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Fibrinogen is essential for aggregating platelets with adenosine diphosphate (ADP) and was recently shown to bind to platelets stimulated with ADP. The present work confirms the specific and saturable nature of the platelet-fibrinogen interaction. Binding of 125iodine-labeled fibrinogen to human gel-filtered platelts was maximal at 1 min, and the receptors were saturated when the fibrinogen concentration in the suspending medium approached 0.8 mg/ml. Assuming that one fibrinogen molecule interacts with a single receptor, experiments with 9 normal donors revealed the presence of 12,896 +/- 2456 receptors per platelet. Much of the bound material dissociated from platelets after incubation with apyrase or EDTA. Binding was markedly inhibited at pH 6.5, in the presence of EDTA, and with platelets from 3 thrombasthenic patients but not with those from a patient with the Bernard-Soulier syndrome. Fibrinogen binding was also virtually absent with platelets that had been incubated with EDTA for 8 min at 37 degrees C and pH 7.8. These platelets could not aggregate when mixed with ADP and adequate CaCl2 and fibrinogen, although they could still change their shape. Thus, ADP-induced binding of fibrinogen correlates with platelet aggregability.

The distribution of surface sialic acid and its possible alteration after ADP-induced shape change and aggregation were studied. Gel-filtered platelets that were aggregated, disaggregated, and centrifuged showed decreased total protein and a significant decrease in sialic acid/mg protein. Platelet surface sialic acid was labeled by periodate oxidation followed by borotritiide reduction. Tritium was apparently incorporated only into sialic acid, since the label was removed by mild acid hydrolysis, had a mobility similar to that of the seven-carbon derivative of sialic acid, and was not taken up when periodate was omitted. When reduced platelet proteins were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, at least three radioactive peaks (1, 2, and 3) were consistently observed, with apparent molecular weights of about 200,000, 150,000, and 100,000 daltons, respectively. Peak 2 had the greatest radioactivity. A shoulder of slightly lower molecular weight than peak 2, or a small peak between peaks 2 and 3, was also seen. Peaks 2 and 3 corresponded to glycoproteins I and III identified with periodic acid-Schiff (PAS) staining, and the material between them to glycoprotein II. Peak 1, and two to four smaller radioactive peaks with apparent molecular weights between 25,000 and 60,000 daltons, were not usually associated with PAS staining. All radioactive peaks were much smaller in platelets labeled after treatment with neuraminidase, indicating that much of the labeled sialic acid was on the platelet surface. The amount of tritium incorporated and its labeling pattern were unaffected when shape change alone was induced with ADP. However, platelets that were aggregated and disaggregated with ADP had reduced tritium uptake with a uniformly decreased labeling pattern, suggesting that the decrease in sialic acid content/mg protein resulted from a generalized loss of surface sialic acid or glycoprotein.

ADP-induced platelet aggregation is associated with the specific binding of fibrinogen to receptors. Since fibrinogen is also a cofactor for the spontaneous aggregation of chilled human platelets during rewarming, we examined the ability of these platelets to bind purified 126l-labeled fibrinogen. Specific binding similar to ADP-induced binding at room temperature was observed after the platelets had been chilled for 20 min at 0–2°C. Aggregation, however, occurred only when the ambient temperature was increased and was followed within 15–30 sec by loss of most of the bound fibrinogen and disaggregation. ADP- and cold-induced fibrinogen binding and aggregation were inhibited by 0.3 µM PGE“ 1 mM dibutyryl cyclic AMP, and 2.5 mM EDTA, unaffected by 208 µM colchicine, and absent from thrombasthenic platelets. Platelets change shape from discs to spiny spheres when they are exposed to ADP or cold. PGE1 and dibutyryl cyclic AMP prevented the shape change induced by ADP but not by chilling. Shape change, fibrinogen binding, and aggregation induced by ADP. but not by chilling, were inhibited by incubating platelets with 7.3 µM antimycin A and 4.9 mM 2-deoxy-D-glucose. Fixed platelets bound fibrinogen only if they were exposed to ADP before fixation but they failed to aggregate. Studies with fixed platelets showed that EDTA and a low pH prevent the interaction of fibrinogen with exposed receptors, whereas platelets stimulated with ADP in the absence of divalent cations or at low pH prior to fixation bound fibrinogen at physiologic pH, and this binding was inhibited by EDTA. The present studies provide the following clues about the requirements for fibrinogen receptor exposure and platelet aggregation. (1) ADP and chilling activate platelets by exposing fibrinogen receptors via a common pathway that is blocked by elevating cyclic AMP. (2) Chilling can bypass the seemingly ATP-dependent step for ADP-induced fibrinogen receptor exposure. (3) Receptor exposure by both ADP and chilling is independent of platelet shape. (4) Increased cyclic AMP inhibits the shape change induced by ADP but not that caused by chilling. (5) EDTA and low pH prevent aggregation by interfering with the binding of fibrinogen to exposed receptors. (6) Receptor mobility may be as important as fibrinogen binding in supporting platelet aggregation.