Paratope and Epitope Mapping of the Antithrombotic Antibody 6B4 in Complex with Platelet Glycoprotein Ibα

Marc De Maeyer,Tadashi Matsushita,Mayo Yamashita,Hans Deckmyn,Bauke De Maeyer,Alexandre Fontayne

doi:10.1074/jbc.m701826200

Marc De Maeyer, Tadashi Matsushita + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m701826200

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Abstract

The monoclonal antibody 6B4 has a potent antithrombotic effect in nonhuman primates by binding to the flexible loop, also known as the beta-switch region (amino acids 230-242), of glycoprotein Ibalpha (GPIbalpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIbalpha and von Willebrand factor suppressing platelet deposition to the damaged vessel wall, a key event in the pathogenesis of arterial thrombosis. To understand the interactions between this antibody and its antigen at the amino acid level, we here report the identification of the paratope and epitope in 6B4 and GPIbalpha, respectively, by using computer modeling and site-directed mutagenesis. The docking programs ZDOCK (rigid body docking) and HADDOCK (flexible docking) were used to model the interaction of 6B4 with GPIbalpha and to delineate the respective paratope and epitope. 6B4 and GPIbalpha mutants were constructed and assayed for their capacity to bind GPIbalpha and 6B4, respectively. From these data, it is found that the paratope of 6B4 is mainly formed by five residues: Tyr(27D), Lys(27E), Asp(28), and Glu(93) located in light chain CDR1 and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIbalpha flexible loop can bind via residues Asp(235) and Lys(237). The experimental results were finally used to build a more accurate docking model. Taken together, this information provides guidelines for the design of new derivatized lead compounds with antithrombotic properties.

Highlights

Von Willebrand factor (VWF), which is bound to the collagen matrix exposed to the flowing blood upon vessel damage
The structure of GPIb␣ consists of a globular N-terminal region, a sialomucin core, an anionic sequence, a transmembrane region, and a cytoplasmic tail
The GPIb␣ gain-of-function mutations G233V and M239V found in platelet-type von Willebrand disease stabilize the ␤-hairpin conformation and increase the affinity of GPIb␣ for von Willebrand factor (VWF) 5– 6-fold [4, 6]

Summary

Introduction

Von Willebrand factor (VWF), which is bound to the collagen matrix exposed to the flowing blood upon vessel damage. Platelets present the flexible loop (residues 230 –242) within the N-terminal domain of GPIb␣ into a ␤-switch conformation, which changes upon binding of VWF into a ␤-hairpin conformation, extending the existing VWF antiparallel ␤-sheet [5]. The GPIb␣ gain-of-function mutations G233V and M239V found in platelet-type von Willebrand disease stabilize the ␤-hairpin conformation and increase the affinity of GPIb␣ for VWF 5– 6-fold [4, 6]. We prepared and characterized a murine monoclonal antibody (mAb) targeting the human GPIb␣, designated as 6B4 [9]. This mAb inhibits platelet adhesion under high shear stress conditions, as was shown in flow chambers [10].

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