide gel electrophoresis. Chromatographic separation into peak 1 and peak 2 subfractions was verified by DEAE-cellulose ion exchange chromatography using the gradient elution system described by Siebenlist et al. (32). Fibrinogen 325 (des-Bbeta 1-42 fibrinogen) that lacks the first 42 amino acids of Bbeta -chains (33) was produced from fraction I-2 fibrinogen as described by Pandya et al. (28) and Pandya and Budzynski (29). Its structure was verified by SDS-polyacrylamide gel electrophoresis.

Table I. Composition of fibrinogen forms

+, contains the part of the molecule; -, missing the part of the molecule. gamma A contains the gamma -chain platelet binding region. gamma ' does not contain the gamma -chain platelet binding region but has a run-on sequence in place of the gamma -chain carboxyl terminal AGDV. AGDV corresponds to the final four residues (408-411) of the gamma -chain, which is important in fibrinogen binding. rFibrinogen, recombinant fibrinogen.

Fibrinogen Aalpha -chain 572-575 (RGDS) Bbeta -chain 1-42 (QGV...GYR)  gamma -chain 408-411 (AGDV)

Fraction I-2 peak1 + + ++ (gamma Agamma A)
Fraction I-9 peak1  - + ++ (gamma Agamma A)
Fraction I-9 peak2  - + +- (gamma Agamma ')
Fibrinogen 325 +  - ++ (gamma Agamma A, 92%)
rFibrinogen + + ++ (gamma Agamma A)
rFibrinogen gamma 407 + +  -- (-AGDV)

Normal recombinant fibrinogen and recombinant fibrinogen gamma 407 (which lacks residues 408-411 on both gamma A-chains) were synthesized by transfected Chinese hamster ovary cells, and purification was monitored as described (9). Briefly, samples were run on SDS-polyacrylamide gel electrophoresis under reduced conditions according to the method of Laemmli (34) and appeared as three bands corresponding to the Aalpha -, Bbeta -, and gamma -chains. Western blot analysis was performed as described (35) using 4A5. Normal recombinant and plasma fibrinogen developed bands corresponding to the gamma -chain; however, the gamma -chain from gamma 407 fibrinogen was undetectable, consistent with previous results (9).

Peptides

Peptide inhibitors of fibrinogen binding to platelets were tested as inhibitors of endothelial cell-mediated clot retraction. The peptide LGGAKQAGDV, a gamma A-chain carboxyl-terminal fibrinogen peptide mimetic, GRGDSP, and a control scrambled version of GRGDSP, PGRSGD, were tested in the clot retraction assay at different concentrations. Quantity and sequences were verified by St. Jude Children's Research Hospital Biotechnology Center laboratories. The methods used for the synthesis, purification, and characterization of these peptides have been described (19).


RESULTS

Endothelial cells retracted clots formed from thrombin-treated peak 1 fibrinogen gamma Agamma A, peak 1 fibrinogen fraction I-9, and peak 2 fibrinogen fraction I-9 (Fig. 1). The rates of clot retraction mediated by the HUVECs were approximately the same for all three types of fibrinogen used in these experiments (Fig. 2). The ability of peak 1 fibrinogen fraction I-9 to support clot retraction mediated by the endothelial cells means that the Aalpha -chain RGD (572-574) sequence was not required for retraction. Furthermore, the fact that clot retraction was also supported by peak 2 fibrinogen fraction I-9 demonstrates that if the carboxyl terminus of the gamma A-chain is required to support clot retraction, a single gamma -chain is sufficient.


Fig. 1. Clot retraction for human umbilical vein endothelial cells. Fg gamma Agamma A, peak 1 fibrinogen; Fr I-9, fibrinogen peak 1 fraction I-9, which lacks the Aalpha -chain 572-574 RGD sequences; Fg gamma Agamma ', fibrinogen peak 2 fraction I-9, which lacks one gamma A-chain carboxyl-terminal platelet binding region as well as the same Aalpha -chain sequences as fraction I-9; rFg, recombinant normal fibrinogen; rFg 407, recombinant fibrinogen missing the gamma A-chain carboxyl-terminal 408-411 AGDV sequences. All fibrinogens were at 250 µg/ml concentration, and thrombin concentration was 1 unit/ml.
[View Larger Version of this Image (86K GIF file)]


Fig. 2. Time course of endothelial cell-mediated clot retraction. Fg gamma Agamma A, peak 1 fibrinogen; Fr I-9, fibrinogen peak 1 fraction I-9, which lacks the Aalpha -chain 572-574 RGD sequences; Fg I-9 gamma Agamma ', fibrinogen peak 2 fraction I-9, which lacks one gamma A-chain carboxyl-terminal platelet binding region as well as the same Aalpha -chain sequences as fraction I-9. All fibrinogens were at 250 µg/ml concentration, and thrombin concentration was 1 unit/ml. n = 1.
[View Larger Version of this Image (12K GIF file)]

