title: Purification and preliminary characterization of the glycoprotein Ib complex in the human platelet membrane.
abstract: Human platelet glycoprotein Ib (GP Ib) is a major integral membrane protein that has been identified as the platelet-binding site mediating the factor VIII/von Willebrand-factor-dependent adhesion of platelets to vascular subendothelium. Recent evidence suggests that GP Ib is normally complexed with another platelet membrane protein, GP IX. In this study, human platelet plasma membranes were selectively solubilized with a buffer containing 0.1% (v/v) Triton X-100. The GP Ib complex (GP Ib plus GP IX) was purified to homogeneity in approximately 30% yield by immunoaffinity chromatography of the membrane extract using the anti-(glycoprotein Ib complex) murine monoclonal antibody, WM 23, coupled to agarose. GP Ib and GP IX were subsequently isolated as purified components by immunoaffinity chromatography of the GP Ib complex using a second anti-(glycoprotein Ib complex) monoclonal antibody, FMC 25, coupled to agarose. As assessed by dodecyl sulphate/polyacrylamide gel electrophoresis, purified GP Ib was identical to the molecule on intact platelets and had an apparent relative molecular mass of 170 000 under nonreducing conditions and 135 000 (alpha subunit) and 25 000 (beta subunit) under reducing conditions. GP IX had an apparent Mr of 22 000 under both nonreducing and reducing conditions. Purified Gb Ib complex and GP Ib inhibited the ristocetin-mediated, human factor VIII/von Willebrand-factor-dependent and bovine factor VIII/von Willebrand-factor-dependent agglutination of washed human platelets suggesting the proteins had been isolated in functionally active form. GP Ib alpha had a similar amino acid composition to that previously reported for its proteolytic degradation product, glycocalicin. The amino acid compositions of GP Ib beta and GP IX were similar but showed marked differences in the levels of glutamic acid, alanine, histidine and arginine. The N-termini of GP Ib alpha and GP IX were blocked; GP Ib beta had the N-terminal sequence, Ile-Pro-Ala-Pro-. On crossed immunoelectrophoresis, both GP Ib and GP IX were found to occur in the same immunoprecipitin arc(s) whether the platelets had been solubilized in the absence or presence of the calcium-dependent protease inhibitor, leupeptin. Binding studies in platelet-rich plasma indicated a similar number of binding sites (means +/- SD) for three anti-(glycoprotein Ib complex) monoclonal antibodies: AN 51, epitope on GP Ib alpha (22 000 +/- 2700, n = 3), WM 23, epitope on GP Ib alpha (21 000 +/- 3400, n = 3), FMC 25, epitope on GP IX (20 100 +/- 2700, n = 3), and FMC 25 (Fab')2 (27 100 +/- 800, n = 2).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.forename: M C
authors.initials: MC
authors.lastname: Berndt
authors.forename: C
authors.initials: C
authors.lastname: Gregory
authors.forename: A
authors.initials: A
authors.lastname: Kabral
authors.forename: H
authors.initials: H
authors.lastname: Zola
authors.forename: D
authors.initials: D
authors.lastname: Fournier
authors.forename: P A
authors.initials: PA
authors.lastname: Castaldi
mesh_terms: D000328:Adult; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000032:analysis*; D011232:Chemical Precipitation / Q000379:methods; D002846:Chromatography, Affinity; D005169:Factor VIII; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification*; D006801:Humans; D007120:Immunochemistry; D007123:Immunoelectrophoresis, Two-Dimensional; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification*; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor
chemical_list: D000596:Amino Acids; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D014841:von Willebrand Factor; D005169:Factor VIII