Abstract
The antithrombotic monoclonal antibody 82D6A3 is directed against amino acids Arg-963, Pro-981, Asp-1009, Arg-1016, Ser-1020, Met-1022, and His-1023 of the von Willebrand factor A3-domain (Vanhoorelbeke, K., Depraetere, H., Romijn, R. A., Huizinga, E., De Maeyer, M., and Deckmyn, H. (2003) J. Biol. Chem. 278, 37815-37821). By this, it potently inhibits the interaction of von Willebrand factor to collagens, which is a prerequisite for blood platelet adhesion to the injured vessel wall at sites of high shear. To fully understand the mode of action of 82D6A3 at the molecular level, we resolved its crystal structure in complex with the A3-domain and fine mapped its paratope by construction and characterization of 13 mutants. The paratope predominantly consists of two short sequences in the heavy chain CDR1 (Asn-31 and Tyr-32) and CDR3 (Asp-99, Pro-101, Tyr-102 and Tyr-103), forming one patch on the surface of the antibody. Trp-50 of the heavy and His-49 of the light chain, both situated adjacent to the patch, play ancillary roles in antigen binding. The crystal structure furthermore confirms the epitope location, which largely overlaps with the collagen binding site deduced from mutagenesis of the A3-domain (Romijn, R. A., Westein, E., Bouma, B., Schiphorst, M. E., Sixma, J. J., Lenting, P. J., and Huizinga, E. G. (2003) J. Biol. Chem. 278, 15035-15039). We herewith further consolidate the location of the collagen binding site and reveal that the potent action of the antibody is due to direct competition for the same interaction site. This information allows the design of a paratope-mimicking peptide with antithrombotic properties.
Highlights
Platelet adhesion, activation, and aggregation are the three major steps in primary hemostasis, a process that is necessary for the cessation of bleeding at sites of vascular injury
von Willebrand factor (VWF) interacts with the fibrillar collagens mainly via its A3-domain [13, 14], whereas its A1-domain has been implicated in binding to collagen type VI [15, 16]
We further demonstrated that 82D6A3 has antithrombotic effects in a baboon arterial thrombosis model [31], proving for the first time that the VWF-collagen interaction is involved in thrombus formation in vivo [32]
Summary
Activation, and aggregation are the three major steps in primary hemostasis, a process that is necessary for the cessation of bleeding at sites of vascular injury. The antithrombotic effect was not associated with significantly increased bleeding times, even at high doses, demonstrating that administration of 82D6A3 results in a broad therapeutic window These benefits were observed in in vivo studies with saratin, another inhibitor of the VWFcollagen interaction [33, 34], and with monoclonal antibodies 6B4 [35, 36] and AjvW-2 [37, 38], which inhibit VWF-GPIb binding. Paratope Determination of the Antithrombotic Antibody 82D6A3 inhibitors of platelet aggregation (GPIIb/IIIa-receptor blockers) or platelet activation (aspirin) Treatment with the latter agents results in smaller therapeutic windows and is frequently associated with bleeding problems and requires close monitoring of the patients (39 – 41). Binding studies with alanine mutants of the single chain variable fragment of 82D6A3 (scFv-82D6A3) resulted in the identification of the paratope
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