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• W2040335095 abstract "Background: Drugs that block platelet–platelet and platelet–fibrin interactions via the αIIbβ3 (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. Objectives: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the αIIbβ3 receptor in an activated state. It compared the effects on platelet function and αIIbβ3 conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine‐glycine‐aspartate (RGD) disintegrin. Methods: The integrin antagonist concentrations required to saturate platelets and to block platelet–platelet and platelet–fibrin interactions were determined by flow cytometery, aggregometry, and clot‐based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of αIIbβ3. Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. Results: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing αIIbβ3 conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating αIIbβ3:echistatin complex. Conclusions: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the αIIbβ3 receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists. Background: Drugs that block platelet–platelet and platelet–fibrin interactions via the αIIbβ3 (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks. Objectives: This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the αIIbβ3 receptor in an activated state. It compared the effects on platelet function and αIIbβ3 conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine‐glycine‐aspartate (RGD) disintegrin. Methods: The integrin antagonist concentrations required to saturate platelets and to block platelet–platelet and platelet–fibrin interactions were determined by flow cytometery, aggregometry, and clot‐based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of αIIbβ3. Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions. Results: Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing αIIbβ3 conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating αIIbβ3:echistatin complex. Conclusions: Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the αIIbβ3 receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists. The intravascular climate balances anticoagulation to allow blood flow and hemostasis to prevent bleeding. Many disease states, such as myocardial infarction, venothrombotic events, and strokes, develop when this balance is perturbed. Life‐saving percutaneous coronary interventions, such as angioplasty with intraluminal stent placement, can stress the intravascular climate by exposing the coagulation system to prothrombotic materials, such as atherosclerotic plaque, collagen, and metal. Currently, standard clinical practice to avoid thrombosis involves anticoagulant therapy with aspirin, heparin, and clopridogel, as well as glycoprotein (GP) IIb/IIIa inhibitors that block platelet αIIbβ3 and, thereby, aggregation. Unfortunately, GPIIb/IIIa inhibitors are not without risk. Tirofiban (Aggrastat®; Merck, West Point, PA, USA) and eptifibatide (Integrilin®; Schering Corporation, Kenilworth, NJ, USA) are potent, selective αIIbβ3 inhibitors, shown to be effective at reducing mortality after percutaneous coronary interventions [1Karvouni E. Katritsis D.G. Ioannidis J.P. Intravenous glycoprotein IIb/IIIa receptor antagonists reduce mortality after percutaneous coronary interventions.J Am Coll Cardiol. 2003; 41: 26-32Crossref PubMed Scopus (0) Google Scholar, 2Kong D.F. Hasselblad V. Harrington R.A. White H.D. Tcheng J.E. Kandzari D.E. Topol E.J. Califf R.M. Meta‐analysis of survival with platelet glycoprotein IIb/IIIa antagonists for percutaneous coronary interventions.Am J Cardiol. 