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(Circulation Research. 2006;99:25.)
© 2006 American Heart Association, Inc.

Molecular Medicine

Conformation-Specific Blockade of the Integrin GPIIb/IIIa

A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

Meike Schwarz, Gerardene Meade, Patrick Stoll, Jari Ylanne, Nicole Bassler, Yung Chih Chen, Christoph E. Hagemeyer, Ingo Ahrens, Niamh Moran, Dermot Kenny, Desmond Fitzgerald, Christoph Bode, Karlheinz Peter

From the Department of Cardiology (M.S., P.S., I.A., C.B.), University of Freiburg, Germany; Department of Clinical Pharmacology (G.M., I.A., N.M., D.K., D.F.), Royal College of Surgeons in Ireland, Dublin; Department of Biochemistry and Biocenter Oulu (J.Y.), University of Oulu, Finland; and Baker Heart Research Institute (N.B., Y.C.C., C.E.H., K.P.), Melbourne, Australia.

Correspondence to Meike Schwarz, MD, Department of Cardiology, University of Freiburg, Breisacherstr. 33, 79106 Freiburg, Germany. E-mail schwarz{at}med1.ukl.uni-freiburg.de

Platelet activation causes conformational changes of integrin GPIIb/IIIa (_IIbß₃), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Key Words: thrombosis • platelets • adhesion molecules • GPIIb/IIIa

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