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Stimulation of platelets by thrombin induces protein kinase C (PKC) activation, phosphorylation of pleckstrin, aggregation and serotonin release. Here, we demonstrate that, in human platelets, thrombin stimulation also induced phosphorylation of the myristoylated alanine-rich C kinase substrate (MARCKS) and serotonin release in intact and digitonin-permeabilized platelets. MARCKS is known to bind actin and cross-link actin filaments, and this is inhibited by PKC-evoked MARCKS phosphorylation. MARCKS phosphorylation and serotonin release in response to increasing concentrations of thrombin have a similar EC50 and time course and, in permeabilized platelets, peptide MPSD, with an amino acid sequence corresponding to the phosphorylation site domain of MARCKS, blocked both responses. However, pleckstrin and myosin light chain phosphorylations were not modified. Ala-MPSD, in which the four serine residues of MPSD were substituted by alanines was ineffective. The results suggest a role for MARCKS in platelet secretion. The fact that pleckstrin phosphorylation has a different time course and was not modified in the presence of MPSD when MARCKS phosphorylation and serotonin release were inhibited would suggest either that pleckstrin phosphorylation is unrelated to secretion or that it might only be involved upstream in the events leading to secretion.

In a 13 d fetal mouse liver tissue culture system for assay of erythropoietin, stimulation of incorporation of 3H-thymidine and of 125I-iodo-2'-deoxy uridine into DNA and of 59Fe iron into haem were compared as metameters of erythropoietin effect. With both DNA and haem tracers, log-dose response curves given by two preparations of human urinary erythopoietin purified to different extents (respectively 2 and 1300 IU, by bioassay in vivo, per mg of protein) were essentially identical at low dose levels although at high dose levels the less pure preparation decreased stimulation of both DNA and haem synthesis. Pre-incubation of erythropoietin with an antiserum to human erythropoietin abolished the effects of the hormones on both DNA and haem synthesis.

Iron bound to human transferrin but not apotransferrin, increases the effect of erythropoietin in stimulating incorporation of 3H-thymidine into DNA in fetal mouse liver cells in vitro. The effect of erythropoietin, with or without transferrin-iron is blocked by pre-incubation of the erythropoietin with rabbit anti erythropoietin serum. Human sera contain factors in addition to erythropoietin and transferin-iron which may modify the stimulation of incorporation of 3H-thymidine into fetal mouse liver DNA induced by erythropoietin. Heat treatment of sera at 56 degrees C for 30 min does not necessarily destroy these factors. Acid heat treatment of sera (pH 5.5 and 100 degrees C for 5 min) may destroy inhibitory factors and can result in an apparent increase in serum erythropoietin activity assessed in this fetal mouse liver system.