Transition of platelet receptor shedding platelet-specific collagen receptor GPVI Al-Tamimi, Mohammad 10.4225/03/58d1d7b238db6 https://bridges.monash.edu/articles/thesis/Transition_of_platelet_receptor_shedding_platelet-specific_collagen_receptor_GPVI/4775104 The platelet collagen receptor, glycoprotein (GP)VI, plays a central role in thrombus formation following collagen exposure due to injury or atherothrombotic disease. GPVI on normal circulating human platelets undergoes metalloproteinase-mediated ectodomain shedding in response to collagen or other ligands in vitro, generating an ~55-kDa soluble fragment (sGPVI). However, GPVI shedding from human platelets in vivo in thrombotic or autoimmune disease has not been previously investigated. To begin developing new assays for analysing GPVI shedding, a panel of eight murine anti-human GPVI monoclonal antibodies (mAbs) was characterised for their effects on GPVI-dependent platelet aggregation and GPVI shedding. All mAbs raised against a purified recombinant GPVI ectodomain fragment encompassing two immunoglobulin domains (residues 21-234, excluding the signal sequence) recognised GPVI in human platelet lysates by western blotting. The majority of mAbs induced aggregation of human platelets, independently of the Fc receptor, FcgammaRIIa (not inhibited by the anti-FcgammaRIIa mAb, IV.3). One mAb neither induced aggregation nor inhibited aggregation in response to collagen or collagen-related peptide (CRP). In contrast, Fab fragment of mAb 12A5 strongly blocked collagen- or CRP-, but not convulxin-induced aggregation. Activating anti-GPVI mAbs also induced metalloproteinase-dependent GPVI shedding generating sGPVI. One anti-GPVI mAb, 1A12, was subsequently used to develop a new assay for quantitative measurement of sGPVI in plasma. A sandwich enzyme-linked immunosorbent assay (ELISA) to measure sGPVI in plasma used rabbit anti-GPVI polyclonal antibody in the solid-phase, the murine anti-GPVI mAb, 1A12, in the fluid-phase and horseradish peroxidase (HRP)-coupled anti-mouse IgG antibody and enhanced chemiluminescence (ECL) for detection. The ELISA was optimised for sensitivity, reproducibility, inter- and intra-assay precision, addition and recovery and detected GPVI in plasma with a lower detection limit of ~1 ng/ml. Purified GPVI ectodomain serially diluted in GPVI-deficient plasma (normal plasma immunodepleted using the anti-GPVI mAb, 1G5) was used to construct a standard curve (0-100 ng/ml). Effects of different anti-coagulants (trisodium citrate, acid-citrate-dextrose or EDTA) were negligible. sGPVI measurements were not due to platelet-derived microparticles, since levels were unaffected by centrifugation at 100,000g for 1 h. In 10 healthy donors (5 female) plasma sGPVI levels were 18.9 ± 4.1 ng/ml (range 11-24 ng/ml). Treating normal platelet-rich plasma with CRP, calmodulin inhibitor W7 (induces GPVI shedding) or N-ethylmaleimide (directly activates sheddases), under conditions previously shown to induce GPVI shedding in washed platelets, also increased plasma sGPVI levels by up to ~7-fold (inhibitable by the metalloproteinase inhibitor, GM6001). The ELISA for quantitation of sGPVI in human plasma was used to identify new mechanisms for inducing GPVI shedding in the absence of collagen. First, it was shown that metalloproteinase-dependent GPVI shedding was induced by pathological shear rates (>3,000 s-1) without a requirement for VWF/GPIbα interaction, αIIbβ3 engagement, or platelet activation (normal shedding in VWF-deficient plasma, or in the presence of inhibitory anti-VWF or anti-GPIbα mAbs, or αIIbβ3 inhibitors). However, shear-induced shedding was enhanced by the cholesterol-depleting agent methyl-β-cyclodextrin (MβCD), which activates sheddases on other cells. Second, metalloproteinase-mediated GPVI shedding was induced by coagulation with or without added tissue factor. Shedding was mediated by Factor Xa (FXa) as indicated by the ability of a FXa-selective inhibitor to block coagulation-induced GPVI shedding, the ability of Russell viper venom (RVV) that activates FX to generate sGPVI, impaired GPVI shedding during coagulation of washed platelets resuspended in FX-deficient plasma (restored by adding FX), and the ability of purified FXa to induce GPVI shedding from washed platelets. Based on the ability of collagen or anti-GPVI mAbs activating platelets via GPVI, and murine anti-platelet mAbs or heparin-induced thrombocytopenia (HIT)-related human autoantibodies activating platelets via FcgammaRIIa to induce GPVI shedding in vitro, sGPVI was assessed in thrombotic and autoimmune diseases. In 159 community-based donors, sGPVI was 19.6 ± 8.1 ng/ml, and not linked to age or gender. sGPVI measured in thrombotic diseases including 159 patients with acute ischaemic stroke and 10 patients with acute coronary syndrome (ACS) was significantly increased compared to matched controls. In autoimmune disease, including one case of idiopathic thrombocytopenia purpura with an anti-GPVI autoantibody that mediated GPVI shedding, sGPVI was increased ~10-fold compared to healthy controls. In 159 suspected HIT patients, and 92 patients with thrombocytopenia not related to heparin, sGPVI levels were significantly higher in HIT-positive patients compared to thrombocytopenia or HIT-negative controls, and there was a strong correlation with levels of HIT-related antibody and capacity of patient plasma + heparin to induce aggregation of healthy donor platelets. Importantly, sGPVI levels >59 ng/ml were predictive of HIT diagnosis with 83% sensitivity and 71% specificity. Moreover, using the ability of HIT-positive plasma to induce heparin-dependent release of sGPVI from normal platelets, a novel ‘sGPVI release’ assay was optimised and found to have similar sensitivity (75%), but higher specificity (96%). In summary, an ELISA for measuring sGPVI in human plasma has been developed, and three novel mechanisms for inducing GPVI shedding identified (anti-GPVI antibodies, pathological shear rates, and activation of coagulation/FXa). GPVI shedding was demonstrated for the first time in vivo in acute ischaemic stroke and autoimmune disease. The sGPVI ELISA and release assays, have been validated experimentally and should be useful research tools for many future clinical applications. The basic knowledge of GPVI downregualtion by shedding has been applied to develop new assays that identified new mechanisms mediating GPVI shedding in human in vitro, and confirmed the occurrence of shedding in vivo in pathological conditions. Translation of basic knowledge to identify the patho-phsysiological mechanisms mediating human diseases can provide new tools and targets for improving the clinical outcome of human diseases. 2017-05-19 02:30:21 Shedding Collagen HIT monash:64606 GPVI thesis(doctorate) ethesis-20110523-152055 Platelets Thrombosis Restricted access and full embargo Stroke Coagulation 1959.1/488928 2011 ITP