Platelets are small anucleate blood cells that survey the integrity of the vessel wall and are key players of blood clotting. Beyond their classical role in haemostasis, they have emerged as key effectors of innate immunity and thrombo-inflammation. These thrombo-inflammatory activities differ fundamentally from their haemostatic functions. Similarly, platelets are essential in the prevention of ‘inflammatory bleeding’ caused by loss of vascular integrity in the inflamed tissue, a process that is mechanistically not understood. Furthermore, the molecular mechanisms driving platelet-mediated haemostasis and thrombo-inflammation can differ between organs and involve partly different receptor-ligand interactions and signalling pathways. We postulate that the balance of immunoreceptor tyrosine-based activation/ inhibition motif (ITAM/ITIM) receptors (notably CLEC-2, GPVI, G6b-B)-ligand interactions and signalling largely controls these processes, but the underlying mechanisms and involved ligands have remained elusive.

P01 aims to better understand why and how the main ITAM/ITIM-coupled receptors and their signalling pathways are differentially employed and alter their functional crosstalk in different (patho-)physiological settings. To this end, antibody-based G6b-B as well as CLEC-2 inhibitors (monovalent) and stimulators (presumably di- or polyvalent) will be developed and used to modulate their function in platelets in vitro and in vivo. We aim to i) identify the postulated vascular CLEC-2/G6b-B ligand(s) and their expression/regulation, ii) dissect the functional crosstalk of CLEC-2, G6b-B and the main platelet adhesion receptor, GPIIb/IIIa, in experimental cerebral venous thrombosis (CVT), and iii) identify functional redundancy/ crosstalk of CLEC 2/GPVI/G6b-B in (inflammatory) haemostasis.

ECR.P01.01 will (i) generate (hybridoma technology) and characterize function modulating monoclonal antibodies (mAbs) against human/mouse CLEC-2 and G6b-B, (ii) use biochemical and cell biological approaches to identify blood-borne ligands of the two receptors, (iii) investigate their regulation and crosstalk in vitro and in genetic mouse models in vivo, and collaborate with the Bioimaging Center at the Rudolf Virchow Center (Head: Prof. Dr. Katrin Heinze) in the development of mAb/Fab-based labelling protocols for ITAM-ITIM receptors as well as custom imaging pipelines to study their localisation and cellular regulation in platelets.