2003; 92: 651-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. However, they carry a 10–12% risk of minor bleeding, a 1.4–2.2% risk of major intracranial, retroperitoneal, or gastrointestinal bleeding [3Atar S. Cannon C.P. Murphy S.A. Rosanio S. Uretsky B.F. Birnbaum Y. Statins are associated with lower risk of gastrointestinal bleeding in patients with unstable coronary syndromes: analysis of the Orbofiban in Patients with Unstable coronary Syndromes‐Thrombolysis In Myocardial Infarction 16 (OPUS‐TIMI 16) trial.Am Heart J. 2006; 151: 976Crossref Scopus (37) Google Scholar], and a 1.5% risk of rapidly reversible thrombocytopenia [4The Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM‐PLUS) Study InvestigatorsInhibition of the platelet glycoprotein IIb/IIIa receptor with tirofiban in unstable angina and non‐Q‐wave myocardial infarction.N Engl J Med. 1988; 338: 1488-97Google Scholar, 5The Platelet Receptor Inhibition in Ischemic Syndrome Management (PRISM) Study Investigators.A comparison of aspirin plus tirofiban with aspirin plus heparin for unstable angina.N Engl J Med. 1998; 338: 1498-505Crossref PubMed Scopus (990) Google Scholar]. These drugs have been associated with increased prothrombotic risk if administered without the anticoagulant heparin [6Bhatt D.L. Topol E.J. Current role of platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes.JAMA. 2000; 284: 1549-58Crossref PubMed Scopus (248) Google Scholar]. They also induce neo‐antigenic sites on platelet αIIbβ3receptors, suggesting that the conformational changes they induce in integrins explain their thrombocytopenic effects in some patients [7Aster R.H. Curtis B.R. Bougie D.W. Dunkley S. Greinacher A. Warkentin T.E. Chong B.H. Thrombocytopenia associated with the use of GPIIb/IIIa inhibitors: position paper of the ISTH working group on thrombocytopenia and GPIIb/IIIa inhibitors.J Thromb Haemost. 2006; 4: 678-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Huxtable L.M. Tafreshi M.J. Rakkar A.N. Frequency and management of thrombocytopenia with the glycoprotein IIb/IIIa receptor antagonists.Am J Cardiol. 2006; 97: 426-9Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar]. While these drugs were designed to be rapidly reversible, a recent in vitro study has shown that eptifibatide has persistent receptor‐perturbing effects at therapeutic levels [9Schwarz M. Katagiri Y. Kotani M. Bassler N. Loeffler C. Bode C. Peter K. Reversibility versus persistence of GPIIb/IIIa blocker‐induced conformational change of GPIIb/IIIa (αIIbβ3, CD41/CD61).J Pharmacol Exp Ther. 2004; 308: 1002-11Crossref PubMed Scopus (0) Google Scholar]. Creating a readily bioavailable and safe form of an i.v. drug is challenging, but platelet activation is even more severe with orally active integrin antagonists. Three phase III clinical trials were stopped when safety committees noted excess mortality in the treated groups [10Heeschen C. Hamm C.W. Difficulties with oral platelet glycoprotein IIb/IIIa receptor antagonists.Lancet. 2000; 335: 330-1Abstract Full Text Full Text PDF Scopus (40) Google Scholar, 11Quinn M.J. Plow E.F. Topol E.J. Platelet glycoprotein IIb/IIIa inhibitors: recognition of a two‐edged sword.Circulation. 2003; 106: 379-85Crossref Scopus (133) Google Scholar, 12Chew D.P. Bhatt D.L. Sapp S. Topol E.J. Increased Mortality With Oral Platelet Glycoprotein IIb/IIIa Antagonists: a meta‐analysis of phase III multicenter randomized trials.Circulation. 2001; 103: 201-6Crossref PubMed Google Scholar]. Subsequent meta‐analyses revealed a 31–35% increased risk of death for patients treated with xemilofiban, sibrafiban, lotrafiban, or orbofiban [11Quinn M.J. Plow E.F. Topol E.J. Platelet glycoprotein IIb/IIIa inhibitors: recognition of a two‐edged sword.Circulation. 