Eptifibatide and 7E3, but Not Tirofiban, Inhibit αvβ3 Integrin–Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin
Background Our objective was to determine whether abciximab, eptifibatide, or tirofiban inhibited ligand binding to αvβ3 integrins on human aortic smooth muscle cells (HASMCs) or human umbilical vein endothelial cells (HUVECs). Abciximab binds αIIbβ3 on platelets and αvβ3 on HUVECs with similar affinity, whereas eptifibatide and tirofiban are thought to be highly specific for αIIbβ3. The conclusion that eptifibatide does not bind vascular αvβ3 integrins may be premature, however, because recent studies have demonstrated that the affinity of αvβ3 for various ligands, including antagonists, is subject to modulation.
Methods and Results Abciximab and 7E3, the anti–β3 integrin monoclonal antibody from which abciximab was derived, bound αvβ3 on HASMCs in a specific and saturable manner and with an affinity similar to binding to αIIbβ3 on platelets. 7E3 and eptifibatide inhibited αvβ3-mediated attachment of HASMCs to thrombospondin (TSP) and prothrombin but had no effect on αvβ5- or β1-mediated HASMC attachment to vitronectin-, collagen-, or fibronectin-coated or noncoated tissue culture plates. The inhibitory effect of eptifibatide was similar in magnitude and not additive to that of 7E3. Eptifibatide and 7E3 inhibited αvβ3-mediated attachment of HUVECs. Tirofiban had only nonspecific effects on HASMC attachment to extracellular matrix proteins. In cell proliferation assays, eptifibatide inhibited αvβ3-mediated responses to soluble TSP by HASMCs and β3 integrin–expressing HEK cells.
Conclusions Eptifibatide and 7E3, but not tirofiban, specifically inhibit αvβ3-mediated binding of human smooth muscle and endothelial cells.
Received January 26, 2001; revision received April 6, 2001; accepted April 9, 2001.
The β3 integrins play an important role in vascular disease. αIIbβ3 integrins are critically important in platelet aggregation and thrombus formation, whereas αvβ3 integrins have been implicated in mediating vascular repair. Vascular injury is a potent stimulus for expression of β3 integrins,1,2 and treatment with αvβ3 antagonists reduces (neo)intima formation after injury of rat carotid,3 rabbit carotid,4 hamster carotid,5 pig coronary,2 pig carotid and femoral,6 and rabbit iliac7,8 arteries.
The central role of αIIbβ3 in thrombosis led to the development of pharmaceutical agents that block interactions between αIIbβ3 and fibrinogen. All 3 currently available agents (tirofiban, abciximab, and eptifibatide) exhibit high-affinity binding to αIIbβ3, inhibit ex vivo platelet aggregation, and reduce clinical thrombotic events.9 Abciximab is an antigen-binding fragment (c7E3) derived from monoclonal anti–β3 integrin antibody 7E3 that binds αIIbβ3 and αvβ3 with equal affinity.10 In contrast, both eptifibatide and tirofiban are small-molecule inhibitors that are thought to be highly specific for αIIbβ3. The conclusion that eptifibatide does not interact with αvβ3 is based on studies showing that eptifibatide did not inhibit vitronectin binding to purified αvβ3 at concentrations 50-fold higher than the IC50 for inhibition of fibrinogen binding to αIIbβ3.11 Recent work has shown that αvβ3 binding is a dynamic process with (1) activation-dependent and activation-independent ligands, (2) variable basal affinity state of αvβ3 among different cell types, and (3) acute modulation of the affinity of αvβ3 for ligands by “inside-out” signals.12–14 Thus, the finding that eptifibatide did not inhibit binding of vitronectin to purified αvβ3 in a cell-free system does not necessarily exclude all interactions between eptifibatide and αvβ3 on the surface of vascular cells.
