AGGREGATION-DEPENDENT SIGNALING IN HUMAN PLATELETS IS SENSITIVE TO PROTEIN SERINE THREONINE PHOSPHATASE INHIBITORS/

When platelets are stimulated by the addition of thrombin, a series of temporally linked signaling events are initiated. Some of the early e vents are needed to engage the integrin glycoprotein (GP) IIb-IIIa in a high-affinity state. This in turn leads to aggregation, which initia tes a wave of events distinct from those triggered by thrombin. Platel et responses are sensitive to protein serine/threonine phosphatase inh ibitors, but which events are dependent on protein phosphatase activit y is not known. In the present studies, the effect of the phosphatase inhibitor calyculin A on aggregation-induced signaling was examined. T he addition of 0.2 unit/mL thrombin caused aggregation-dependent redis tribution of cytoskeletal proteins (actin binding protein, talin, vinc ulin, and alpha-actinin), glycoproteins (GPIIb-IIIa, PECAM), and signa ling molecules (PI3-kinase, pp60(c-src)) to the cytoskeletal fraction of platelets. Addition of 1-2 mu M calyculin A blocked the ability of 0.2 unit/mL thrombin to induce aggregation and the association of thes e molecules with the cytoskeleton. Aggregation (60-80% of control) was restored if 1 unit/mL thrombin was added, but there was no correspond ing redistribution of actin binding protein, talin, vinculin, alpha-ac tinin, GPIIb-IIIa, PECAM, PD-kinase, and pp60(c-src) to the cytoskelet on. Treatment of platelets with calyculin A resulted in an increase in the phosphorylation state of a membrane skeletal protein of 50 kDa. T hese data strongly suggest that platelet aggregation is dissociable fr om aggregation-induced signaling, which is dependent on type 1 and 2A phosphatase activities.