A MOLECULAR MECHANISM FOR THE DITHI0THREIT0L-MEDIAT5D PLATELET AGGREGATION

Abstract

Biochemical mechanisms of stimulus response coupling is an intricate problem in platelet biochemistry. Recently we obtained evidence that support the view that conformational changes of an (unsaturated fatty acid – and U46619-binding) haemoprotein induced by the binding of arachidonic acid, H2O2 or PGH2 liberated in apparently different platelet compartments in response to different stimuli could constitute a mechanism (L.K. Krishnan … M.P. Jamaluddin, FEBS Lett, in the press). We investigated the effect of dithiothreitol (DTT), a platelet agonist whose mechanism of action is unknown, on the purified haemoprotein. DTT was found by spectral measurements and gelfiltration experiments to bring about a slow time-dependent conformational .change and oligomerization of the protein concomitantly with its oxidation. Oxidised DTT (trans-4,5-dihy-droxy-1,2-dithiane) was found to induce a similar conformational change by binding to the protein (halfsaturation cancn. 2 mM). Oligomerization changed the charge characteristics of the protein, from net positive to net negative, ait pH 7.4. Protein-protein association is associated with large volume increases. Excluded volume effects and changes in charge distribution at the side of protein conformational change could trigger actin polymerization, pseudopod formation and aggregation, modulated by protein phosphorylation and Ca2+ concentration. In conformity with these ideas oxidized DTT near its half-maximal saturation concentration for the protein, was found to aggregate gelfiltered calf platelets. Presumably it functions as a thioanalogue of PGH2. Oxidized glutathione or oxidized 2-mercaptoethanol could also bring about protein conformational change and platelet aggregation.