STAUROSPORINE UP-REGULATES THE EXPRESSION OF PHORBOL DIBUTYRATE BINDING-SITES IN HUMAN PLATELETS

Tumor-promoting phorbol esters bind to and activate protein kinase C ( PKC). Staurosporine, a potent PKC inhibitor, interferes with PKC catal ytic activity without altering phorbol ester binding sites in cell-fre e systems. We found that, unlike cell-free systems, treatment of intac t platelets with staurosporine enhances the expression of phorbol 12,1 3-dibutyrate (PDBu) binding sites. Incubation of platelets at 37 degre es with staurosporine (25 nM to 1 mu M and 2 nM tritiated PDBu ([H-3]P DBu) increased the amount of [H-3]PDBu specifically bound to intact pl atelets by approximately 10 to 200% of control values. This effect was rapid and plateaued after 10 min of cell treatment. Scatchard analysi s of the data showed that staurosporine (500 nM) significantly increas ed the total binding capacity B-max from 42.9 +/- 15.4 x 10(3) to 78 /- 7.3 x 10(3) sites per platelet and reduced the apparent dissociatio n constant value Kd from 30.8 +/- 8.6 nM to 9.4 +/- 3.4 nM. Enhanced P DBu binding capacity and affinity were also observed with human mononu clear and polymorphonuclear leukocytes. Fractionation of staurosporine -treated platelets showed an increased binding capacity of the particu late fraction (102%) and decreased binding capacity of the soluble fra ction (60%) compared to controls, with no change in the affinity of PD Bu binding to these fractions. Chelation of internal calcium with BAPT A did not significantly attenuate the staurosporine-mediated rise in P BDu binding but prevented the platelet-activating factor-induced respo nse, indicating that cytosolic calcium does not play an important role in these staurosporine effects. These results show that, in addition to interfering with PKC protein-phosphorylating activity, staurosporin e enhances PDBu binding affinity and capacity in intact platelets. Thi s latter effect appears to be due to translocation of soluble PDBu bin ding sites, presumably PKC units.