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

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Abstract
Background: A disintegrin and metalloprotease 17 (ADAM17) catalyzes platelet glycoprotein (GP) Iba ectodomain shedding, thereby releasing glycocalicin in plasma. The spatiotemporal control over the enzyme-substrate interaction and the biological consequences of GPIba shedding are poorly understood. Objectives: This study aimed to determine the spatiotemporal control over GPIba shedding by ADAM17. cipitation, 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 a- and dense granules. Mild activation by platelet adhesion to immobilized fibrinogen did not cause GPIba 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 GPIba 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 GPIba 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 GPIba shedding, whereas membrane permeable small molecule ADAM inhibitors can. Conclusion: The data show that platelets harbor 2 distinct GPIba 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.
Keywords
ADAM17, ectodomain shedding, enzyme, GPIb & alpha, platelet, GLYCOPROTEIN-IB, IN-VITRO, METALLOPROTEINASE INHIBITORS, MEMBRANE-GLYCOPROTEINS, IIB-IIIA, IX-V, ACTIVATION, REDISTRIBUTION, LOCALIZATION, GPIbα

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MLA
Six, Katrijn, et al. “GPIbα Shedding in Platelets Is Controlled by Strict Intracellular Containment of Both Enzyme and Substrate.” JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 21, no. 8, 2023, pp. 2223–35, doi:10.1016/j.jtha.2023.03.020.
APA
Six, K., Debaene, C., Van den Hauwe, M., De Rycke, R., Gardiner, E. E., Compernolle, V., & Feys, H. (2023). GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate. JOURNAL OF THROMBOSIS AND HAEMOSTASIS, 21(8), 2223–2235. https://doi.org/10.1016/j.jtha.2023.03.020
Chicago author-date
Six, Katrijn, Caitlin Debaene, Michelle Van den Hauwe, Riet De Rycke, Elizabeth E. Gardiner, Veerle Compernolle, and Hendrik Feys. 2023. “GPIbα Shedding in Platelets Is Controlled by Strict Intracellular Containment of Both Enzyme and Substrate.” JOURNAL OF THROMBOSIS AND HAEMOSTASIS 21 (8): 2223–35. https://doi.org/10.1016/j.jtha.2023.03.020.
Chicago author-date (all authors)
Six, Katrijn, Caitlin Debaene, Michelle Van den Hauwe, Riet De Rycke, Elizabeth E. Gardiner, Veerle Compernolle, and Hendrik Feys. 2023. “GPIbα Shedding in Platelets Is Controlled by Strict Intracellular Containment of Both Enzyme and Substrate.” JOURNAL OF THROMBOSIS AND HAEMOSTASIS 21 (8): 2223–2235. doi:10.1016/j.jtha.2023.03.020.
Vancouver
1.
Six K, Debaene C, Van den Hauwe M, De Rycke R, Gardiner EE, Compernolle V, et al. GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate. JOURNAL OF THROMBOSIS AND HAEMOSTASIS. 2023;21(8):2223–35.
IEEE
[1]
K. Six et al., “GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate,” JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 21, no. 8, pp. 2223–2235, 2023.
@article{01HK76T5Y8VRK7XKP41KPFR4XX,
  abstract     = {{Background: A disintegrin and metalloprotease 17 (ADAM17) catalyzes platelet glycoprotein (GP) Iba ectodomain shedding, thereby releasing glycocalicin in plasma. The spatiotemporal control over the enzyme-substrate interaction and the biological consequences of GPIba shedding are poorly understood. 

Objectives: This study aimed to determine the spatiotemporal control over GPIba shedding by ADAM17. cipitation, 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 a- and dense granules. Mild activation by platelet adhesion to immobilized fibrinogen did not cause GPIba 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 GPIba 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 GPIba 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 GPIba shedding, whereas membrane permeable small molecule ADAM inhibitors can. 

Conclusion: The data show that platelets harbor 2 distinct GPIba 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.}},
  author       = {{Six, Katrijn and Debaene, Caitlin and Van den Hauwe, Michelle and De Rycke, Riet and  Gardiner, Elizabeth E. and Compernolle, Veerle and Feys, Hendrik}},
  issn         = {{1538-7933}},
  journal      = {{JOURNAL OF THROMBOSIS AND HAEMOSTASIS}},
  keywords     = {{ADAM17,ectodomain shedding,enzyme,GPIb & alpha,platelet,GLYCOPROTEIN-IB,IN-VITRO,METALLOPROTEINASE INHIBITORS,MEMBRANE-GLYCOPROTEINS,IIB-IIIA,IX-V,ACTIVATION,REDISTRIBUTION,LOCALIZATION,GPIbα}},
  language     = {{eng}},
  number       = {{8}},
  pages        = {{2223--2235}},
  title        = {{GPIbα shedding in platelets is controlled by strict intracellular containment of both enzyme and substrate}},
  url          = {{http://doi.org/10.1016/j.jtha.2023.03.020}},
  volume       = {{21}},
  year         = {{2023}},
}

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