Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.
Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), 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.
Schwarz
Meike
M
Department of Cardiology, University of Freiburg, Freiburg, Germany. schwarz@med1.ukl.uni-freiburg.de
Meade
Gerardene
G
Stoll
Patrick
P
Ylanne
Jari
J
Bassler
Nicole
N
Chen
Yung Chih
YC
Hagemeyer
Christoph E
CE
Ahrens
Ingo
I
Moran
Niamh
N
Kenny
Dermot
D
Fitzgerald
Desmond
D
Bode
Christoph
C
Peter
Karlheinz
K
D020816:Amino Acid Motifs; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D001760:Bleeding Time; D001792:Blood Platelets / Q000378:metabolism; D002340:Carotid Artery Diseases / Q000139:chemically induced / Q000517:prevention & control; D002712:Chlorides; D022801:Complementarity Determining Regions; D000077542:Eptifibatide; D005290:Ferric Compounds; D005340:Fibrinogen / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000494:pharmacology; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008968:Molecular Conformation; D010455:Peptides / Q000494:pharmacology; D015539:Platelet Activation; D010975:Platelet Aggregation Inhibitors / Q000276:immunology* / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology*; D013927:Thrombosis / Q000139:chemically induced / Q000517:prevention & control; D000077466:Tirofiban; D014443:Tyrosine / Q000031:analogs & derivatives / Q000494:pharmacology
D000906:Antibodies; D002712:Chlorides; D022801:Complementarity Determining Regions; D005290:Ferric Compounds; D005343:Fibrinolytic Agents; D010455:Peptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D014443:Tyrosine; D005340:Fibrinogen; D000077466:Tirofiban; D000077542:Eptifibatide; C024555:ferric chloride