W0359

The interaction of the amino terminal domain of glycoprotein (GP) Ibα with the von Willebrand factor (VWF) A1 domain is essential for platelet adhesion under high shear stress. GP Ibα also binds α-thrombin, and may thus contribute to platelet activation and expression of procoagulant activity. To clarify the structural bases of the ligand binding function of GP Ibα, we have determined the crystal structure of its amino terminal domain (residues 1-290 of the mature protein) in complex with human α-thrombin. The complex was formed at a 1:1 stoichiometric ratio of the two components and the occurrence of the interaction was confirmed by the observation of a mobility shift in native polyacrylamide gel electrophoresis. Crystals grew in the P4₃2₁2 space group with cell parameters a=67.65 Å, c=328.58 Å. The x-ray diffraction data extended to 2.3 Å resolution. The three dimensional structure of the complex was determined using molecular replacement techniques based on the independently determined structures of GP Ibα and α-thrombin. Structural and functional evidence indicates that α-thrombin binds to the amino terminal domain of platelet glycoprotein (GP) Ibα through a mechanism that involves two distinct ligand and receptor sites. The initial contact between the two molecules occurs through the exosite II of α-thrombin and involves negatively charged residues in GP Ibα including the sulfated Tyr276 and Asp277. The first interaction leads to the exposure of a second site on the receptor that is initially cryptic, owing to internal contacts between the sulfated Tyr279 and Arg217, but eventually binds to an extended surface of a second thrombin molecule including exosite I. This dual mode of interaction has the potential to mediate receptor clustering on the platelet surface, thus promoting signaling and activation, but at the same time limits the prothrombotic function of α-thrombin by markedly reducing its fibrinogen clotting activity through blockade of exosite I.