Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins

Abstract

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

Novel therapeutics and emerging technology in haemostasis and thrombosis: highlights from the British society for haemostasis and thrombosis annual meeting

“…Dr Magdalena Gierula (Imperial College London, UK) described the role of the C-terminal tail of TFPIα on regulation of coagulation. Platelets were also the focus of the best poster prize which went to Dr Joanne Clark (University of Birmingham, UK) for her poster on their latest work on CLEC-2 clustering and CLEC-2 nanobody development ( 21 , 22 ) ( Figure 1B ).…”