Chlorpromazine is unable to block the protein kinase C translocation to human platelet plasma membranes stimulated by phorbol ester.

A well-characterized protein kinase C (PKC) activity, associated with the secretion and aggregation functions, has been previously described in human platelets. T h e phenothiazine, chlorpromazinc, is an amphiphilic cationic drug which interferes with the organization o f biological membranes and is known as a non-specific inhibitor (at SO0 p ~ ) of 47 kDa protein (P47) phosphorylation and aggrcgation induced by thrombin in washed platelets (iti vivo), as well as a good PKC activity inhibitor in partially purified or crude extracts (iti vitro) [ I I. T h e tumour promoter, phorbol cstcr (TPA), activates platelet PKC, as monitored by activity determination or by phosphorylation o f the P47, and a TPAdependent dense-granule secretion in platelets, independent o f C a 2 + mobilization has also bccn shown [ 1, 21. However, increasing concentrations o f chlorpromazine (30-500 p ~ ) caused a progressive lysis of gel-filtered platelets; the lysis was markedly enhanced by thrombin and TPA at 100 p ~ chlorpromazine. At non-lytic concentration (below 30 p ~ ) , the drug caused an increase in the thrombino r TPAmediated phosphorylation of phosphoinositides and had n o effect on the P47 phosphorylation, but inhibited the ATP+ A D P secretion [3,4]. In view of these results, we decided to invcstigatc the ability of chlorpromazine t o interfere with the PKC translocation and/or activation in intact platelets stimulated with TPA. T h e cells, 1.5-2 x IO''/ml, were prepared by the method of Baenziger & Majerus 1.51; the PKC translocation was determined as previously described [2]. In the presence o f leupcptin and other proteinase inhibitors, PKC activity was measured in soluble (S) and particulate ( P ) fractions using [ y3'P]ATP (3000 Ci/mmol) as described [ 61. T h e number o f cells in each translocation assay was S x 10x/ml. Protein concentrations in the assay of S and P fractions were routinely 200 pg/ml and I 0 0 pg/ml, respectively. Previously, we determined (Fig. l a ) the effect o f TPA o r chlorpromazine on the PKC activity in platelet crude extract ( in vilro). T h e dose-rcsponsc curve for PKC activation by