The mAb 4A5 was used to determine if endothelial cell-mediated clot retraction is dependent on the carboxyl terminus of the fibrinogen gamma A-chain. This mAb antibody was used because it binds to the carboxyl terminus of the fibrinogen gamma A-chain (19) and thereby inhibits cross-linking of fibrinogen by Factor XIIIa (19) and the adhesion of platelets to immobilized fibrinogen (6). As shown in Fig. 3, the mAb 4A5 inhibited the retraction of clots formed from both peak 1 fibrinogen and fibrinogen fraction I-9 compared with mIgG controls. This inhibition of endothelial cell-mediated clot retraction by the mAb 4A5 indicated either a requirement for the gamma A-chain carboxyl terminus or an indirect inhibitory effect on clot retraction by the antibody. To distinguish between these alternatives, recombinant fibrinogen containing normal Aalpha - and Bbeta -chains, but lacking residues gamma A408-411 (9) was used in the clot retraction assay. Recombinant normal fibrinogen was used as a control. The recombinant normal fibrinogen supported the adhesion of normal and resting platelets (data not shown) and clot retraction in HUVECs (Fig. 1). Likewise, recombinant fibrinogen gamma 407, though it did not support the adhesion of resting platelets (data not shown), supported clot retraction mediated by HUVECs without any apparent impairment of function (Figs. 1 and 4).


Fig. 3. Inhibition of clot retraction by mAb 4A5. Bars represent the percentage of control retraction (fibrinogen/mIgG) for fibrinogen 4A5 and fibrinogen fraction I-9 4A5 after 7.5 h. gamma Agamma AFg/mIgG, peak 1 fibrinogen; Fr I-9, fibrinogen peak 1 fraction I-9, which lacks the Aalpha -chain 572-574 RGD sequences. mAb 4A5 binds the carboxyl-terminal of the fibrinogen gamma A-chain. All fibrinogens were at 250 µg/ml concentration, thrombin was at 1 unit/ml, and 4A5 was 50 µg/ml. Data are presented as the mean ± standard error. Fg, fibrinogen; Fr, fraction. n = 3.
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Fig. 4. Endothelial cell-mediated clot retraction with recombinant fibrinogen and recombinant gamma 407 fibrinogen that lacks residues gamma 408-411. gamma Agamma A Fg, peak 1 fibrinogen (n = 3); Fr I-9, fibrinogen peak 1 fraction I-9 (n = 3), which lacks the Aalpha -chain 572-574 RGD sequences; rFg, recombinant normal fibrinogen (n = 2); rFg407, recombinant fibrinogen missing the gamma A-chain carboxyl-terminal 408-411 AGDV sequences (n = 2). All fibrinogens were at 250 µg/ml concentration, and thrombin was 1 unit/ml. Data represent retraction 5 h after the addition of thrombin. The data is presented as the mean ± standard deviation.
[View Larger Version of this Image (49K GIF file)]

Fibrinogen 325, which lacks the first 42 amino acid residues of the Bbeta -chains was also used in the clot retraction assay. It has been shown that fibrin prepared from fibrinogen molecules lacking residues 1-42 of the Bbeta -chains failed to support endothelial cell spreading (36, 37). Fibrinogen 325 in this study supported HUVEC clot retraction (data not shown).

Two peptides that inhibit platelet-mediated clot retraction were tested in the HUVEC system (10). Although gamma A-chain carboxyl-terminal mimetic peptide L10 (LGGAKQAGDV) inhibited platelet-mediated clot retraction (data not shown), it did not inhibit retraction in the HUVEC system at the concentrations up to 6 mM (Fig. 5). In contrast, the RGD peptide GRGDSP, but not a scrambled control peptide (PGRSGD), inhibited clot retraction mediated by platelets (10) (data not shown) and endothelial cells (Fig. 5).


