Expression and Purification of a Soluble Functional form of the Platelet αIIbβ3 Integrin

Abstract

Platelet glycoproteins aIIb and p3 are membrane proteins that associate to form a Ca'+-dependent heterodimer which constitutes an inducible member of the integrin family at the surface of the cell. To produce a soluble form of this complex, aIIb and p3 were both deleted of their transmembrane and cytoplasmic domains and were expressed in COS cells. Production of the truncated subunits and their mode of assembly were examined by immunoprecipitation experiments and compared to those of wildtype aIIbfl. Synthesis and processing of the truncated heterodimer proceeded via a pathway similar to that observed for the wild-type aIIbp3 in COS cells or in human megakaryocytes. The truncated p3 subunit associated with the Pro-truncated form of the aIIb subunit. This precursor form was not secreted. After proteolytic cleavage of the Pro-truncated aIIb, the mature heterodimer was secreted into the culture supernatant. To quantify the molar ratio of the various secreted soluble forms, an immunocapture assay was designed. All secreted tr-aIIb subunits associated with tr-p3. In contrast, tr-p3 was produced and secreted in excess as the free form. Immunoreactivity of the wild-type and soluble truncated complexes was identical since all the monoclonal antibodies used reacted with surface-located epitopes on both complexes. This indicated that the soluble truncated heterodimer adopted a native conformation. To purify this soluble heterodimer, tr-aIIbp3-containing culture supernatant was adsorbed on an RGDW-affinity column and eluted with a solution of the free peptide RGDW. In the RGD-eluted material, the amount of each subunit was stoichiometric, suggesting that the complex was not disrupted during purification. The capacity of the wild-type and truncated RGD-eluted complexes to interact with soluble fibrinogen was compared using a solid-phase immunocapture assay. tr-aIIbp3 and platelet aIIbp3 exhibited similar fibrinogen-binding capacity. For both complexes, these interactions were mediated by RGD and y fibrinogen signals.Keywords. Integrin, aIIbp3 ; soluble form; aIIbp3 integrin.Platelet glycoprotein IIbIIIa is a member of the p3 integrin subfamily which functions as a receptor for adhesive proteins at the surface of platelet. The molecule has a loose specificity as it interacts with various multivalent adhesive proteins including fibrinogen [l,21, von Willebrand factor [3], fibronectin [4] and vitronectin [5]. The glycoproteins IIb (or aIIb) and IIIa (or 83) subunits are synthesized separately in the megakaryocyte which is the precursor cell of a platelet in the bone marrow. The p3 subunit is produced in excess and stored, while the aIIb subunit is produced as a precursor form and is immediately associated with the p3 subunit prior to its proteolytic maturation and its expression at the surface of the cell [6]. Neither of the subunits alone is surface-exposed or secreted. They form a calcium-dependent heterodimer and are exclusively implicated in platelet adhesion and aggregation.aIIb/33 exemplifies a typical integrin which is ...

Disruption of the β3 663-687 disulfide bridge confers constitutive activity to β3 integrins

“…15 The underlying molecular mechanism for the thrombasthenic phenotype was the failure of ␤ 3 ⌬616 to complex ␣ IIb . This observation appeared to be in conflict with previous reports in which ␤ 3 -truncated proteins complex ␣ subunits 14,18,19 and the mutated complexes reached the cell surface. 14 Because the apparent discrepancy could be due to differences in the lengths of the deleted fragments, we performed a transient transfection analysis of progressive carboxy-terminal or internally deleted forms of ␤ 3 .…”

Identification of a region in glycoprotein IIIa involved in subunit association with glycoprotein IIb: further lessons from Iraqi-Jewish Glanzmann thrombasthenia

“…Moreover, cells transfected with cDNA constructs that excluded the transmembrane and cytoplasmic domains of both GPIIIa and GPIIb still produced GPIIb/IIIa complexes, which were secreted from the cells but were not surface expressed. 16,17 Another study showed that cells transfected with normal GPIIb cDNA and ⌬616-762 GPIIIa cDNA with a predictable exclusion of the transmembrane, the intracellular domains, and a segment of the extracellular domain did not form GPIIb/IIIa complexes. 20 Our data demonstrate that the lack of an even smaller segment of the extracellular domain (⌬657-762) results in the lack of intracellular GPIIb/IIIa complex formation.…”

“…GPIIIa from cells containing IJ-1 and Stop657 migrated more rapidly, consistent with the presence of truncated forms of GPIIIa ( Figure 6A). Given that previous studies showed that truncated forms of GPIIb and GPIIIa still formed soluble complexes and that these complexes as well as truncated GPIIIa were secreted by cells, 16,17 we examined the culture medium of IJ-1 and Stop657 cells for GPIIIa. Neither by immunoprecipitation and Western blotting nor by metabolic labeling of the cells and immunoprecipitation with AP5 antibody could we detect any secreted form of GPIIIa in the medium (data not shown).…”

A plant surface protein sharing structural properties with animal integrins

“…5 B). Such an immunological cross-reactivity between a 70Ϫ72-kDa soybean plasma membrane component and the β3 subunit has Characterization of AcAt2 a polyclonal antibody raised against A. thaliana glycoproteins purified by RGD peptide already been observed (Schindler et al, 1989) and recently a 58-kDa polypeptide present in total protein extracts of Arabiaffinity. AcAt2, a polyclonal antibody, was raised against Arabidopsis glycocoproteins purified by affinity chromatography on a dopsis shoots and roots has been shown to cross-react with the antibody against chicken β1 polypeptide ( Katembe et al, 1997).…”

“…A. thaliana and Rubus protoplasts generated by enzymatic digestion of the cell wall Evidence for the presence of integrin-like proteins relies on the use of exogenous RGD-containing peptides that induce aber-were probed using a 1:100 dilution of rabbit polyclonal antibodies P23 in protoplast buffer (Gamborg's medium containing rant cell wall-plasma membrane interactions and enhanced growth rate of soybean suspension culture (Schindler et al, sucrose 0.06 M and mannitol 0.56 M) or, as a control, rabbit non-immune serum. All samples were then washed with proto-1989), inhibit gravisensing in Characean algae (Wayne et al, 1992) and block the clumping normally seen in protoplasts of plast buffer and incubated with a goat anti-(rabbit IgG)-fluorescein conjugate and viewed with a Nikon Microphot-FXA microsalt-adapted tobacco cells (Zhu et al, 1993), or on immunoblot cross-reactivity with antisera to β3 (Schindler et al, 1989) and scope (Nikon Europe B.V.). The presence of cell wall in the protoplast preparation was examined with the dye Calcofluor, a β1 integrin subunit (Quatrano et al, 1991;Kaminskyj and Heath, 1995;Gens et al, 1996;Katembe et al, 1997).…”