2003; 106: 379-85Crossref Scopus (133) Google Scholar, 13Newby L.K. Califf R.M. White H.D. Harrington R.A. Van de W.F. Granger C.B. Simes R.J. Hasselblad V. Armstrong P.W. The failure of orally administered glycoprotein IIb/IIIa inhibitors to prevent recurrent cardiac events.Am J Med. 2002; 112: 647-58Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Moreover, the incidence of myocardial infarction significantly increased in a subset of patients with acute coronary syndromes [13Newby L.K. Califf R.M. White H.D. Harrington R.A. Van de W.F. Granger C.B. Simes R.J. Hasselblad V. Armstrong P.W. The failure of orally administered glycoprotein IIb/IIIa inhibitors to prevent recurrent cardiac events.Am J Med. 2002; 112: 647-58Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar]. Fluctuating plasma concentrations and platelet‐activating effects that left unoccupied αIIbβ3 receptors in a prothrombotic state [13Newby L.K. Califf R.M. White H.D. Harrington R.A. Van de W.F. Granger C.B. Simes R.J. Hasselblad V. Armstrong P.W. The failure of orally administered glycoprotein IIb/IIIa inhibitors to prevent recurrent cardiac events.Am J Med. 2002; 112: 647-58Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 14Nurden A.T. Nurden P. GPIIb/IIIa antagonists and other anti‐integrins.Semin Vasc Med. 2003; 3: 123-30Crossref PubMed Scopus (0) Google Scholar] may have contributed to these deaths; clinical trial data document enhanced platelet reactivity [15Holmes M.B. Sobel B.E. Cannon C.P. Schneider D.J. Increased platelet reactivity in patients given orbofiban after an acute coronary syndrome: an OPUS‐TIMI 16 substudy. Orbofiban in Patients with Unstable coronary syndromes. Thrombolysis In Myocardial Infarction.Am J Cardiol. 2000; 85: 491-3Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar] and αIIbβ3 activation in patients treated with orbofiban [16Cox D. Smith R. Quinn M. Theroux P. Crean P. Fitzgerald D.J. Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes.J Am Coll Cardiol. 2000; 36: 1514-9Crossref PubMed Scopus (107) Google Scholar] and roxifiban [17Serebruany V.L. Malinin A.I. O'Connor C.M. Gurbel P.A. Effects of roxifiban on platelet aggregation and major receptor expression in patients with coronary artery disease for the Roxifiban Oral Compound Kinetics Evaluation Trial‐I (ROCKET‐I Platelet Substudy).Am Heart J. 2003; 146: 91-8Crossref PubMed Scopus (0) Google Scholar]. The mechanisms responsible for the failure of oral integrin antagonists remain controversial, disrupting new development [18Leclerc J.R. Platelet glycoprotein IIb/IIIa antagonists: lessons learned from clinical trials and future directions.Crit Care Med. 2002; 30: S332-40Crossref PubMed Google Scholar], and efforts to extend the benefits of i.v. agents to patients undergoing medical management for acute coronary syndromes yield only modest improvements [19Boersma E. Westerhout C.M. Intravenous glycoprotein IIb/IIIa inhibitors in acute coronary syndromes: lessons from recently conducted randomized clinical trials.Curr Opin Investig Drugs. 2004; 5: 313-9PubMed Google Scholar, 20Rebeiz A.G. Roe M.T. Alexander J.H. Mahaffey K.W. Granger C.B. Peterson E.D. Califf R.M. Harrington R.A. Integrating antithrombin and antiplatelet therapies with early invasive management for non‐ST‐segment elevation acute coronary syndromes.Am J Med. 2004; 116: 119-29Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Intense clinical interest is currently focused on therapies that use integrin antagonists after clopridogel treatment [21Gurbel P.A. Bliden K.P. Zaman K.A. Yoho J.A. Hayes K.M. Tantry U.S. Clopidogrel loading with eptifibatide to arrest the reactivity of platelets: results of the Clopidogrel Loading With Eptifibatide to Arrest the Reactivity of Platelets (CLEAR PLATELETS) study.Circulation. 2005; 111: 1153-9Crossref PubMed Scopus (350) Google Scholar] or combine them with monoparin [22Day J.R. Malik I.S. Weerasinghe A. Poullis M. Nadra I. Haskard D.O. Taylor K.M. Landis R.C. Distinct yet complementary mechanisms of heparin and glycoprotein IIb/IIIa inhibitors on platelet activation and aggregation: implications for restenosis during percutaneous coronary intervention.Heart. 