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GPIbα shedding is restricted to the inner platelet membrane

1Belgian Red Cross Flanders, Ghent, Oost-Vlaanderen, Belgium, 21. Transfusion Research Center, Belgian Red Cross Flanders, Ghent, Oost-Vlaanderen, Belgium, 32. Department of Biomedical Molecular Biology and Expertise Centre for Transmission Electron Microscopy, Ghent University - 3. Center for Inflammation Research and BioImaging Core, VIB, Ghent, Oost-Vlaanderen, Belgium, 44. Department of Biological Regulation, Weizmann Institute of Science, Rehovot, HaMerkaz, Israel, 5John Curtin School of Medical Research, The Australian National University, Canberra, Australian Capital Territory, Australia, 61. Transfusion Research Center, Belgian Red Cross Flanders - 6. Blood services, Belgian Red Cross Flanders - 7. Faculty of Medicine and Health Sciences, Ghent University, Ghent, Oost-Vlaanderen, Belgium, 71. Transfusion Research Center, Belgian Red Cross Flanders - 7. Faculty of Medicine and Health Sciences, Ghent University, Ghent, Oost-Vlaanderen, Belgium

Abstract Number: PB0385

Meeting: ISTH 2022 Congress

Theme: Platelets and Megakaryocytes » Platelet Receptors

Background: GPIbα is an abundant transmembrane receptor on the platelet cytoplasmic membrane. Following strong and sustained platelet activation, ADAM17 catalyzes GPIbα proteolysis thereby releasing the soluble glycocalicin ectodomain.

Aims: We aimed to understand the enzyme-substrate relationship in space and time.

Methods: ADAM17 and GPIbα were detected using labeled monoclonal antibodies in human resting platelets or after activation via GPVI and PARs, using immunofluorescence and western blot analysis.

Results: To chase glycocalicin, resting platelets were preincubated with anti-GPIbα monoclonal antibody before activation. After activation, the majority of antibody was detected in the platelet pellet with only 11.2±4.0% released in supernatant despite 82.1±4.9% of GPIbα effectively shed. Furthermore, preincubation of resting platelets with anti-GPIbα antibody 5G6, targeting the ADAM17 cleavage site, did not inhibit glycocalicin release (93±6% relative to control without 5G6). When 5G6 was present during activation, glycocalicin release was partially inhibited (59±11% relative to control, p=0.02; n=5). Finally, pretreatment of platelets with the membrane impermeable O-sialoglycoprotein endopeptidase removed a 45kDa N-terminal fragment of surface-exposed GPIbα but had no effect on the molecular weight or amount of glycocalicin released in platelet supernatant following activation-dependent shedding. Statistically, >80% of GPIbα was an ADAM17 substrate, while < 2% of released GPIbα was surface-exposed. This agrees with our transmission electron microscopy findings using anti-ADAM17 immunogold labeling, showing ADAM17 resides almost exclusively (96±2% of label; n≥25 platelets) within the inner membranes of both resting and activated platelets. This intracellular detection was confirmed by immunofluorescence microscopy and flow cytometry where successful ADAM17-antigen detection required permeabilization with saponin. In addition, only membrane-permeable sheddase inhibitors marimastat, GM6001 and KP-457 significantly inhibited GPIbα shedding (88±5%, 88±4% and 95±1%, respectively). In contrast, membrane-impermeable ADAM17 inhibitors D1A12 (11±11%) and recombinant prodomain (7±10%; ≥6) could not inhibit metalloproteolysis of GPIbα.

Conclusion(s): The majority of GPIbα is a substrate for ADAM17, but surface-exposed GPIbα is not.

To cite this abstract in AMA style:

Six K, Van den Hauwe M, De Rycke R, Sagi I, Gardiner E, Compernolle V, Feys H. GPIbα shedding is restricted to the inner platelet membrane [abstract]. https://abstracts.isth.org/abstract/gpib%ce%b1-shedding-is-restricted-to-the-inner-platelet-membrane/. Accessed September 29, 2023.

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