Alboaggregin A activates platelets by a mechanism involving glycoprotein VI as well as glycoprotein Ib

The snake venom C-type lectin alboaggregin A (or 50-kd alboaggregin) from Trimeresurus albolabris was previously shown to be a platelet glycoprotein (GP) Ib agonist. However, investigations of the signal transduction induced in platelets showed patterns of tyrosine phosphorylation that were different from those of other GPIb agonists and suggested the presence of an additional receptor. In this study, the binding of biotinylated alboaggregin A to platelet lysates, as well as affinity chromatography evaluations of platelet lysates on an alboaggregin A-coated column, indicated that this other receptor is GPVI. Additional experiments with reagents that inhibit either GPIb or GPVI specifically supported this finding. These experiments also showed that both GPIb and GPVI have a role in the combined signaling and that the overall direction this takes can be influenced by inhibitors of one or the other receptor pathway.

Platelet GPIb complex as a target for anti-thrombotic drug development

Specific inhibition of platelet function is a major target of anti-thrombotic drug research. Platelet receptors are both accessible and specific but have multiple functions often linked to a wide range of ligands. GPIb complex is best known as a major platelet receptor for von Willebrand factor essential for platelet adhesion under high shear conditions found in arteries and in thrombosis. Recent animal studies have supported inhibition of GPIb as a good candidate for anti-thrombotic drug development with several classes of proteins showing important specific effects and the required discrimination between roles in haemostasis and thrombosis is important to protect against bleeding complications. These include antibodies, several classes of snake venom proteins, mutant thrombin molecules and peptides affecting subunit interactions. However, due to the nature of its receptor-ligand interactions involving large protein-protein interfaces, the possibility of developing classic pharmaceutical inhibitors for long term (and perhaps oral) treatment is still unclear, and additional information about structural interactions and signalling mechanisms is essential.

The crystal structure of the platelet activator aggretin reveals a novel (alphabeta)2 dimeric structure

Aggretin is a C-type lectin purified from Calloselasma rhodostoma snake venom. It is a potent activator of platelets, resulting in a collagen-like response by binding and clustering platelet receptor CLEC-2. We present here the crystal structure of aggretin at 1.7 A which reveals a unique tetrameric quaternary structure. The two alphabeta heterodimers are arranged through 2-fold rotational symmetry, resulting in an antiparallel side-by-side arrangement. Aggretin thus presents two ligand binding sites on one surface and can therefore cluster ligands in a manner reminiscent of convulxin and flavocetin. To examine the molecular basis of the interaction with CLEC-2, we used a molecular modeling approach of docking the aggretin alphabeta structure with the CLEC-2 N-terminal domain (CLEC-2N). This model positions the CLEC-2N structure face down in the "saddle"-shaped binding site which lies between the aggretin alpha and beta lectin-like domains. A 2-fold rotation of this complex to generate the aggretin tetramer reveals dimer contacts for CLEC-2N which bring the N- and C-termini into the proximity of each other, and a series of contacts involving two interlocking beta-strands close to the N-terminus are described. A comparison with homologous lectin-like domains from the immunoreceptor family reveals a similar but not identical dimerization mode, suggesting this structure may represent the clustered form of CLEC-2 capable of signaling across the platelet membrane.