Platelet-collagen adhesion: evidence for participation of antigenically distinct entities.

Abstract

Univalent antibody fragments prepared from a rabbit antiserum raised against whole human platelets completely inhibited adhesion of platelets to immobilized trimeric collagen in a defined, Mg2+-dependent, adhesion assay. An octylglucoside extract of whole platelets completely neutralized this antibody, and all neutralizing activity bound to immobilized wheat germ agglutinin. Further fractionation on concanavalin A gave rise to subfractions that each neutralized only partially at saturation, when tested against antibody concentrations that inhibit 50% of platelet-collagen adhesion. When tested against higher antibody concentrations that completely inhibited adhesion, each subfraction had no detectable neutralizing effect, although the combined subfractions neutralized completely. This and other evidence suggests that more than one platelet entity participates in platelet-collagen adhesion. Although distinct, they appear to play interdependent roles in a single adhesion process.

Divalent cation regulation of the surface orientation of platelet membrane glycoprotein IIb. Correlation with fibrinogen binding function and definition of a novel variant of Glanzmann's thrombasthenia.

“…The lack of difference in tryptic peptide maps does not exclude qualitative difference in the GP in that only one protease was employed, only radiolabeled peptides were visualized, and minor sequence changes may not (30), and cross-linking studies (42) has implicated GPIIb in platelet adhesion to collagen. 'The work of Shadle and Barondes (43) suggests that GPIIb is one of a multiplicity of platelet proteins that neutralize the ability of antiplatelet antibodies to inhibit Mg-dependent platelet adhesion to trimeric chick skin type I collagen. In addition, PMI-1, an antiGPIIb, blocked adhesion (25).…”

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

“…Immunoaffinity chromatography was performed essentially as described by McEver et al (l 3). Detergent extracts of outdated platelets were prepared using 1% octylglucoside or 1% Triton X-100 as described (5). Samples were dialyzed against Tris saline (15 mN Tris-HC1, pH 7.4, 126 mM NaC1, 5.4 mM KCI, 5 x 10 -3 M CaC12, 1 mM MgCl2, 0.1% glucose) to reduce detergent concentration, applied to the column, and recirculated through the column for 24 h at 4"C to maximize specific binding.…”

“…This was performed as described previously (5,14). Screening the effects of tissue culture supernatants from hybridoma clones was done by preincubating 5JCr-labeled platelets with dilutions of the supernatants that had been heat inactivated at 56"C for 20 min, then measuring platelet-collagen adhesion in the normal way.…”

“…Screening the effects of tissue culture supernatants from hybridoma clones was done by preincubating 5JCr-labeled platelets with dilutions of the supernatants that had been heat inactivated at 56"C for 20 min, then measuring platelet-collagen adhesion in the normal way. Measurement of the effects of Fab prepared from monoclonal antibodies on platelet-coUagen adhesion, and neutralizing effects of various antigens was done as described previously (5).…”

“…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.…”