Acute vascular injury leads to the concentration of numerous extracellular matrix proteins in the vessel wall. Among these proteins are thrombospondin 1 (TSP), which binds endothelial cells and smooth muscle cells (SMCs) via αvβ3 integrins.15 We have been particularly interested in TSP because of recent evidence that (1) treatment with an anti-TSP antibody inhibited SMC proliferation and reduced neointimal formation after carotid injury in the rat,16 and (2) TSP-induced proliferation of SMCs is mediated by interactions with αvβ3 integrins.1,17 Because the efficacy of αvβ3 antagonists varies depending on the cell type and ligand studied, the aim of the present studies was to examine the effects of 7E3, eptifibatide, and tirofiban on vascular cell interactions with TSP.
Vitronectin, collagen, fibronectin, prothrombin, echistatin, and GRGES peptides were obtained from Sigma Chemical Co. TSP was purified from recently outdated human platelets by a modification of the method described by Santoro and Frazier.1 The level of transforming growth factor-β in TSP preparations was <0.15 ng/mL. Eptifibatide was obtained from Cor Therapeutics and 7E3, c7E3, and 10E5 from Centocor.
Cell Culture, Proliferation Assays, and Flow Cytometric Analysis
Human aortic SMCs (HASMCs) were obtained from Clonetics and maintained in culture as previously described.1 Ca2+ was present at a concentration of 1.60×10−3 mol/L in the medium. Human umbilical vein endothelial cells (HUVECs) were a gift from C. Patterson, MD (University of North Carolina, Chapel Hill) and were cultured by standard techniques. Fluorescence-activated cell sorter analysis was performed as described by Pampori et al.14
Cell Adhesion Assay
Twenty-four–well plates were coated and then blocked with 3% BSA. HASMCs or HUVECs in suspension were treated with peptides or antibodies as indicated and then added to plates and incubated for 60 minutes at 37°C. Nonadherent cells were removed by washing, and PBS containing 0.5% crystal violet was added. The cells were then washed 3 times in PBS and visualized by light microscopy. Cell binding was quantified by measurement of optical density. Adhesion in the absence of any inhibitors was assigned the value of 100%.
Receptor Binding Studies
These studies were performed as previously described.1 Briefly, c7E3 or 7E3 was radiolabeled and diluted in growth-arrest medium containing 0.02% azide to prevent internalization. HASMCs were incubated with antibodies for 4 hours at 37°C, and bound radioactivity was quantified with a gamma counter. For competition binding, the ability of unlabeled antibodies to compete with 0.2 μg/mL 125I-labeled 7E3 binding was determined.
Transfection and Selection of Stable β3 Integrin–Expressing HEK Cells
pcDNA-1neo constructs encoding full-length β3 subunits were a gift of D. Cheresh (Scripps Research Institute, La Jolla, Calif) and have been described previously.18 β3 integrin–deficient HEK 293 cells (ATCC) were cultured in DMEM supplemented with 10% heat-inactivated horse serum. When the cells were semiconfluent, the medium was changed to DMEM without serum, and transfection was performed with the FuGENE Transfection Reagent (Boehringer Mannheim). Six hours later, the medium was changed to DMEM with 10% serum for 72 hours. G418 (500 μg/mL) was then added until stable cell lines were established.
Results are presented as mean±SEM from ≥3 independent experiments. The data were analyzed by ANOVA followed by the Tukey multiple range teSt. A value of P≤0.05 was considered statistically significant.
Abciximab Binds HASMCs in a Specific and Saturable Manner
Saturation binding experiments revealed that the affinity of abciximab (c7E3) for HASMCs (Kd=6.9±2.8 nmol/L; Figure 1A) was similar to that for HUVECs, platelets, and purified αvβ3.10 The affinity of c7E3 for HASMCs was less than that of 7E3 (Kd=3.8±0.4 nmol/L), but at concentrations >10 μg/mL, 7E3 and c7E3 had similar effects in competing for binding to αvβ3 on HASMCs (Figure 1B). The binding affinities of c7E3 and 7E3 were both significantly less than that of LM609 (Kd=0.18±0.01 nmol/L; Figure 1A), a monoclonal antibody that is highly specific for αvβ3. There were 132 000±28 000 binding sites for c7E3 and 91 000±6000 binding sites for 7E3 per HASMC, consistent with the monovalent binding of abciximab and bivalent binding of 7E3 observed previously for platelets and HUVECs.10
β3 Subunits Form Heterodimers Primarily, if Not Solely, With αv Subunits in HASMCs
Binding of 7E3 to HASMCs was almost completely inhibited by LM609 (Figure 1B). The monoclonal antibody 10E5, which binds αIIbβ3, had no effect on 7E3 binding. The fluorescence patterns on flow cytometry observed with LM609 and 7E3 were nearly identical (Figure 1C). Minimal staining was observed with 10E5 (Figure 1C) or with HIP8, a monoclonal IgG1 that reacts with the calcium-dependent complex of αIIbβ3 expressed on human platelets (data not shown). These results demonstrate that β3 subunits form heterodimers primarily, if not solely, with αv subunits in HASMCs.
