Conformation-Specific Blockade of the Integrin GPIIb/IIIa: A Novel Antiplatelet Strategy That Selectively Targets Activated Platelets

• Published in: Circulation research 2006-07, Vol.99 (1), p.25-33
• Main Authors: Schwarz, Meike, Meade, Gerardene, Stoll, Patrick, Ylanne, Jari, Bassler, Nicole, Chen, Yung Chih, Hagemeyer, Christoph E, Ahrens, Ingo, Moran, Niamh, Kenny, Dermot, Fitzgerald, Desmond, Bode, Christoph, Peter, Karlheinz
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Animals; Antibodies - immunology; Bleeding Time; Blood Platelets - metabolism; Carotid Artery Diseases - chemically induced; Carotid Artery Diseases - prevention & control; Chlorides; Complementarity Determining Regions; Ferric Compounds; Fibrinogen - metabolism; Fibrinolytic Agents - pharmacology; Humans; Mice; Mice, Inbred C57BL; Molecular Conformation; Peptides - pharmacology; Platelet Activation; Platelet Aggregation Inhibitors - immunology; Platelet Aggregation Inhibitors - pharmacology; Platelet Glycoprotein GPIIb-IIIa Complex - antagonists & inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex - chemistry; Platelet Glycoprotein GPIIb-IIIa Complex - immunology; Thrombosis - chemically induced; Thrombosis - prevention & control; Tyrosine - analogs & derivatives; Tyrosine - pharmacology
• ISSN: 0009-7330; 1524-4571
• DOI: 10.1161/01.RES.0000232317.84122.0c

Platelet activation causes conformational changes of integrin GPIIb/IIIa (αIIbβ3), 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.