Temperature-dependent effects of EDTA on the membrane glycoprotein IIb-IIIa complex and platelet aggregability.
In agreement with previous studies, we observed that incubation of washed human platelets with EDTA at 37 degrees C for short periods caused an irreversible loss of their aggregation response to adenosine diphosphate and markedly diminished their capacity to bind fibrinogen. AP-2 is a monoclonal antibody that reacts with a determinant specific to the glycoprotein (GP) IIb-IIIa complex. We now report that in a direct binding assay, the number of sites for AP-2 on platelets incubated with EDTA at 37 degrees C fell to approximately 30% of those present on control platelets. This effect of EDTA was not observed at room temperature. Analysis of the treated platelets by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed normal amounts of GP IIb and GP IIIa. However, studies using crossed immunoelectrophoresis with 125I-AP-2, 125I-Tab (anti-GP IIb), or 125I-AP-3 (anti-GP IIIa) in intermediate gels showed that at 37 degrees C, EDTA was inducing an irreversible change in GP IIb-IIIa complexes. A reduction in size and probable dissociation of the GP IIb-IIIa precipitate was accompanied by the appearance of precipitates having the characteristics of those given by free GP IIb and free GP IIIa and the location of a major new cathodal precipitate, which bound Tab and AP-3 but not AP-2. Membrane modifications associated with the loss of antigenic determinants on GP IIb-IIIa may explain EDTA-induced loss of platelet aggregability at 37 degrees C.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001792:Blood Platelets / Q000032:analysis*; D011232:Chemical Precipitation; D004492:Edetic Acid / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007123:Immunoelectrophoresis, Two-Dimensional; D010974:Platelet Aggregation / Q000187:drug effects*; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000187:drug effects / Q000276:immunology; D013696:Temperature
D000911:Antibodies, Monoclonal; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D000244:Adenosine Diphosphate; D004492:Edetic Acid