Eptifibatide and Tirofiban Inhibited HASMC Adhesion to TSP
Eptifibatide and tirofiban inhibited attachment of HASMCs to TSP-coated plates in a dose-dependent manner (Figure 2A). At a concentration of 30 μmol/L, eptifibatide inhibited attachment by 37%. Tirofiban was ≈1 order of magnitude less potent than eptifibatide in this assay. Echistatin, a disintegrin found in the venom of Echis carinatus that binds αvβ3 on SMCs with high specificity,19 inhibited HASMC attachment to TSP with a potency ≈100-fold and 1000-fold greater than those of eptifibatide and tirofiban, respectively (Figure 2B). Treatment with GRGES peptides had no effect on HASMC attachment.
Incubation with 7E3 (30 μg/mL) inhibited HASMC attachment to TSP by 31% (P=NS, eptifibatide 30 μmol/L versus 7E3). Higher concentrations of 7E3 had the same inhibitory effect as 30 μg/mL (P=NS, 7E3 30 μg/mL versus 7E3 100 μg/mL), and thus this concentration was used for the remainder of our studies (Figure 2C). Combined treatment with eptifibatide and 7E3 had the same inhibitory effect as eptifibatide or 7E3 alone. Treatment with the monoclonal anti-αIIbβ3 antibody 10E5, at a dose 15-fold greater than that necessary to block attachment of genetically manipulated CHO cells expressing αIIbβ3 to fibrinogen,20 had no effect on HASMC attachment to TSP.
Inhibitory Effects of 7E3 and Eptifibatide, but Not Tirofiban, Were Specific for αvβ3 Integrins
HASMCs attach to various matrix proteins via different integrins, and we examined adhesion to prothrombin-, vitronectin-, collagen-, and fibronectin-coated and noncoated plates to determine the specificity of the inhibitory effects of 7E3, eptifibatide, and tirofiban. Prothrombin and vitronectin support αvβ3-mediated13 and αvβ5-mediated21,22 attachment of HASMCs, respectively, whereas attachment to collagen- and fibronectin-coated and noncoated plates is mediated by various β1 integrins.23
Eptifibatide inhibited HASMC attachment to prothrombin in a dose-dependent manner (Figure 3A). At a concentration of 30 μmol/L, eptifibatide inhibited HASMC attachment to prothrombin by 38%, an effect similar in magnitude to that observed with 7E3. 10E5 had a small, statistically nonsignificant effect on attachment to prothrombin.
Neither 7E3 nor eptifibatide inhibited HASMC attachment to vitronectin-coated plates (Figure 3B). These results are consistent with previous studies of human aortic21 and human iliac22 SMCs in which 7E3 at concentrations of 20 and 60 μg/mL had no effect on adhesion to vitronectin. Similarly, neither 7E3 nor eptifibatide had any effect on HASMC attachment to collagen- or fibronectin-coated or noncoated tissue culture plates (Figure 3B).
Tirofiban at a concentration of 30 μmol/L inhibited HASMC attachment to TSP-, prothrombin-, collagen-, vitronectin-, and fibronectin-coated and noncoated plates by 5% to 20% (Figures 2 and 3⇑). The ability of tirofiban to inhibit attachment of HASMCs to vitronectin-, collagen-, and fibronectin-coated and noncoated plates demonstrates that tirofiban has inhibitory effects independent of αvβ3 integrins.
