Molecular Identification of the Cross-reacting Epitope on αMβ2 Integrin I Domain Recognized by Anti-αIIbβ3 Monoclonal Antibody 7E3 and Its Involvement in Leukocyte Adherence

Janet Plescia,Michael S Conte,Guy Vanmeter,Grazia Ambrosini,Dario C Altieri

doi:10.1074/jbc.273.32.20372

Abstract

The monoclonal antibody (mAb) 7E3 directed to the platelet integrin alphaIIb beta3 was tested for its cross-reactivity with the homologous leukocyte integrin alphaM beta2. Nested recombinant fragments of alphaM I domain were expressed as glutathione S-transferase fusion proteins and analyzed for antibody recognition. In enzyme-linked immunosorbent assay, mAb 7E3 bound alphaM I domain fragments containing the amino-terminal sequence Cys128-Ser172, whereas the carboxyl-terminal region Leu173-Pro291 was ineffective. A synthetic peptide designated R1.1 and duplicating the alphaM sequence G127CPQEDSDIAFLIDGSGSIIPHDF150 bound mAb 7E3. In contrast, the adjacent alphaM region F150RRMKEFVSTVMEQLKKSKTLFS172 or a control peptide with a scrambled R1.1 sequence was not recognized by mAb 7E3. Binding of mAb 7E3 to alphaM I domain blocked monocyte and neutrophil adhesion to immobilized fibrinogen and fibrinogen-dependent leukocyte-endothelium bridging, indistinguishably from bona fide anti-beta2 mAb IB4. In contrast, leukocyte binding to stable transfectants expressing intercellular adhesion molecule-1 was not affected by mAb 7E3. Balloon-mediated injury of iliofemoral arteries in rabbits resulted in prominent deposition of fibrinogen and increased monocyte adhesion to the injured vessel, in a reaction inhibited by mAb 7E3, but unaffected by control mAb 14E11. Through its cross-reactivity between alphaIIb beta3 and alphaM beta2, mAb 7E3 may initiate a new class of integrin antagonists, capable of simultaneously targeting platelet and leukocyte adhesion mechanisms in vascular injury.

Highlights

Hemostasis and immune-inflammatory responses [1] are maintained by the adhesive interactions mediated by integrins ␣IIb␤3 (GPIIb/IIIa) on platelets [2] and ␣M␤2 (Mac-1) on leukocytes [3]
We have shown that anti-␣IIb␤3 mAb 7E3, an integrin antagonist currently used in clinical practice [11], binds a discrete region of ␣M I domain and inhibits fibrinogenmediated leukocyte adhesion in vitro, and in balloon-injured arteries of rabbits, ex vivo
Of paramount importance for normal hemostasis [23], platelet aggregation mechanisms maintained by ␣IIb␤3 may precipitate thrombus formation and acute ischemic cardiovascular emergencies [5, 6]

Summary

Introduction

Hemostasis and immune-inflammatory responses [1] are maintained by the adhesive interactions mediated by integrins ␣IIb␤3 (GPIIb/IIIa) on platelets [2] and ␣M␤2 (Mac-1) on leukocytes [3] Despite their critical role in vascular cell homeostasis and signaling [4], platelet and leukocyte adhesion mechanisms participate in the pathogenesis of vascular injury. Erable effort has been devoted to the identification of molecular antagonists of ␣IIb␤3 [9] and ␣M␤2 [10], capable of disrupting aberrant platelet and monocyte adherence mechanisms. It was reported that mAb 7E3 unexpectedly cross-reacted with the active conformation of ␣M␤2, induced on monocytes by inflammatory stimuli [14] or Mn2ϩ ions [15]. We found that mAb 7E3 recognizes a discrete region in ␣M I domain [17], which is critically involved in monocyte adherence to fibrinogen in vitro and in balloon-injured arteries, ex vivo

Methods

Results

Conclusion