Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets

M Schwarz, G Meade, P Stoll, J Ylanne… - Circulation …, 2006 - Am Heart Assoc
M Schwarz, G Meade, P Stoll, J Ylanne, N Bassler, YC Chen, CE Hagemeyer, I Ahrens…
Circulation research, 2006Am Heart Assoc
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 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.
Am Heart Assoc