Eptifibatide Inhibited Proliferative Responses to Soluble TSP
In previous studies,1 we found that TSP-induced proliferation of HASMCs was mediated by αvβ3, and we used this assay to determine the functional significance of eptifibatide/αvβ3 interactions. For these studies, HASMCs were plated on noncoated tissue culture dishes, grown to subconfluence, and then growth-arrested. Soluble TSP (25 μg/mL) was added and stimulated a modest proliferative response (125±6% of vehicle-treated groups). Treatment with eptifibatide inhibited 84±8% of this response, similar to the effect of 7E3 (71±9%, P=NS, eptifibatide versus 7E3; Figure 4). Eptifibatide did not inhibit proliferative responses to platelet-derived growth factor BB (PDGF-BB) or 10% serum. Tirofiban and GRGES peptides had no effect on proliferative responses to TSP, PDGF-BB, or serum.
Antiproliferative Effect of Eptifibatide Was Mediated by β3 Integrins
To determine whether the antiproliferative effect of eptifibatide was due to specific interactions with β3 integrins, we used human embryonic kidney (HEK) cells that express αv subunits but not β3 subunits. These cells were transfected with pcDNA-1neo constructs18 encoding β3 integrin subunits. Stable expression of β3 integrins and formation of αvβ3 complexes was confirmed by immunoprecipitation with LM609 followed by Western analysis (Figure 5A).
HEK cells that expressed β3 subunits proliferated at a rate that was 65% greater than mock-transfected HEK cells when grown in 10% serum on standard tissue culture plates. After growth arrest, β3-transfected, but not mock-transfected, HEK cells proliferated in response to TSP. The proliferative response to TSP in β3-transfected HEK cells was 88% inhibited by eptifibatide (Figure 5B).
Eptifibatide Inhibited HUVEC Attachment to TSP and Vitronectin
Eptifibatide inhibited the attachment of HUVECs to TSP in a dose-dependent manner (Figure 6). The inhibitory effects of eptifibatide (30 μmol/L) and 7E3 were similar in magnitude (54% and 57%, respectively) and not additive. Endothelial cells, in contrast to SMCs, adhere to vitronectin via αvβ3,15,22 and we found that eptifibatide inhibited endothelial cell attachment to vitronectin by 30±9% (P<0.05 compared with control), an effect that was the same in magnitude as that observed with 7E3 (40±8%; P<0.05 compared with control; P=NS, eptifibatide versus 7E3).
Our results demonstrate that eptifibatide can inhibit ligand binding to αvβ3 under specific conditions. This conclusion is based on findings that (1) eptifibatide inhibited HASMC and HUVEC attachment to TSP, events mediated by αvβ315; (2) the inhibitory effect of eptifibatide was quantitatively similar to and not additive to that observed with 7E3; (3) eptifibatide inhibited HASMC adhesion to prothrombin and HUVEC adhesion to vitronectin, events mediated by αvβ3; and (4) eptifibatide inhibited proliferative responses of HASMCs and β3 integrin–expressing HEK cells to soluble TSP.
The inhibitory effects of eptifibatide on HASMC adhesion and proliferation were specific for αvβ3. High doses of eptifibatide did not interfere with HASMC adhesion to vitronectin, fibronectin, collagen, or standard tissue culture plates, events that are mediated primarily by αvβ521 or β1 integrins.23 Furthermore, the inhibitory effect of eptifibatide on HASMC attachment to TSP and prothrombin cannot be attributed to eptifibatide binding to αIIbβ3, for 3 reasons. First, Western analysis and flow cytometry confirmed that HASMCs do not express αIIb subunits. Second, attachment to TSP or prothrombin was not inhibited by high concentrations of a function-blocking anti-αIIbβ3 antibody (10E5). And third, the inhibitory effects of eptifibatide were similar to and not additive to those of 7E3. As shown in Figure 1, binding of 7E3 to human aortic HASMCs is mediated via αvβ3 and not αIIbβ3.
