TMB-8 and dibucaine induce tyrosine phosphorylation and dephosphorylation of a common set of proteins in platelets

A. Oda, B. J. Druker, H. Ariyoshi, M. Smith and E. W. Salzman Department of Surgery, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA. Dibucaine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), which are local anesthetics, affect diverse functions of many cell types. For example, platelet aggregation is inhibited by both, and both cause changes in platelet morphology and structure. Little is known of the mechanisms. We found that both dibucaine (0.125-0.5 mM) and TMB-8 (0.25-1.0 mM) induced rapid tyrosine phosphorylation of several platelet proteins (160, 70-75, and 40 kDa) and dephosphorylation of a 62- to 64-kDa protein detectable by a specific antiphosphotyrosine monoclonal antibody (4G10). Platelet aggregation induced by alpha-thrombin (10 nM) was inhibited by the local anesthetics in approximately the same dose range. Neither dibucaine nor TMB-8 induced activation of protein kinase C (PKC) or myosin light-chain kinase. Their activation was not essential for tyrosine phosphorylation induced by local anesthetics. However, an increase in tyrosine phosphorylation of several proteins (95-130 kDa) induced by alpha-thrombin (10 nM) was inhibited by dibucaine (0.5 mM) or TMB-8 (0.5 mM). Furthermore, when dibucaine (0.5 mM) was added 1 min after addition of alpha-thrombin (10 nM), disaggregation was paralleled to dephosphorylation of many proteins, including those mentioned. Tyrosine phosphorylation and dephosphorylation of specific proteins may account for some of the diverse effects of local anesthetics on platelets and other cells. Addition of TMB-8 (0.5 mM) or dibucaine (0.5 mM) also inhibited activation of PKC, induced by alpha-thrombin (10nM), suggesting that some of the inhibitory effects of dibucaine or TMB-8 may be due to inhibitory effects of local anesthetics on PKC.