Pharmacological Inhibition of Glycoprotein VI- and Integrin alpha 2 beta 1-Induced Thrombus Formation Modulated by the Collagen Type

Background In secondary cardiovascular disease prevention, treatments blocking platelet-derived secondary mediators pose a risk of bleeding. Pharmacological inter-ference of the interaction of platelets with exposed vascular collagens is an attractive alternative, with clinical trials ongoing. Antagonists of the collagen receptors, glyco-protein VI (GPVI), and integrin alpha 2131, include recombinant GPVI-Fc dimer construct Revacept, 9O12 mAb based on the GPVI-blocking reagent Glenzocimab, Syk tyrosine-kinase inhibitor PRT-060318, and anti-alpha 2131 mAb 6F1. No direct comparison has been made of the antithrombic potential of these drugs.Methods Using a multiparameter whole-blood microfluidic assay, we compared the effects of Revacept, 9O12-Fab, PRT-060318, or 6F1 mAb intervention with vascular collagens and collagen-related substrates with varying dependencies on GPVI and alpha 2131. To inform on Revacept binding to collagen, we used fluorescent-labelled anti-GPVI nanobody-28.Results and Conclusion In this first comparison of four inhibitors of platelet-collagen interactions with antithrombotic potential, we find that at arterial shear rate: (1) the thrombus-inhibiting effect of Revacept was restricted to highly GPVI-activating surfaces; (2) 9O12-Fab consistently but partly inhibited thrombus size on all surfaces; (3) effects of GPVI-directed interventions were surpassed by Syk inhibition; and (4) alpha 2131-directed intervention with 6F1 mAb was strongest for collagens where Revacept and 9O12-Fab were limitedly effective. Our data hence reveal a distinct pharma-cological profile for GPVI-bind ing competition (Revacept), GPVI receptor blockage (9O12-Fab), GPVI signaling (PRT-060318), and alpha 2 beta 1 blockage (6F1 mAb) in flow dependent thrombus formation, depending on the platelet-activating potential of the collagen substrate. This work thus points to additive antithrombotic action mechanisms of the investigated drugs

Galectin-9 activates platelet ITAM receptors glycoprotein VI and C-type lectin-like receptor-2

Background Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. Objective In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2). Methods Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6(-/-)), and GPVI and CLEC-2-double knockout (Gp6(-/-)/Gp1ba-Cre-Clec1b(fl)(/)(fl)) mice were used. Results We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets. Conclusions We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation.

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity