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(Circulation. 2001;104:582.)
© 2001 American Heart Association, Inc.

Basic Science Reports

Eptifibatide and 7E3, but Not Tirofiban, Inhibit _vß₃ Integrin–Mediated Binding of Smooth Muscle Cells to Thrombospondin and Prothrombin

Manjiri Lele, MD; Mansoor Sajid, MD, PhD; Nadeem Wajih, PhD; George A. Stouffer, MD

From the Program in Molecular Cardiology, University of North Carolina, Chapel Hill.

Correspondence to George A. Stouffer, MD, 324 Burnett-Womack Building, CB 7075, University of North Carolina, Chapel Hill, NC 27599-7075. E-mail rstouff{at}med.unc.edu

	Abstract

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Background— Our objective was to determine whether abciximab, eptifibatide, or tirofiban inhibited ligand binding to _vß₃ integrins on human aortic smooth muscle cells (HASMCs) or human umbilical vein endothelial cells (HUVECs). Abciximab binds _IIbß₃ on platelets and _vß₃ on HUVECs with similar affinity, whereas eptifibatide and tirofiban are thought to be highly specific for _IIbß₃. The conclusion that eptifibatide does not bind vascular _vß₃ integrins may be premature, however, because recent studies have demonstrated that the affinity of _vß₃ for various ligands, including antagonists, is subject to modulation.

Methods and Results— Abciximab and 7E3, the anti–ß₃ integrin monoclonal antibody from which abciximab was derived, bound _vß₃ on HASMCs in a specific and saturable manner and with an affinity similar to binding to _IIbß₃ on platelets. 7E3 and eptifibatide inhibited _vß₃-mediated attachment of HASMCs to thrombospondin (TSP) and prothrombin but had no effect on _vß₅- or ß₁-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 _vß₃-mediated attachment of HUVECs. Tirofiban had only nonspecific effects on HASMC attachment to extracellular matrix proteins. In cell proliferation assays, eptifibatide inhibited _vß₃-mediated responses to soluble TSP by HASMCs and ß₃ integrin–expressing HEK cells.

Conclusions— Eptifibatide and 7E3, but not tirofiban, specifically inhibit _vß₃-mediated binding of human smooth muscle and endothelial cells.

Key Words: receptors • vitronectin • thrombin • angioplasty • muscle, smooth

	Introduction

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The ß₃ integrins play an important role in vascular disease. _IIbß₃ integrins are critically important in platelet aggregation and thrombus formation, whereas _vß₃ integrins have been implicated in mediating vascular repair. Vascular injury is a potent stimulus for expression of ß₃ integrins,^1,2 and treatment with _vß₃ 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 iliac^7,8 arteries.

The central role of _IIbß₃ in thrombosis led to the development of pharmaceutical agents that block interactions between _IIbß₃ and fibrinogen. All 3 currently available agents (tirofiban, abciximab, and eptifibatide) exhibit high-affinity binding to _IIbß₃, inhibit ex vivo platelet aggregation, and reduce clinical thrombotic events.⁹ Abciximab is an antigen-binding fragment (c7E3) derived from monoclonal anti–ß₃ integrin antibody 7E3 that binds _IIbß₃ and _vß₃ with equal affinity.¹⁰ In contrast, both eptifibatide and tirofiban are small-molecule inhibitors that are thought to be highly specific for _IIbß₃. The conclusion that eptifibatide does not interact with _vß₃ is based on studies showing that eptifibatide did not inhibit vitronectin binding to purified _vß₃ at concentrations 50-fold higher than the IC₅₀ for inhibition of fibrinogen binding to _IIbß₃.¹¹ Recent work has shown that _vß₃ binding is a dynamic process with (1) activation-dependent and activation-independent ligands, (2) variable basal affinity state of _vß₃ among different cell types, and (3) acute modulation of the affinity of _vß₃ for ligands by "inside-out" signals.^12–14 Thus, the finding that eptifibatide did not inhibit binding of vitronectin to purified _vß₃ in a cell-free system does not necessarily exclude all interactions between eptifibatide and _vß₃ 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 _vß₃ 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 _vß₃ integrins.^1,17 Because the efficacy of _vß₃ 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.

	Methods

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Reagents
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-ß 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 1.60x10^-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 ß₃ Integrin–Expressing HEK Cells
pcDNA-1neo constructs encoding full-length ß₃ subunits were a gift of D. Cheresh (Scripps Research Institute, La Jolla, Calif) and have been described previously.¹⁸ ß₃ 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.

Statistical Analysis
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 P0.05 was considered statistically significant.

	Results

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Abciximab Binds HASMCs in a Specific and Saturable Manner
Saturation binding experiments revealed that the affinity of abciximab (c7E3) for HASMCs (K_d=6.9±2.8 nmol/L; Figure 1A) was similar to that for HUVECs, platelets, and purified _vß₃.¹⁰ The affinity of c7E3 for HASMCs was less than that of 7E3 (K_d=3.8±0.4 nmol/L), but at concentrations >10 µg/mL, 7E3 and c7E3 had similar effects in competing for binding to _vß₃ on HASMCs (Figure 1B). The binding affinities of c7E3 and 7E3 were both significantly less than that of LM609 (K_d=0.18±0.01 nmol/L; Figure 1A), a monoclonal antibody that is highly specific for _vß₃. 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

View larger version (19K): [in this window] [in a new window]   Figure 1. Comparison of binding of monoclonal antibody 7E3 and antigen-binding fragment c7E3 to HASMCs. A, Ability of 125I-7E3, 125I-LM609, and 125I-c7E3 to bind to HASMCs was determined by saturation binding. Specific binding was defined as total binding minus nonspecific binding determined by a 100-fold excess of unlabeled 7E3 or c7E3. B, Competition of 7E3, c7E3, and LM609 with 125I-7E3 for binding to HASMCs. HASMCs were incubated with 0.2 µg/mL 125I-7E3 in presence of increasing concentrations of unlabeled competitor. Data points represent mean±SEM of triplicate determinations from representative experiment performed twice. C, HASMCs were incubated with 7E3, 10E5, LM609, or mouse IgG. Flow cytometry was performed with primary antibody binding to HASMCs detected by FITC–labeled secondary antibody.

ß₃ Subunits Form Heterodimers Primarily, if Not Solely, With _v Subunits in HASMCs
Binding of 7E3 to HASMCs was almost completely inhibited by LM609 (Figure 1B). The monoclonal antibody 10E5, which binds _IIbß₃, 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 _IIbß₃ expressed on human platelets (data not shown). These results demonstrate that ß₃ subunits form heterodimers primarily, if not solely, with _v 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 _vß₃ 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.

View larger version (19K): [in this window] [in a new window]   Figure 2. Effects of 7E3, eptifibatide, and tirofiban on HASMC adhesion to immobilized TSP. HASMCs in suspension were incubated with eptifibatide (A, various concentrations; B and C, 30 µmol/L), tirofiban (A, various concentrations; B, 30 µmol/L), echistatin (100 nmol/L), GRGES (30 µmol/L), 7E3 (various concentrations), or 10E5 (100 µg/mL) and then added to TSP-coated plates. *P<0.05 vs control.

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-_IIbß₃ antibody 10E5, at a dose 15-fold greater than that necessary to block attachment of genetically manipulated CHO cells expressing _IIbß₃ to fibrinogen,²⁰ had no effect on HASMC attachment to TSP.

Inhibitory Effects of 7E3 and Eptifibatide, but Not Tirofiban, Were Specific for _vß₃ 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 _vß₃-mediated¹³ and _vß₅-mediated^21,22 attachment of HASMCs, respectively, whereas attachment to collagen- and fibronectin-coated and noncoated plates is mediated by various ß₁ 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.

View larger version (49K): [in this window] [in a new window]   Figure 3. Effects of 7E3, eptifibatide, and tirofiban on HASMC adhesion to prothrombin, vitronectin (VN), collagen, and fibronectin (FN). HASMCs in suspension were incubated with eptifibatide (A, various concentrations; B, 30 µmol/L), GRGES (30 µmol/L), 7E3 (30 µg/mL), or 10E5 (30 µg/mL) and then added to (A) prothrombin-, or (B) VN-, collagen-, or FN-coated or noncoated plates. *P<0.05 vs control.

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 _vß₃ integrins.

Eptifibatide Inhibited Proliferative Responses to Soluble TSP
In previous studies,¹ we found that TSP-induced proliferation of HASMCs was mediated by _vß₃, and we used this assay to determine the functional significance of eptifibatide/_vß₃ 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.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effects of 7E3, eptifibatide, and tirofiban on proliferative responses of growth-arrested HASMCs. HASMCs were grown on noncoated tissue culture plates until subconfluence and then growth-arrested in 0.5% FBS for 4 days. Soluble TSP (25 µg/mL), PDGF-BB (20 ng/mL), 10% serum, or vehicle was added ± eptifibatide (30 µmol/L), tirofiban (30 µmol/L), GRGES (30 µmol/L), 7E3 (30 µg/mL), or 10E5 (30 µg/mL). Cell number assays were performed 72 hours later. Proliferative responses to TSP, PDGF-BB, and FBS were 125%, 233%, and 259% of vehicle-treated controls, respectively. Data are percentage of proliferative response elicited by various agents in absence of any peptides. *P<0.05 vs control.

