Glycoprotein Ibalpha Inhibitor Complex Crystal Structure at High Resolution.

The platelet Glycoprotein Ib/V/IX (GpIb/V/IX) complex is considered a major target for anticoagulant therapy. The primary function of the receptor is to mediate platelet adhesion to von Willebrand factor (VWF) bound to damaged sub-endothelium. This represents the first critical step for platelet adhesion under conditions of high fluid shear stress. GpIb/V/IX is implicated in a number of thrombotic pathological processes such as stroke or myocardial infarction and the bleeding disorders Bernard-Soulier syndrome, platelet type von Willebrand disease (Pt-VWD) and thrombotic thrombocytopenic purpura. We have successfully determined the structure of the GpIbalpha N-terminal domain in complex with a potent (sub nM) 11meric peptide inhibitor (OS1) of the interaction with VWF.

We have determined the crystal structure to 1.8Å resolution using molecular replacement. Results. The peptide sequence CTERMALHNLC was readily identifiable bound to GpIbalpha between the extended regulatory (R) loop and the concave surface of the leucine rich repeats. The peptide adopts one and a half turns of an alpha-helix and contacts three subsites (S1, S2 and S3). S1 and S2 reside within the leucine rich repeats and S3 has a unique feature as this subsite involves contact with the regulatory R-loop stabilizing it in a well defined conformation with helical character. This loop alters conformation between an extended beta-hairpin in the VWF-A1 bound structure and a more compact largely disordered structure in the unliganded structure. In this regard, the Pt-VWD mutations of GpIbalpha, G233V and M239V, which reside in the R-loop act by inducing a beta-conformation and thus result in a high affinity form of the receptor.

These studies provide a strategy for targeting the GpIbalpha-VWF interaction using small molecules or alpha-helical peptides exploiting the GpIbalpha subsites described here and acting allosterically to stabilise a low affinity conformation of the receptor with an alpha helical R-loop. Ligand mimetic peptide complex crystal structures for the platelet receptors integrin aIIbb3 with RGD, and alpha2beta1 with a collagen peptide have been described and the former are currently in therapeutic use for treatment of thromboemboletic disorders. Targeting the GpIbalpha-VWF interaction may provide anti-thrombotic drugs which affect platelet adhesion under high shear stress without compromising normal processes of platelet adhesion and aggregation which may be required for normal hemostasis to function. Targeting protein-protein interactions is considered one of the great contemporary challenges in drug discovery. The understanding of how the S1S2S3 subsites provide very effective inhibition of a large protein-protein interaction has wide applicability. LRR proteins are an extended family mediating protein-protein interactions involved in a variety of disease processes such as sepsis, asthma, immunodeficiencies, atherosclerosis, alzheimers (leucine rich repeat kinase) and leukaemia (leucine rich repeat phosphatase). The structural fit of the helical curvature of the peptide with the arc of the leucine rich repeats may provide a basis for further development of alpha-helical peptide mimetics targeting other members of the LRR family which utilize the concave face.

No relevant conflicts of interest to declare.