Fig. 5. Peptide dose response of endothelial cell-mediated clot retraction. L10, LGGAKQAGDV mimetic of the fibrinogen gamma A-chain platelet binding region. GRGDSP contains the RGD sequence that mimics the fibrinogen 572-574 Aalpha -chain binding region. PGRSGD is a scrambled control version of GRGDSP. Inhibition after 4 h is shown for endothelial cells. approx 170,000 cells, 250 µg/ml fibrinogen, and 1 unit/ml thrombin per tube were used.
[View Larger Version of this Image (30K GIF file)]

Anti-integrin mAbs also provided useful information concerning the details of endothelial cell-mediated clot retraction. First, the alpha vbeta 3-specific mAb LM609 partially inhibited clot retraction (Fig. 6A). The alpha v-specific mAb CLB-706 also inhibited clot retraction (Fig. 6A). These results confirm observations made by others (3, 4) indicating a role for the alpha vbeta 3 integrin in clot retraction mediated by a variety of nucleated cells. Similarly, the mAb 7E3, but not A2A9, inhibited clot retraction in HUVECs (Fig. 6B). In contrast, both of those mAbs inhibited platelet-mediated clot retraction, presumably reflecting a role for alpha IIbbeta 3 in the process (Fig. 6B). These data demonstrate that clot retraction mediated by HUVECs is alpha vbeta 3-dependent since LM609, CLB-706, and 7E3 but not A2A9 or JB1a (which blocks the function of beta 1 integrin subunits) can inhibit alpha vbeta 3-dependent functions. Finally, the AP3 mAb, which binds to the beta 3 integrin subunit and can inhibit platelet-mediated clot retraction (10, 28), also partially inhibited (at a concentration of 100 µg/ml) clot retraction in the HUVEC system (Fig. 6A). The alpha IIbbeta 3-specific mAb Tab, which also inhibits platelet-mediated clot retraction (10), did not inhibit endothelial cell-mediated clot retraction (50 µg/ml) compared with a mouse IgG control (data not shown).


Fig. 6. Antibody inhibition of platelet and endothelial cell-mediated clot retraction. A, LM609 (50 µg/ml) binds to the alpha vbeta 3 receptor, CLB-706 (50 µg/ml) binds to alpha v, AP3 (100 µg/ml) binds the beta 3 subunit, and JB1a (1:10 dilution from ascites fluid) binds the beta 1 subunit (n = 1). B, 7E3 (100 µg/ml) binds both alpha vbeta 3 and alpha IIbbeta 3 integrins, and A2A9 (50 µg/ml) is alpha IIbbeta 3-specific. mIgG, a mouse control IgG, concentration is 50 µg/ml. Data represent retraction 4 h after the addition of 1 unit/ml thrombin. The data are presented as the mean ± standard deviation. (n = 3)
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DISCUSSION

Studies by Cheresh et al. (5) and others (16) provide evidence implicating the fibrinogen Aalpha -chain RGD 572-574 sequence as a putative endothelial cell binding site recognized by alpha vbeta 3 (22). These studies also demonstrated that the fibrinogen Aalpha -chain 95-97 RGD residues do not play a significant role in HUVEC adhesion to fibrinogen (5, 16). mAb data indicated that endothelial cell adhesion to this region of immobilized fibrinogen was mediated by the integrin alpha vbeta 3 (5, 22). Consequently, the possibility was tested that the fibrinogen Aalpha -chain 572-574 sequence is required to support alpha vbeta 3-mediated clot retraction by HUVECs. This possibility was tested by using fibrinogen fragment fraction I-9 in an endothelial cell-supported clot retraction assay. Surprisingly, the fibrinogen fraction I-9 (Table I) supported apparently normal clot retraction. These results mean that the Aalpha -chain RGD (572-574) sequence is not required for clot retraction mediated by HUVECs.

The finding that HUVECs-mediated clot retraction is not dependent on the fibrinogen Aalpha -chain RGD 572-574 sequence resulted in the evaluation of the role of the fibrinogen gamma A-chain carboxyl-terminal sequences in the endothelial cell clot retraction system. Four approaches were used for this aspect of the study. Proteolytic fragments of fibrinogen, an anti-fibrinogen mAb, recombinant fibrinogens, and peptides were used in these studies. Peak 2 fibrinogen fraction I-9 (Table I) was found to support HUVEC-mediated clot retraction normally (Fig. 1), demonstrating that if a fibrinogen gamma -chain carboxyl terminus is required, a single functional gamma A-chain carboxyl terminus is sufficient even in the absence of the Aalpha -chain 572-574 RGD sequences. The mAb 4A5 results reflected either a requirement for the gamma A-chain carboxyl-terminal platelet binding site or indirect inhibitory effects caused by 4A5 (Fig. 3). Use of recombinant human fibrinogen, which does not support platelet aggregation (9) or the adhesion of resting platelets (data not shown), supported normal clot retraction in the endothelial cell clot retraction system (Fig. 4). These results demonstrate that the inhibition of clot retraction by mAb 4A5 apparently resulted from indirect effects (steric hindrance or the induction of an incompatible conformational change of the fibrinogen), since clot retraction was not dependent on the presence of the AGDV sequence of the fibrinogen gamma A-chain. The gamma A-chain carboxyl-terminal mimetic peptide L10 has been shown to inhibit platelet-supported clot retraction (10) but did not inhibit clot retraction at concentrations up to 6 mM mediated by endothelial cells in our assays (Fig. 5). The data from the proteolytic fragments of fibrinogen, an anti-fibrinogen mAb, recombinant fibrinogens, and peptides demonstrate that HUVEC-mediated clot retraction is not dependent on the 408-411 AGDV sequence of the fibrinogen gamma A-chain. Thus, the fibrinogen cell binding sites required to support endothelial cell-mediated clot retraction are different than those required to support platelet aggregation (8, 9) and adhesion (6, 7, 16, 17).

