Summary
Monobromobimane (mBBr, bimane), a compound that penetrates cells and forms a fluorescent adduct with thiol groups, was used to assess the significance of thiols in platelet function. Exposure of washed platelets for 1 min to 100 μM mBBr abolished ADP-induced aggregation; shape change was not inhibited by 500 μM mBBr. The nonpenetrating compound monobromotrimethylammoniobimane was ineffective. Established ADP-induced aggregation was reversed by bimane, and fibrinogen binding to ADP-stimulated platelets was inhibited, an effect mainly due to decreased number of binding sites. Aggregation stimulated by A23187 and arachidonate was less effectively inhibited whereas epinephrine- and collagen-induced aggregation were abolished by 50 μM mBBr. Similar effects on aggregation and secretion were observed in platelet-rich plasma except that higher mBBr concentrations were usually necessary. Aggregation and 14C-serotonin secretion stimulated by 0.1 U/ml thrombin were partially inhibited by pretreatment with bimane. With lower thrombin concentrations, they were often enhanced, as was 3H-arachidonate release. Bimane inhibited epinephrine-induced arachidonate release in gel-filtered platelets, possibly because it abolished the primary aggregation necessary for this release. mBBr did not elevate cyclic AMP but enhanced the increase induced by PGEi and prevented the subsequent decrease typically caused by ADP. Examination of SDS polyacrylamide gels with ultraviolet light showed that mBBr reacted with many platelet proteins but not with GP lib or Ilia. This observation, and the fact that bimane did not inhibit the fibrinogen-induced aggregation of DTT- or chymotrypsin-treated platelets suggest that it reacts with thiol group(s) that are involved in “exposing” the fibrinogen receptor.
Key words
Platelets - Bimane - Thiol group label
