Abstract/Summary

Glycoprotein receptor VI (GPVI) is the major collagen receptor in platelets. Ligand binding induces GPVI clustering, which initiates a tyrosine kinase-based signalling cascade via an immunoreceptor tyrosine-based activation motif (ITAM). GPVI has been shown to play roles in both the initiation and growth of thrombi, although GPVI deletion is not associated with significant bleeding. Therefore, targeting the GPVI pathway is a potential route to overcome the bleeding risk associated with current therapies. G6b-B is a glycoprotein receptor with restricted expression to platelets membrane surface that inhibits platelet activation by ITAM receptors. Crosslinking with G6b-B antibodies prevents platelet activation. We hypothesize that it will be possible to overcome the bleeding risk of current antithrombotics, which target other platelet activation pathways, by inhibiting GPVI-mediated pathway molecules or activating G6b-B using novel biologics, such as Affimers. To inhibit the GPVI pathway we generated novel anti-human GPVI monoclonal antibodies (mAbs) and their F(ab) fragments (developed prior to the start of this project by Emfret Analytics Würzburg, Germany). The aim was to determine their mode of action and to which epitopes or regions of the protein they bind. Four new anti-GPVI mAbs and their F (ab) fragments were characterised. Among the mAbs, E7 was the only antibody to fully block GPVI activity. A9 caused a minor inhibition suggestive of either direct competition for the CRP binding site, binding close enough to cause steric hindrance to its binding. GPVI-mediated platelet activation was inhibited by all four F(ab) fragments suggesting these have potential as a novel α-GPVI therapy. Structural characterization of these anti-GPVI mAbs (E12, E7, E2, D3 and A9) with GPVI was assessed with three complementary approaches, namely bio-layer interferometry (BLI), crystallography, and epitope mapping. BLI showed that none of the mAbs are monomer or dimer specific. Crystallographic studies were not successful and will need further future optimisation. GPVI chimeras were generated to identify that the mAbs were binding to the GPVI D1 domain, which is the ligand binding domain. Additional studies will be needed for a full structural characterization of these mAbs bound to GPVI which protein-based therapeutics are required to demonstrate during their development phase. Our other aim was to develop new biologics (Affimers) to target and activate the ITIM-receptor G6b-B, which constitutively inhibits platelet activation by ITAM-like receptors. The potential antithrombotic effect of G6b-B and whether G6b-B stimulation could lead to less reactive platelets, reducing the risk, or severity of thrombosis has not been extensively studied until now. Here we targeted for the first time an inhibitory pathway to downregulate GPVI by targeting G6b-B. Three Affimers were identified to bind G6b-B. Preliminary functional studies showed that these Affimers did not induce G6b-B to inhibit platelet activation through the GPVI activation pathway in classical in vitro platelet function assays (namely aggregometry). However, preliminary in vitro flow studies with Affimer 24 showed some potential to influence thrombus size on CRP coated surfaces. In conclusion, in this thesis we provide an insight of the first functional and structural characterization of new mAbs targeting human GPVI, with some showing potential as good candidates for antiplatelet therapy. Additionally, we show the first attempt to target an inhibitory pathway as anti-platelet therapy by developing Affimers against G6b-B. Further research is needed to explore whether G6b-B stimulation could lead to less reactive platelets reducing the risk, or severity of thrombotic disease without causing substantial bleeding.