GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate.

Background: A disintegrin and metalloprotease 17 (ADAM17) catalyzes platelet glycoprotein (GP) Ibα ectodomain shedding, thereby releasing glycocalicin in plasma. The spatiotemporal control over the enzyme-substrate interaction and the biological consequences of GPIbα shedding are poorly understood.
Objectives: This study aimed to determine the spatiotemporal control over GPIbα shedding by ADAM17.
Methods: Transmission electron microscopy with immunogold staining, immunoprecipitation, and quantitative western blotting were used.
Results: Immunogold staining showed that all ADAM17 antigen is expressed intracellularly, irrespective of platelet activation. ADAM17 clustered in patches on a tortuous membrane system different from α- and dense granules. Mild activation by platelet adhesion to immobilized fibrinogen did not cause GPIbα shedding, whereas strong and sustained stimulation using thrombin and collagen (analogs) did. Glycocalicin release kinetics was considerably slower than typical hemostasis, starting at 20 minutes and reaching a plateau after 3 hours of strong stimulation. Inhibition of the ADAM17 scissile bond specifically in GPIbα receptors that reside on the platelet's extracellular surface did not prevent shedding, which is in line with the strict intracellular location of ADAM17. Instead, shedding was restricted to a large GPIbα subpopulation that is inaccessible on resting platelets but becomes partially accessible following platelet stimulation. Furthermore, the data show that proteinaceous, water-soluble ADAM17 inhibitors cannot inhibit GPIbα shedding, whereas membrane permeable small molecule ADAM inhibitors can.
Conclusion: The data show that platelets harbor 2 distinct GPIbα subpopulations: one that presents at the platelet's surface known for its role in primary hemostasis and one that provides substrate for proteolysis by ADAM17 with kinetics that suggest a role beyond hemostasis.
Competing Interests: Declaration of competing interests There are no competing interests to disclose.
(Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)