Fibrinogen-Binding Properties of the Human Platelet Glycoprotein IIb–IIIa Complex: A Study Using Crossed-Radioimmunoelectrophoresis

Fibrinogen-binding platelet proteins have been examined by crossed-immunoeloctrophoresis of solubilized, washed platelets followed by the incubation of the immunoplates with 125l-fibrinogen and exposure to x-ray films. Incubation with 0.1 mg/ml of 125l-fibrinogen revealed the binding of fibrinogen to the immunoprecipitates representing the glycoprotein IIb–IIIa complex, factor XIIIa chain, a granule membrane protein termed G4, fibrinogen, and albumin. Only the glycoprotein IIb–IIIa precipitate and the fibrinogen precipitate showed significant binding when the concentration of 125l-fibrinogen was lowered to 0.01 mg/ml. The binding to all the immunoprecipitates was completely abolished in the presence of a 20 fold excess of unlabeled fibrinogen, but not when unlabeled fibrinogen was replaced by the same amounts of bovine serum albumin or human hemoglobin. This indicates that the binding of fibrinogen is specific. Furthermore, 125l-fibronectin and 125l-thrombin did not bind to the glycoprotein IIb–IIIa precipitate. The binding of 125l-fibrinogen to the precipitates representing the glycoprotein IIb–IIIa complex, the factor XIIIa chain, and G4, but not to the albumin precipitate, was significantly lowered in the presence of EDTA. This effect of EDTA increased with increasing pH, with no binding at pH 8.7. Immunoprecipitates of dissociated glycoprotein lIb and IIIa did not bind 125l-fibrinogen. Binding of 125l-fibrinogen to the glycoprotein IIb–IIIa precipitate was dependent on the presence of either Ca2+or Mg2+, the binding to the factor XIIIa chain required Ca2+, and the binding to G4 required Mg2+. The results indicate that the glycoprotein IIb–IIIa complex, but not the separate glycoproteins IIb and IIIa, can act as Ca2+or Mg2+-dependent fibrinogen receptor, under proper physiologic conditions.