Platelet-collagen adhesion: inhibition by a monoclonal antibody that binds glycoprotein IIb.

Abstract

TO identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr ~ 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein Ilba, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins lib and III form a complex.Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited >80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein lib participates in some aspect of plateletcollagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of ~25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein lib is not the only molecule involved in this process.Antibodies are a major tool for identification of molecules that participate in cell adhesion. Iterative absorption of polyclonal antibodies has permitted identification of several cell adhesion molecules (1). An additional approach has employed monoclonal antibodies, which offer the advantage of exquisite immunologic specificity. These have been used to identify molecules involved in the adhesion of myoblasts (2) and melanoma cells (3) to surfaces in tissue culture, and of Dictyostelium discoideum cells to each other (4).In the preceding article, we described a polyclonal antiserum raised against whole human blood platelets that was employed to identify and partially purify neutralizing antigens involved in platelet-collagen adhesion (5). This work was, however, greatly complicated by the finding that the adhesion that was measured in this assay apparently involved several immunologically distinct molecular entities, each of which only partially neutralized the polyclonal antiserum. Because of the complexities of the system, we turned to monoclonal antibodies to develop monospecific reagents that might react with the relevant entities individually, permitting their clear identification and independent characterization. We describe here a monoclonal antibody that reacts specifically with platelet membrane glycoprotein IIb and blocks platelet-collagen adhesion. The platelet antigen purified by immunoaffinity chromatography, using immobilized monoclonal antibody, only partly neutralizes the polyclonal antiserum described in the preceding article (5), supporting the conclusion that multiple entities are involved in platelet-collagen adhesio...

Isolation of a subpopulation of glycoprotein IIb-III from platelet membranes that is bound to membrane actin.

“…The mouse monoclonal antibody (PMi-l) was prepared and thoroughly characterized elsewhere by Shadle et al (38) and was kindly provided by Dr. Mark H. Ginsberg (Scripps Clinic and Research Foundation, La Jolla, CA). In brief, this monoclonal antibody was derived from mice immunized with platelet plasma membranes and was selected on the basis of its ability to inhibit platelet adhesion to collagen.…”

Factor XIII is not involved in human platelet-collagen interaction.

“…Several platelet membrane proteins have been reported as the receptor for the collagen which is a major component in the vessel wall (Shadle et al 1984;Nieuwenhuis et al 1986 ;Chiang et al 1987 ;Sugiyama et al 1987). Platelet FXIII was recently described as collagen receptor for bovine platelet-collagen aggregation interaction (Saito et al 1986).…”

“…Collagen is one of the important components among the vascular subendothelium and several reports on the platelet membrane receptors which are involved in platelet-collagen interaction have been described in recent years (Shadle et al 1984; Tsunehisa et al 1984;Chiang et al 1987; Kotite and Cunningham 1986;Nieuwenhuis et al 1986 ; Sugiyama et al 1987). Saito et al (1986) have reported that bovine factor XIII (FXIII) may be a platelet receptor for collagen.…”

A Novel Anti-platelet Monoclonal Antibody (3C7) Specific for the Complexof Integrin αIIbβ3 Inhibits PlateletAggregation and Adhesion

“…Our strategy allowed recognition of epitopes not exposed or inaccessible on the resting integrin. In contrast, all other anti-LIBS or activation-dependent mAbs were produced using either whole platelets (27), platelet membranes (26,28), purified ␣ IIb ␤ 3 (5), purified ␣ IIb ␤ 3 mixed with RGDS (4), or synthetic ␣ IIb ␤ 3 peptides (29) as the immunogen. In addition, a single-chain variable fragment specific for activated ␣ IIb ␤ 3 was obtained using phage display against activated platelets (30).…”

“…Three types of anti-LIBS antibodies against ␣ IIb or ␤ 3 are described in the literature (a) with no effect on platelet aggregation (PMI-1, ␣ IIb -specific) (25,26), (b) with inhibition of platelet aggregation (anti-LIBS-1, ␤ 3 -specific) (4), and (c) with activation of ␣ IIb ␤ 3 (D3GP3, anti-LIBS-2, -3, -6, ␤ 3 -specific) (4,5,22). To our knowledge, 3C7 is the first anti-LIBS mAb against the complex of ␣ IIb ␤ 3 .…”