The amino-terminal region of the fibrinogen Bbeta -chain is a presumptive endothelial cell interaction site. Since this region of the Bbeta -chain appears to affect the interaction of HUVECs with fibrin, a protease-treated form of fibrinogen that lacks the first 42 amino acid residues of the Bbeta -chains (fibrinogen 325) (28, 29) was tested and shown to support clot retraction (data not shown). Even though this region has been shown to interact with HUVECs, it is not necessary to support HUVECs clot retraction. The amino-terminal 42 residues of the Bbeta -chains are therefore not required to support clot retraction mediated by HUVECs.

The results of this study reveal that HUVECs can mediate clot retraction in a manner that is not dependent on the Aalpha -chain 572-574 RGD sequences, the 408-411 AGDV sequence of the gamma A-chain carboxyl-terminal platelet binding sites, or residues 1-42 of the fibrinogen Bbeta -chains. In summary, the data mean that (a) either of these fibrinogen cell binding sites may be sufficient to support clot retraction mediated by HUVECs or that (b) another region(s) of fibrinogen alone or in conjunction with either of the above mentioned fibrinogen platelet or nucleated cell binding sites can be utilized by endothelial cells to support clot retraction.

The data presented here for HUVECs agree with those reported by others for the role of the fibrinogen gamma A-chain in clot retraction mediated by platelets (9, 18). Rooney et al. (9) report that the alpha IIbbeta 3 binding sites on fibrinogen that mediate platelet aggregation appear to differ from the alpha IIbbeta 3 binding site(s) on fibrin that are used during clot retraction (9). Their conclusion that the gamma A-chain 408-411 AGDV sequence is not required for clot retraction was supported by the results of Holmback et al. (18) obtained using homozygous mutant mice. The results presented here extend the observation that the AGDV sequence is not required to support clot retraction to a new system, the endothelial cell clot retraction system. Additionally, these results demonstrate that clot retraction in this system is not dependent on either the Aalpha -chain 572-574 RGD sequence or the Bbeta -chain 1-42 residues.

A role for the alpha vbeta 3 integrin in endothelial cell-mediated clot retraction is supported by the facts that the alpha vbeta 3-specific mAb LM609, the alpha v-specific mAb CLB-706, and the mAb 7E3, which blocks both alpha vbeta 3 and alpha IIbbeta 3, inhibited endothelial cell-mediated clot retraction, whereas the alpha IIbbeta 3-specific A2A9 and the anti-beta 1 function blocking mAb, JB1a, did not inhibit clot retraction in this system. The fact that the mAb AP3 partially inhibited clot retraction may indicate a role for residues within the beta 3 348-426 sequence, which encompass the AP3 binding site, in clot retraction mediated by endothelial cells as well as platelets, since AP3 has also been shown to inhibit platelet-mediated clot retraction (10, 38). Alternatively, the inhibitory effect of AP3 may be indirect.

Finally, the observations that sites other than the gamma A-chain 408-411 AGDV sequences and the Aalpha -chain 572-574 sequences appear to be required for both platelet (fibrinogen fraction I-9 supports platelet-mediated clot retraction, data not shown) and HUVEC-mediated clot retraction may mean that receptor sites other than those used for platelet aggregation and platelet and endothelial cell adhesion are used to bind fibrin during clot retraction mediated by either platelets or HUVECs.


FOOTNOTES

*   This work was partially funded by National Institutes of Health Grant HL56369.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed: University of Tennessee-Campbell Clinic, Dept. of Orthopaedic Surgery, 956 Court Ave., Room A302, Memphis, TN 38163. Tel.: 901-448-5880; Fax: 901-448-6062; E-mail: rsmith{at}utmem1.utmem.edu.
1   The abbreviations used are: mAb, monoclonal antibody; mIgG, mouse IgG; HUVEC, human umbilical vein endothelial cell.

ACKNOWLEDGEMENTS

We thank Jason Holcomb, Stephanie Long, Tursha Hamilton, and Robert Loudon for their invaluable technical assistance with this project.


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