2004; 90: 794-9Crossref PubMed Google Scholar], enoxaparin [23Topol E.J. A contemporary assessment of low‐molecular‐weight heparin for the treatment of acute coronary syndromes: factoring in new trials and meta‐analysis data.Am Heart J. 2005; 149: S100-6Crossref PubMed Scopus (0) Google Scholar], bivalrudin [24Exaire J.E. Butman S.M. Ebrahimi R. Kleiman N.S. Harrington R.A. Schweiger M.J. Bittl J.A. Wolski K. Topol E.J. Lincoff A.M. Provisional glycoprotein IIb/IIIa blockade in a randomized investigation of bivalirudin versus heparin plus planned glycoprotein IIb/IIIa inhibition during percutaneous coronary intervention: predictors and outcome in the Randomized Evaluation in Percutaneous coronary intervention Linking Angiomax to Reduced Clinical Events (REPLACE)‐2 trial.Am Heart J. 2006; 152: 157-63Crossref PubMed Scopus (0) Google Scholar], tenecteplase [25Gibson C.M. Jennings L.K. Murphy S.A. Lorenz D.P. Giugliano R.P. Harrington R.A. Cholera S. Krishnan R. Califf R.M. Braunwald E. Association between platelet receptor occupancy after eptifibatide (integrilin) therapy and patency, myocardial perfusion, and ST‐segment resolution among patients with ST‐segment‐elevation myocardial infarction: an INTEGRITI (Integrilin and Tenecteplase in Acute Myocardial Infarction) substudy.Circulation. 2004; 110: 679-84Crossref PubMed Scopus (0) Google Scholar], a fibrinolytic agent [26Goodman S. Enoxaparin and glycoprotein IIb/IIIa inhibition in non‐ST‐elevation acute coronary syndrome: insights from the INTERACT trial.Am Heart J. 2005; 149: S73-80Crossref PubMed Scopus (0) Google Scholar], or paclitaxel‐eluting stents [27Teirstein P.S. Kao J. Watkins M. Tannenbaum M.A. Laufer N. Chang M. Mehran R. Dangas G. Russell M.E. Ellis S.G. Stone G.W. Impact of platelet glycoprotein IIb/IIIa Inhibition on the paclitaxel‐eluting stent in patients with stable or unstable angina pectoris or provocable myocardial ischemia (a TAXUS IV substudy).Am J Cardiol. 2005; 96: 500-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. We tested the hypothesis that therapeutic interventions designed to block fibrinogen binding to the platelet αIIbβ3 receptor are inextricably linked to ligand‐induced conformational changes that shift the integrin from quiescence to an activated state. We compared the effects on platelet function and αIIbβ3 solution structure of the orally active compounds orbofiban and the roxifiban metabolites XV549 and XP280 with the i.v. agents eptifibatide and tirofiban. The resultant steady‐state and dynamic parameters for each pharmaceutical integrin antagonist were compared to recombinant echistatin (rEch), a potent arginine‐glycine‐aspartate (RGD) disintegrin [28Gan Z.R. Gould R.J. Jacobs J.W. Friedman P.A. Polokoff M.A. Echistatin. A potent platelet aggregation inhibitor from the venom of the viper, Echis carinatus.J Biol Chem. 1988; 263: 19827-32Abstract Full Text PDF PubMed Google Scholar, 29Hantgan R.R. Stahle M.C. Connor J.H. Lyles D.S. Horita D.A. Rocco M. Nagaswami C. Weisel J.W. McLane M.A. The disintegrin echistatin stabilizes integrin αIIbβ3’s open conformation and promotes its oligomerization.J Mol Biol. 2004; 342: 1625-36Crossref PubMed Scopus (0) Google Scholar], which served as a benchmark. Our unique approach to examining the strengths and weaknesses of integrin antagonists applies biophysics and molecular modeling to characterize the structure of the platelet αIIbβ3 receptor [29Hantgan R.R. Stahle M.C. Connor J.H. Lyles D.S. Horita D.A. Rocco M. Nagaswami C. Weisel J.W. McLane M.A. The disintegrin echistatin stabilizes integrin αIIbβ3’s open conformation and promotes its oligomerization.J Mol Biol. 2004; 342: 1625-36Crossref PubMed Scopus (0) Google Scholar, 30Hantgan R.R. Stahle M.C. Connor J.H. Horita D.A. Rocco M. McLane M.A. Yakovlev S. Medved L.V. Integrin αIIbβ3:ligand interactions are linked to binding‐site remodeling.Prot Sci. 2006; 15: 1893-906Crossref PubMed Scopus (0) Google Scholar]. We recognize the limitations in extrapolating to the complex clinical arena from a system of