Paratope and epitope mapping of the antithrombotic antibody 6B4 in complex with platelet glycoprotein Ibα

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 Ib alpha(GPIb alpha). This interaction blocks, in high shear stress conditions, the specific interaction between GPIb alpha 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 GPIb alpha, 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 GPIb alpha mutants were constructed and assayed for their capacity to bind GPIb alpha 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 CDR1and -3, respectively, and Tyr(100C) of the heavy chain CDR3. These residues form a valley, where the GPIb alpha 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.