Antagonism of αvβ3 integrins may have important antithrombotic and anti-inflammatory effects in patients with acute coronary syndromes and/or undergoing percutaneous coronary intervention. αvβ3 is a receptor for soluble prothrombin,13 and combined blockade of αvβ3 and αIIbβ3 was significantly more effective at reducing platelet-mediated thrombin generation than blockade of either αvβ3 or αIIbβ3 alone.24 Furthermore, in a study25 that used plasma from patients with acute myocardial infarction, the adhesion of platelets to the luminal surface of activated HUVECs was inhibited ≈50% by various αvβ3 antagonists. There is also evidence that αvβ3 functions in adherence of leukocytes to endothelial cells and matrix proteins, fibrin clot retraction by nucleated cells, SMC migration and proliferation, vascular cell apoptosis, vascular remodeling, and intimal formation.2–8,26
An important, unresolved question is whether either abciximab or eptifibatide has effects on vascular cells in patients with unstable angina or undergoing percutaneous coronary intervention. The average steady-state plasma concentrations in patients receiving FDA-approved doses were 1.8 to 2.4 μmol/L with eptifibatide (A. True, PharmD, Cor Therapeutics, personal communication) and 0.2 to 0.4 μg/mL with abciximab.27 There was wide individual variation, however, in plasma levels. Furthermore, plasma concentrations of abciximab may not reflect concentrations in the injured artery, because the drug is largely platelet-bound, with continuous redistribution among αvβ3 and αIIbβ3 in vitro10 and also possibly between platelets and αvβ3 in the vessel wall in vivo. Thus, although we found that eptifibatide had effects on cultured vascular cells at concentrations closer to those obtained in plasma during clinical use than did abciximab, an understanding of whether abciximab or eptifibatide interacts with αvβ3 in injured blood vessels must await direct study, including determination of local concentrations of the drugs within the arterial wall and time course of expression and accessibility of αvβ3 receptors.
Our results provide further evidence that αvβ3 integrins exist in multiple conformations and are subject to modulation. Scarborough et al11 found that eptifibatide did not block vitronectin binding to purified αvβ3, whereas we found that eptifibatide blocked TSP binding to αvβ3 on HASMCs and HUVECs, and Pampori et al14 reported that low concentrations of eptifibatide (1 μmol/L) partially blocked binding of β3 integrin ligands to genetically manipulated CHO cells expressing αvβ3. αvβ3 conformation in cultured cells is regulated by PMA, Mn2+, and ADP,13 but factors that control conformational state in vivo are unknown.
We found that abciximab bound to HASMCs with an affinity (Kd=6.9±2.8 nmol/L) similar to that for binding to HUVECs (Kd=9.8±2.7 nmol/L), human coronary SMCs (Kd=14±8.4 nmol/L), purified αvβ3 (Kd=11.0±3.2 nmol/L), and αIIbβ3 (Kd=6.2±2.7 nmol/L) on platelets.10 The affinity of abciximab for HASMCs was more than an order of magnitude less than that of LM609 (Kd=0.18±0.01 nmol/L), a monoclonal antibody that is highly specific for αvβ3. c7E3 was slightly less efficient at competing for binding to HASMCs than 7E3, similar to previous results in HUVECs.10
We found no evidence that tirofiban specifically interacts with αvβ3, even at concentrations 300-fold greater than those obtained in plasma during clinical usage (≈100 nmol/L28). Tirofiban had the same inhibitory effect on HASMC attachment to vitronectin- or collagen-coated or noncoated plates, events not mediated by αvβ3, as it did on HASMC attachment to TSP or prothrombin. Moreover, tirofiban did not inhibit TSP-induced proliferation and had no effect on HUVEC attachment to TSP, events that are mediated by αvβ3. Our results are consistent with those of previous studies that found that tirofiban at a concentration of 120 μmol/L did not block HUVEC attachment to or spreading on vitronectin.22 The different affinities of eptifibatide and tirofiban for αvβ3 are not surprising, given that eptifibatide is a synthetic, cyclic peptide with a Lys-Gly-Asp (KGD) sequence, whereas tirofiban is a nonpeptide derivative of tyrosine.
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