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...

No abstract

Embryonic chick muscle contains two developmentally regulated lectins, which may be involved in cell interactions. These endogenous lectins are assayed as agglutinins of appropriate test erythrocytes. One of these, called lectin-2, interacts with specific glycosaminoglycans, especially heparin and dermatan sulfate. Lectin-2 is present at constant levels in both chick fibroblast and chick muscle cells throughout 14 days of culture but is released into the medium of cultured embryonic muscle after 7-8 days of culture, soon after myoblast fusion. Lectin-2 interacts strongly with a component of substrate-attached material in embryonic muscle cultures which is extractable from the cutlure dishes with alkali after the cells have been removed with ethylediaminetetraacetic acid. The active component in the substrate-attached material appears to be a glycosaminoglycan that is a more potent inhibitor of lectin-2 agglutination activity than any of the known glycosaminoglycans that we have tested. The active material is degraded by chondroitinase ABC but not by chondrotinase AC, hyaluronidase, or proteolytic enzymes and thus appears to be similar to dermatan sulfate. The results of these studies raise the possibility that lectin-2 functions by interacting with glycosaminoglycans, either associated with the cell surface or with the extracellular matrix.

Extracts of young rat lung contain a heparin-inhibitable lectin that closely resembles one recently purified from chicken liver. Both lectins interact with heparin and N-acetyl-D-galactosamine, and were purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. They both behave as high molecular weight aggregates that can be dissociated into two peptides with apparent molecular weights of 13,000 and 16,000 by gel electrophoresis in SDS. Samples of purified lectin contained up to 20% DNA by weight, and the degree of lectin aggregation and hemagglutination activity was greatly reduced by treatment with micrococcal nuclease without inhibiting heparin-binding activity. Association of lectin with DNA is an artifact of homogenization in high salt, since only 2% of the lectin is found associated with a purified nuclear fraction.