The glycoprotein Ib complex of human blood platelets

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Abstract
Human glycoprotein Ib (GPIb) is a major integral membrane protein on human blood platelets responsible for the initial attachment of platelets to the exposed vascular subendothelium. In this report we describe an isolation method for a ‘GPIb complex’ as well as for its individual components. A three‐step procedure involving Triton X‐114 phase‐partition, affinity chromatography on wheat germ agglutinin and ion‐exchange chromatography on DEAE‐Sephacel yielded milligram quantities of purified GPIb complex. The single components of the complex were further purified by gel filtration on AcA34 in 0.1% sodium dodecyl sulfate. As well as GPIb, the complex contains GP17, actin‐binding protein, actin and a series of unidentified proteins with different molecular masses. In contrast to GPIbα, which is very rich in O‐linked oligosaccharides, sugar analysis revealed that GPIbβ and GP17 seem to have only N‐linked chains of the lactosamine type. The C‐terminal α‐chain remnant, which probably spans the plasma membrane, was identified and isolated for the first time. Western blotting with polyclonal rabbit anti‐GPIb antibodies and silver‐staining of one‐ or two‐dimensional dodecyl sulfate/polyacryl‐amide gels revealed that it has an apparent molecular mass of 20 kDa and is linked to GPIbβ by a disulfide bridge close to the membrane. The thrombin‐binding site on GPIb is located near the N‐terminus on a 40‐kDa fragment of GPIbα. A disulfide bridge in the N‐terminal region is not essential for thrombin binding to GPIb.

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