Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling

Summary

Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in bloodfilled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following α∥bβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγR∥A receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.

 

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Keywords

Platelet physiology - rheology - lymphatics - adhesion molecules - signal transduction

Video 1 Suppl. Video 1: Src and Syk inhibitors reduce stability of platelet adhesion to LECs. To analyse real-time adhesion of platelets to LECs, heparinized whole blood was incubated with FITC-conjugated anti-human CD41 and CD42b antibodies at 1/100 dilution and inhibitors or vehicle control as appropriate for 10 minutes and then perfused over LEC monolayers at 50 s^-1. Digital images were captured at 500 ms interval with 500 ms exposure time and 2×2 binning settings to create 1 minute videos. Upper left: vehicle; upper right: 9 µM integrilin; lower left: 10 µM dasatinib; lower right: 30 µM PRT060318.

Video 2 Suppl. Video 2: Src and Syk inhibitors reduce stability of platelet adhesion to hFcPDPN. To analyse real-time adhesion of platelets to hFcPDPN, heparinized whole blood was incubated with 4 µM DiOC6, 10µg/ml IV.3 antibody and inhibitors or vehicle control as appropriate for 10 minutes and then perfused over hFcPDPN-coated channels at 50 s^-1. Digital images were captured at 250 ms interval with 200 ms exposure time and 2×2 binning settings to create 1 minute videos. Upper left: vehicle; upper right: 9 µM integrilin; lower left: 10 µM dasatinib; lower right: 30 µM PRT060318.

 

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Conflicts of interest

None declared.

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