purified receptors and ligands, whose concentrations can be controlled and whose interactions can be measured with precision. To narrow the gap, we designed our biophysical studies, based on data from platelet function tests, to focus on therapeutically relevant ranges of receptor blockade. They provide evidence that, while both oral and i.v. integrin antagonists strongly inhibit platelet–platelet and platelet–fibrin interactions, their safety and efficacy may be fundamentally limited. Our results suggest that the same molecular mechanisms that enable naturally occurring disintegrins and pharmaceutical integrin antagonists to bind tightly to the αIIbβ3 receptor and block fibrinogen binding also contribute to their ability to perturb the resting integrin's conformation. COR Therapeutics (San Francisco, CA, USA) provided eptifibatide (Integrilin®; N6‐[aminoiminomethyl]‐N2‐(3‐mercapto‐1‐oxopropyl‐l‐lysylglycyl‐l‐α‐aspartyl‐l‐tryptophanyl‐l‐prolyl‐cysteinamide, cyclic [1Karvouni E. Katritsis D.G. Ioannidis J.P. Intravenous glycoprotein IIb/IIIa receptor antagonists reduce mortality after percutaneous coronary interventions.J Am Coll Cardiol. 2003; 41: 26-32Crossref PubMed Scopus (0) Google Scholar, 2Kong D.F. Hasselblad V. Harrington R.A. White H.D. Tcheng J.E. Kandzari D.E. Topol E.J. Califf R.M. Meta‐analysis of survival with platelet glycoprotein IIb/IIIa antagonists for percutaneous coronary interventions.Am J Cardiol. 2003; 92: 651-5Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 3Atar S. Cannon C.P. Murphy S.A. Rosanio S. Uretsky B.F. Birnbaum Y. Statins are associated with lower risk of gastrointestinal bleeding in patients with unstable coronary syndromes: analysis of the Orbofiban in Patients with Unstable coronary Syndromes‐Thrombolysis In Myocardial Infarction 16 (OPUS‐TIMI 16) trial.Am Heart J. 2006; 151: 976Crossref Scopus (37) Google Scholar, 4The Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM‐PLUS) Study InvestigatorsInhibition of the platelet glycoprotein IIb/IIIa receptor with tirofiban in unstable angina and non‐Q‐wave myocardial infarction.N Engl J Med. 1988; 338: 1488-97Google Scholar, 5The Platelet Receptor Inhibition in Ischemic Syndrome Management (PRISM) Study Investigators.A comparison of aspirin plus tirofiban with aspirin plus heparin for unstable angina.N Engl J Med. 1998; 338: 1498-505Crossref PubMed Scopus (990) Google Scholar, 6Bhatt D.L. Topol E.J. Current role of platelet glycoprotein IIb/IIIa inhibitors in acute coronary syndromes.JAMA. 2000; 284: 1549-58Crossref PubMed Scopus (248) Google Scholar]‐disulfide) [31Phillips D.R. Scarborough R.M. Clinical pharmacology of eptifibatide.Am J Cardiol. 1997; 80: 11B-20BAbstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. Merck (West Point, PA, USA) provided tirofiban hydrochloride (Aggrastat®; l‐tyrosine‐N‐[butylsulfonyl]‐O‐[‐4–4(piperidinebutyl)] monohydrochloride); concentrations were determined spectrally [32Hantgan R.R. Rocco M. Nagaswami C. Weisel J.W. Binding of a fibrinogen mimetic stabilizes integrin αIIbβ3’s open conformation.Protein Sci. 2001; 10: 1614-26Crossref PubMed Scopus (0) Google Scholar, 33Hantgan R.R. Stahle M.C. Jerome W.G. Nagaswami C. Weisel J.W. Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GPIIb/IIIa structure.Thromb Haemost. 2002; 87: 910-7Crossref PubMed Scopus (0) Google Scholar]. DuPont Pharmaceuticals (Wilmington, DE, USA) provided the roxifiban metabolites XV549 (methyl N3‐[2‐{3‐(4‐formamidino‐phenyl)‐isoxazolin‐5(R)‐yl}‐acetyl]‐N2‐(1‐butyloxycarbonyl)‐2,3‐(S)‐diaminopropionate) [34Shi G. Lloyd T.L. Sy S.K. Jiao Q. Wernicki A. Mutlib A. Emm T.A. Unger S.E. Pieniaszek Jr, H.J. Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.J Pharm Biomed Anal. 2003; 31: 937-51Crossref PubMed Scopus (14) Google Scholar], XP280, the benzene sulfonate salt of XV459 [35Pieniaszek Jr, H.J. Davidson A.F. Walton H.L. Pinto D.J. Olson R.E. Reilly T.M. Barrett Y.C. A double antibody radioimmunoassay for the determination of XV459, the active hydrolysis metabolite of roxifiban, in human plasma.J Pharm Biomed Anal. 2003; 30: 1441-9Crossref PubMed Scopus (6) Google Scholar], and orbofiban (ethyl 3‐[[[[1‐[4(aminoiminomethyl)phenyl]‐2‐oxo‐3S pyrrolidinyl]amino]carbonyl]amino]‐propanoate, acetate salt) [36Ogawa T. Sugidachi A. Naganuma H. Asai F. Antiplatelet and antithrombotic effects of orbofiban, a new orally active GPIIb/IIIa antagonist, in guinea pigs.Thromb Res. 2000; 97: 307-15Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]; concentrations were determined by dry weight. Highly purified human fibrinogen was purchased from American Diagnostica (Greenwich, CT, USA) and highly purified human α‐thrombin from Sigma Chemicals (St Louis, MO, USA). cHArGD (cyclo(s,s)‐l‐lysyl‐l‐tyrosyl‐glycyl‐l‐cystinyl‐l‐homoarginyl‐glycyl‐l‐aspartyl‐l‐trytopanyl‐l‐prolyl‐l‐cystine) was synthesized, purified, and characterized as described previously [37Hantgan R.R. Lyles D.S. Mallett T.C. Rocco M. Nagaswami C. Weisel J.W. Ligand binding promotes the entropy‐driven oligomerization of integrin αIIbβ3.J Biol Chem. 2003; 278: 3417-26Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 38Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Effects of ligand‐mimetic peptides Arg‐Gly‐Asp‐X (X = Phe, Trp, Ser) on αIIbβ3 integrin conformation and oligomerization.Biochemistry. 1999; 38: 14461-4Crossref PubMed Scopus (0) Google Scholar]. It was labeled with Oregon Green succinimidyl ethyl ester (Invitrogen Molecular Probes, Carlsbad, CA, USA) in a 45‐min reaction at pH 8.3; the labeled peptide was separated from excess dye by reversed‐phase high performance liquid chromatography on a C18 column [39Knight L.C. Romano J.E. Functional expression of bitistatin, a disintegrin with potential use in molecular imaging of thromboembolic disease.Protein Expr Purif. 2005; 39: 307-19Crossref PubMed Scopus (15) Google Scholar]. The rEch mutant rEch (1–49) D27W M28L was prepared using cloning, site‐directed mutagenesis, expression, and purification protocols, as recently described [29Hantgan R.R. Stahle M.C. Connor J.H. Lyles D.S. Horita D.A. Rocco M. Nagaswami C. Weisel J.W. McLane M.A. The disintegrin echistatin stabilizes integrin αIIbβ3’s open conformation and promotes its oligomerization.J Mol Biol. 2004; 342: 1625-36Crossref PubMed Scopus (0) Google Scholar, 30Hantgan R.R. Stahle M.C. Connor J.H. Horita D.A. Rocco M. McLane M.A. Yakovlev S. Medved L.V. Integrin αIIbβ3:ligand interactions are linked to binding‐site remodeling.Prot Sci. 2006; 15: 1893-906Crossref PubMed Scopus (0) Google Scholar]. rEch (1–49) D27W M28L was labeled with Oregon Green succinimidyl ethyl ester, as described previously [30Hantgan R.R. Stahle M.C. Connor J.H. Horita D.A. Rocco M. McLane M.A. Yakovlev S. Medved L.V. Integrin αIIbβ3:ligand interactions are linked to binding‐site remodeling.Prot Sci. 2006; 15: 1893-906Crossref PubMed Scopus (0) Google Scholar]. Blood was obtained by venipuncture from healthy, adult volunteers (six women/two men) into 1/10 vol. of sodium citrate (110 mmol L−1) anticoagulant, using Wake Forest University Health Sciences procedures approved by the Institutional Review Board. Platelet counts in platelet‐rich plasma (PRP; 280 000 ± 107 000 platelets per microliter; n = 8) were determined with a Coulter MDII Cell Counter (Beckman‐Coulter, Miami, FL, USA) [40Hantgan R.R. A study of the kinetics of ADP‐triggered platelet shape change.Blood. 1984; 64: 896-906Crossref PubMed Google Scholar]. Platelet GpIIb/IIIa occupancy was determined using flow cytometric analysis with a Biocytex Kit BX7001 (Marseilles, France) [33Hantgan R.R. Stahle M.C. Jerome W.G. Nagaswami C. Weisel J.W. Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GPIIb/IIIa structure.Thromb Haemost. 