Mouse podoplanin supports adhesion and aggregation of platelets under arterial shear: A novel mechanism of haemostasis

Abstract

The podoplanin-CLEC-2 axis is critical in mice for prevention of hemorrhage in the cerebral vasculature during mid-gestation. This raises the question as to how platelets are captured by podoplanin on neuroepithelial cells in a high shear environment. In this study, we demonstrate that mouse platelets form stable aggregates on mouse podoplanin at arterial shear through a CLEC-2 and Src kinase-dependent pathway. Adhesion and aggregation are also dependent on the platelet glycoprotein (GP) receptors, integrin αIIbβ3 and GPIb, and the feedback agonists ADP and thromboxane A₂ (TxA₂). CLEC-2 does not bind to von Willebrand factor (VWF) suggesting that the interaction with podoplanin is sufficient to both tether and activate platelets. Consistent with this, the surface plasmon resonance measurements reveal that mouse CLEC-2 binds to mouse podoplanin with nanomolar affinity. The present findings demonstrate a novel pathway of hemostasis in which podoplanin supports platelet capture and activation at arteriolar rates of shear.

Keywords:

• Capillary flow assay
• CLEC-2
• platelet activation
• platelet aggregation
• podoplanin

Acknowledgements

The authors would like to thank Jamie R. McFarlane Webster for work performed in the protein expression facility (PEF) in the University of Birmingham in the production of the recombinant forms of podoplanin, Prof. Bernard Nieswandt for kindly providing p0p/B antibody, Beata Grygielska for technical assistance, Maria Hoellerer for expression plasmids, and Siân Lax for her assistance in preparing the manuscript.

Declaration of interest

The authors declare no known conflicts of interest

Funding

SEL is a Wellcome Trust Prize Student (ref 099850). CEH and AYP were supported by the Wellcome Trust 088410. SPW holds a BHF Chair (CH/03/003).

Supplemental data

Supplemental data for this article can be accessed on the publisher’s website.

Additional information

Funding

SEL is a Wellcome Trust Prize Student (ref 099850). CEH and AYP were supported by the Wellcome Trust 088410. SPW holds a BHF Chair (CH/03/003).