Part of the activating cross-linked immunoglobulin G is internalized by human platelets to sites not accessible for enzymatic digestion

The differential uptake of tritium-labeled immunoglobulin G (IgG) cross- linked with bisdiazonium-benzidine (BDB) (3H-BDB-IgG) by washed, pooled human platelets to sites inaccessible to pronase digestion was tested. Up to 52% of the 3H-BDB-IgG associated with platelets at 37 degrees C resisted pronase treatment, whereas only 23% of the cross-linked IgG associated with platelets at 4 degrees C, or at 37 degrees C but in the presence of deoxyglucose/antimycin A, remained refractory to pronase. This effect was not due to platelet agglutination. Pronase resistance reached a maximum after a 60-minute incubation period at 37 degrees C. With increasing 3H-BDB-IgG input, both the total cross-linked IgG associated with platelets and the fraction resistant to pronase digestion approached saturation at 4 degrees C, but not at 37 degrees C. The proportion of 3H-BDB-IgG bound to platelets at 4 degrees C that was resistant to pronase treatment increased by 13% within five minutes of warming the platelets to 37 degrees C. Pretreatment of platelets with 10 mmol/L acetylsalicylic acid (or 10 mumol/L prostaglandin E1) prior to the addition of 3H-BDB-IgG led to a 74% (95%) inhibition of the 3H-BDB-IgG-induced 14C-serotonin release, but to only a 44% (49%) inhibition of pronase-digestible bound ligand. In contrast, pretreatment with 10 mumol/L cytochalasin B led to a mere 17% reduction of 14C-serotonin release, whereas acquisition of resistance to pronase digestion by the bound 3H-BDB-IgG was inhibited by 90%. Incubation of platelets at 37 degrees C with 3H-BDB-IgG and removal of unbound material prior to the addition of prostaglandin E1 or deoxyglucose/antimycin A had little effect on the susceptibility of platelet-associated 3H-BDB-IgG to pronase, whereas the addition of cytochalasin B to 3H-BDB-IgG-treated platelets resulted in greatly increased susceptibility of the platelet-associated ligand to pronase. Thus, after binding, 3H-BDB-IgG becomes transferred in an energy- dependent process to pronase-resistant cellular sites, most likely to the open canalicular system.