Multistep Reaction in ADP-induced Platelet Aggregation*

The influence of divalent ions and ADP on the induction, relaxation, and stability of the ADP-induced platelet receptor for fibrinogen was investigated. Platelets, isolated by differential sedimentation and gel filtration in calciumand magnesium-free buffer, exhibited a well defined shape change upon ADP stimulation, but aggregation occurred only when calcium or magnesium were added. ‘“I-Fibrinogen did not bind to the platelets in the absence of divalent ions. Either calcium or magnesium at millimolar levels supported fibrinogenxeceptor interactions, but approximately half as many ‘”1-fibrinogen molecules were bound per platelet in the presence of magnesium as in the presence of calcium. The affinity of the receptor for fibrinogen in the presence of either divalent ion was similar, suggesting a single class of receptors with the divalent ions affecting only their number. Under optimal conditions of ADP and divalent ions, the number of fibrinogen molecules bound per platelet ranged from 15,400 to 82,500 (mean = 38,000) in the presence of calcium and from 8,500 to 32,300 (mean = 20,600) in the presence of magnesium. Variability in the number of induced receptors was not only noted among different donors but also in the same donor drawn on multiple occasions. The interaction of ‘”I-fibrinogen with the platelet was progressively stabilized suggesting that the initial reversible binding is followed by a stable interaction. Experiments with apyrase confirmed this evolution of the receptor system and suggested that the continuous presence of ADP is required for the binding of fibrinogen. The aggregation and deaggregation properties of the platelets correlated very well with the binding of fibrinogen to its receptor and with the stability of this interaction. Based on the present study and the available information, the interaction of fibrinogen with platelets appears to involve multiple steps, and a model sequence for these reactions is proposed.