Journal
JOURNAL OF THROMBOSIS AND HAEMOSTASIS
Volume 6, Issue 10, Pages 1789-1795Publisher
WILEY
DOI: 10.1111/j.1538-7836.2008.03088.x
Keywords
GPIb-IX; inside-out regulation; membrane-proximal disulfide bonds; VWF binding
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Funding
- National Institutes of Health [HL082808]
- American Heart Association [0565078Y]
- Welch Foundation [AU-1581]
- Harry S. and Isabel C. Cameron Foundation Fellowship
- Howard Temin Award [CA096706]
- NATIONAL CANCER INSTITUTE [K01CA096706] Funding Source: NIH RePORTER
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL082808] Funding Source: NIH RePORTER
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Background: In the platelet glycoprotein (GP)Ib-IX complex, the binding site for its ligand von Willebrand factor (VWF) is restricted to the N-terminal domain of the GPIb alpha subunit. How the other subunits in the complex, GPIb beta and GPIX, regulate the GPIb alpha-VWF interaction is not clear. Objectives and methods: As GPIb alpha connects with two GPIb beta subunits via disulfide bonds, we tested whether these intersubunit covalent links were important to the proper VWF-binding activity of the GPIb-IX complex by characterizing the structure and VWF-binding activity of a mutant GPIb-IX complex that lacked the GPIb alpha-GPIb beta disulfide bonds. Results: Mutating both Cys484 and Cys485 of GPIb alpha to serine prevents GPIb alpha from forming covalent disulfide bonds with GPIb beta, while maintaining the integrity of the complex in the membrane. The mutations cause two GPIb beta subunits to form a disulfide bond between themselves. As compared to Chinese hamster ovary (CHO) cells stably expressing the wild-type GPIb-IX complex at a comparable level, CHO cells stably expressing the mutant GPIb-IX complex bind to significantly less soluble VWF in the presence of ristocetin and roll on the immobilized VWF under flow at a higher velocity. Conclusions: The disulfide bonds between GPIb alpha and GPIb beta are necessary for optimal GPIb alpha binding to VWF. The structural plasticity around the disulfide bonds may also help to shed light on the inside-out mechanism underlying GPIb beta modulation of VWF binding.
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