Antiproliferative Effect of Eptifibatide Was Mediated by ß₃ Integrins
To determine whether the antiproliferative effect of eptifibatide was due to specific interactions with ß₃ integrins, we used human embryonic kidney (HEK) cells that express _v subunits but not ß₃ subunits. These cells were transfected with pcDNA-1neo constructs¹⁸ encoding ß₃ integrin subunits. Stable expression of ß₃ integrins and formation of _vß₃ complexes was confirmed by immunoprecipitation with LM609 followed by Western analysis (Figure 5A).

View larger version (26K): [in this window] [in a new window]   Figure 5. Effects of eptifibatide on proliferative responses of HEK cells. ß3 integrin–deficient HEK cells were transfected with an empty vector or pcDNA-1neo constructs encoding full-length ß3 integrin subunits. Immunoprecipitation using LM609 followed by Western analysis with an anti–ß3 integrin antibody confirmed expression (A). Cells were grown in serum-containing medium for 5 days and then growth-arrested in 0.5% FBS for 4 days. Soluble TSP (25 µg/mL) or vehicle was added ± eptifibatide (30 µmol/L) or GRGES (30 µmol/L). Cell number assays were performed 72 hours later. All groups were compared with mock-transfected cells that were not exposed to TSP (B). *P<0.05 vs control.

HEK cells that expressed ß₃ 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, ß₃-transfected, but not mock-transfected, HEK cells proliferated in response to TSP. The proliferative response to TSP in ß₃-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 _vß₃,^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).

View larger version (36K): [in this window] [in a new window]   Figure 6. Effects of eptifibatide and tirofiban on HUVEC adhesion to immobilized TSP. HUVECs in suspension were incubated with eptifibatide (various concentrations), GRGES (30 µmol/L), tirofiban (30 µmol/L), 7E3 (30 µg/mL), or 10E5 (30 µg/mL) and then added to TSP-coated plates. *P<0.05 vs control.

	Discussion

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Our results demonstrate that eptifibatide can inhibit ligand binding to _vß₃ under specific conditions. This conclusion is based on findings that (1) eptifibatide inhibited HASMC and HUVEC attachment to TSP, events mediated by _vß₃¹⁵; (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 _vß₃; and (4) eptifibatide inhibited proliferative responses of HASMCs and ß₃ integrin–expressing HEK cells to soluble TSP.

The inhibitory effects of eptifibatide on HASMC adhesion and proliferation were specific for _vß₃. 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 _vß₅²¹ or ß₁ integrins.²³ Furthermore, the inhibitory effect of eptifibatide on HASMC attachment to TSP and prothrombin cannot be attributed to eptifibatide binding to _IIbß₃, for 3 reasons. First, Western analysis and flow cytometry confirmed that HASMCs do not express _IIb subunits. Second, attachment to TSP or prothrombin was not inhibited by high concentrations of a function-blocking anti-_IIbß₃ 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 _vß₃ and not _IIbß₃.

Antagonism of _vß₃ integrins may have important antithrombotic and anti-inflammatory effects in patients with acute coronary syndromes and/or undergoing percutaneous coronary intervention. _vß₃ is a receptor for soluble prothrombin,¹³ and combined blockade of _vß₃ and _IIbß₃ was significantly more effective at reducing platelet-mediated thrombin generation than blockade of either _vß₃ or _IIbß₃ 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 _vß₃ antagonists. There is also evidence that _vß₃ 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 _vß₃ and _IIbß₃ in vitro¹⁰ and also possibly between platelets and _vß₃ 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 _vß₃ 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 _vß₃ receptors.

Our results provide further evidence that _vß₃ integrins exist in multiple conformations and are subject to modulation. Scarborough et al¹¹ found that eptifibatide did not block vitronectin binding to purified _vß₃, whereas we found that eptifibatide blocked TSP binding to _vß₃ on HASMCs and HUVECs, and Pampori et al¹⁴ reported that low concentrations of eptifibatide (1 µmol/L) partially blocked binding of ß₃ integrin ligands to genetically manipulated CHO cells expressing _vß₃. _vß₃ 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 (K_d=6.9±2.8 nmol/L) similar to that for binding to HUVECs (K_d=9.8±2.7 nmol/L), human coronary SMCs (K_d=14±8.4 nmol/L), purified _vß₃ (K_d=11.0±3.2 nmol/L), and _IIbß₃ (K_d=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 (K_d=0.18±0.01 nmol/L), a monoclonal antibody that is highly specific for _vß₃. 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 _vß₃, 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 _vß₃, 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 _vß₃. 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 _vß₃ 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.

Received January 26, 2001; revision received April 6, 2001; accepted April 9, 2001.

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