Calyculin A and okadaic acid inhibit human platelet aggregation by blocking protein phosphatases types 1 and 2A

Abstract

Two potent inhibitors of protein phosphatase type 1 (PP1) and type 2A (PP2A), calyculin A (CAL-A) and okadaic acid (OKA), inhibited human platelet aggregation induced by thrombin, collagen and 9,11-epithio-11,12-methano-thromboxane A 2 (STA 2). IC 50 values of CAL-A and OKA for STA 2-induced aggregation were 53 nM and 3.5 μM, respectively. These drugs also inhibited thrombin-induced [ 14C]serotonin secretion of platelets. CAL-A and OKA elicited phosphorylation of certain proteins with an apparent M r (× 10 −3) of 200, 60, 50 and 20 light chain of myosin (MLC). Agonist-induced 47,000 M r protein phosphorylation was strongly inhibited by these compounds, whereas phosphorylation of 20,000 M r MLC was enhanced. The increase in 50,000 M r protein phosphorylation by CAL-A and OKA was observed in the presence of agonists, and the 50,000 M r phosphorylation may be involved in the inhibition of platelet activation by these compoundes. Subcellular analysis of the phosphatase activity in human platelets showed that MLC phosphatase activity was present mainly (approx. 78%) in the cytosolic fraction. Chromatography of human platelet extract on heparin-Sepharose resolved two peaks of MLC phosphatase activity: PP2A in 0.1 M NaCl eluate and PP1 in 0.5 M NaCl eluate. PP2A and PP1 isozymes (PP1α PP1α and PP1δ) have also been identified in human platelets, by cross-reactivity with polyclonal antibodies against (PP2A and PP1 isozymes, respectively. These results suggest that PP1 and/or PP2A may play an important role in the process of platelet activation by regulating levels of phosphorylation of certain proteins.