title: Anti-glycoprotein VI monoclonal antibodies directly aggregate platelets independently of FcgammaRIIa and induce GPVI ectodomain shedding.
abstract: Adhesion of circulating platelets to the blood vessel wall initiates thrombus formation in haemostasis and thrombotic disease. The platelet collagen receptor, glycoprotein (GP) VI, is critical for thrombus formation at arterial shear rates and is a potential therapeutic target for anti-thrombotic drugs. In this study, we evaluate eight newly-derived, purified murine anti-human GPVI monoclonal antibodies (mAbs) for their effect on GPVI-dependent platelet aggregation and GPVI ectodomain shedding. All mAbs were raised against the ligand-binding GPVI ectodomain encompassing two immunoglobulin domains (residues 21-234, excluding the signal sequence) and recognized full-length GPVI in human platelet lysates by western blotting. The majority of antibodies induced aggregation in both human platelet-rich plasma (PRP) and washed platelets independently of the Fc receptor, FcgammaRIIa (not inhibited by the blocking anti-FcgammaRIIa mAb, IV.3), whereas one mAb (11A7) neither induced aggregation nor inhibited aggregation in response to GPVI ligands, collagen, and collagen-related peptide (CRP). In contrast, Fab fragments of mAb 12A5 strongly blocked collagen- and CRP-, but not convulxin-induced aggregation. In addition, it is shown for the first time in vitro that anti-GPVI mAbs can induce metalloproteinase-dependent ectodomain shedding of human GPVI, generating an approximately 10-kDa remnant that remained platelet-associated and an approximately 55-kDa soluble fragment. In conclusion, this analysis of anti-GPVI mAbs provides useful tools for studying the functional role of platelet GPVI.
authors.affiliation: Department of Immunology, Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Al-Tamimi
authors.forename: Fi-Tjen
authors.initials: FT
authors.lastname: Mu
authors.forename: Jane F
authors.initials: JF
authors.lastname: Arthur
authors.forename: Yang
authors.initials: Y
authors.lastname: Shen
authors.forename: Masaaki
authors.initials: M
authors.lastname: Moroi
authors.forename: Michael C
authors.initials: MC
authors.lastname: Berndt
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Gardiner
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D051379:Mice; D010974:Platelet Aggregation / Q000502:physiology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D017452:Receptors, IgG / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C496124:FCGR3A protein, human; D007140:Immunoglobulin Fab Fragments; D010980:Platelet Membrane Glycoproteins; D017452:Receptors, IgG; C098341:platelet membrane glycoprotein VI