2002; 87: 910-7Crossref PubMed Scopus (0) Google Scholar] with PRP samples incubated with orbofiban or XV549 (0–500 nm). Platelet aggregation profiles were obtained in a Chrono‐Log Model 500 aggregometer following addition of CaCl2 (2 mmol L−1) and ADP (45 μmol L−1) to stirred samples of PRP in the presence/absence of the orbofiban or the roxifiban metabolite XV549 (0–500 nm). Orbofiban (0–500 nm) or XV549 (0–5000 nm) concentrations required to block platelet adhesion to clotted fibrin were determined by a microtiter plate colorimetric assay, as described previously [33Hantgan R.R. Stahle M.C. Jerome W.G. Nagaswami C. Weisel J.W. Tirofiban blocks platelet adhesion to fibrin with minimal perturbation of GPIIb/IIIa structure.Thromb Haemost. 2002; 87: 910-7Crossref PubMed Scopus (0) Google Scholar]. In all cases, data were expressed and analyzed as the ratio of the measured parameter with integrin antagonist to that without, using platelets from the same donor so that each donor served as her/his own control. Milligram quantities of highly purified integrin αIIbβ3 were isolated from outdated human blood platelets (kindly provided by the Blood Bank, North Carolina Baptist Hospital, Winston‐Salem, NC, USA), as described previously [38Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Effects of ligand‐mimetic peptides Arg‐Gly‐Asp‐X (X = Phe, Trp, Ser) on αIIbβ3 integrin conformation and oligomerization.Biochemistry. 1999; 38: 14461-4Crossref PubMed Scopus (0) Google Scholar]. Biophysical measurements were performed on peak integrin fractions obtained by size‐exclusion chromatography at 4 oC on a 0.9 × 85 cm2 column of Sephacryl S‐300 equilibrated in a pH 7.4 buffer (HSCM‐OG) containing 0.13 mol L−1 NaCl, 0.01 mol L−1 HEPES, 0.001 mol L−1 CaCl2, 0.001 mol L−1 MgCl2, 3 × 10−8 mol L−1 basic trypsin inhibitor, 10−6 mol L−1 leupeptin, and 0.03 mol L−1n‐octyl‐β‐d‐glucopyranoside. Sedimentation velocity and equilibrium measurements were performed, as described previously [38Hantgan R.R. Paumi C. Rocco M. Weisel J.W. Effects of ligand‐mimetic peptides Arg‐Gly‐Asp‐X (X = Phe, Trp, Ser) on αIIbβ3 integrin conformation and oligomerization.Biochemistry. 1999; 38: 14461-4Crossref PubMed Scopus (0) Google Scholar], in a Beckman Optima XL‐A analytical ultracentrifuge (Palo Alto, CA, USA) equipped with absorbance optics and an An60 Ti rotor. Fluorescence anisotropy measurements (steady state) were carried out, as described previously [30Hantgan R.R. Stahle M.C. Connor J.H. Horita D.A. Rocco M. McLane M.A. Yakovlev S. Medved L.V. Integrin αIIbβ3:ligand interactions are linked to binding‐site remodeling.Prot Sci. 2006; 15: 1893-906Crossref PubMed Scopus (0) Google Scholar], on a Safire II (Tecan Instruments, Männedorf, Switzerland). Time‐dependent fluorescence anisotropy measurements were carried out in an ISS K2 spectrofluorometer, using vertically polarized excitation (475 nm) and two detectors to enable simultaneous collection of the horizontally (FH) and vertically (Fv) polarized fluorescence emission (525 nm) signals at one point per second. PyMol molecular graphics software [41DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific, 2002Google Scholar] was used to visualize and to measure interatomic distances within the 2.7 Å resolution structure of the αIIbβ3 headpiece, crystallized in the presence of the ligand‐mimetic peptides tirofiban, 1TY5.pdb, and eptifibatide, 1TY6 [42Xiao T. Takagi J. Coller B.S. Wang J.H. Springer T.A. Structural basis for allostery in integrins and binding to fibrinogen‐mimetic therapeutics.Nature. 2004; 432: 59-67Crossref PubMed Scopus (0) Google Scholar]. The conformation of the roxifiban metabolite XP280 was modeled using Alchemy 2000 molecular graphics software (Tripos; St Louis, MO, USA). Comparing the inhibitory properties of the orally active compounds orbofiban [36Ogawa T. Sugidachi A. Naganuma H. Asai F. Antiplatelet and antithrombotic effects of o" @default.
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