Evidence for interaction between platelet fibrinogen receptors

Previous analysis of fibrinogen binding to human aspirin-treated gel-filtered platelets yielded upwardly concave Scatchard plots. To ascertain whether this was due to the presence of independent heterogeneous receptor populations binding fibrinogen with different affinities, the dissociation of purified ¹²⁵I-fibrinogen from ADP-treated gel-filtered platelets was evaluated as a function of receptor occupancy. Dissociation of bound labeled fibrinogen was measured after 50-fold dilution with buffer containing 0, 0.02, 0.8, and 2.0 mg/ml unlabeled fibrinogen. Dissociation of labeled fibrinogen increased with increasing receptor occupancy and was biphasic. With buffer alone, 76.0% ± 5.8% (SD) of labeled fibrinogen dissociated in 30 min, with an initial rate of 0.392 ± 0.175 min^-1; with 0.2 mg/ml fibrinogen, 83.7% ± 3.9% dissociated, with an initial rate of 0.589 ± 0.044 min⁶-¹; with 0.8 mg/ml, 91.8% ± 1.3% of the labelled fibrinogen dissociated, with an initial rate of 0.910 ± 0.028 min^-1; and with 2.0 mg/ml fibrinogen, 97.3% ± 2.3% of label dissociated, with a initial rate of 1.06 ± 0.257 min^-1 (n = 5). The final rates of fibrinogen dissociation were unaffected by unlabeled fibrinogen in the dilution buffer and were not statistically different from the final dissociation rate of 0.015 ± 0.10 min^-1 observed following dilution with buffer alone. These results were neither an artifact of aspirin treatment or gel filtration, as similar observations were made using non-aspirin-treated washed platelets, nor were they an artifact of the purified fibrinogen preparations, because binding studies using whole plasma as the major source of fibrinogen also yielded upwardly concave Scatchard plots. Since the data demonstrate that the initial rate and extent of fibrinogen dissociation are dependent on fibrinogen receptor occupancy, they suggest receptor interactions possibly resulting from receptor clustering or crosslinking. Because the dissociation was biphasic, the results also suggest some heterogeneity among platelet-fibrinogen-interactions.