title: Aggregation-dependent signaling in human platelets is sensitive to protein serine/threonine phosphatase inhibitors.
abstract: When platelets are stimulated by the addition of thrombin, a series of temporally linked signaling events are initiated. Some of the early events are needed to engage the integrin glycoprotein (GP) IIb-IIIa in a high-affinity state. This in turn leads to aggregation, which initiates a wave of events distinct from those triggered by thrombin. Platelet responses are sensitive to protein serine/threonine phosphatase inhibitors, but which events are dependent on protein phosphatase activity is not known. In the present studies, the effect of the phosphatase inhibitor calyculin A on aggregation-induced signaling was examined. The addition of 0.2 unit/mL thrombin caused aggregation-dependent redistribution of cytoskeletal proteins (actin binding protein, talin, vinculin, and alpha-actinin), glycoproteins (GPIIb-IIIa, PECAM), and signaling molecules (PI3-kinase, pp60c-src) to the cytoskeletal fraction of platelets. Addition of 1-2 microM calyculin A blocked the ability of 0.2 unit/mL thrombin to induce aggregation and the association of these molecules with the cytoskeleton. Aggregation (60-80% of control) was restored if 1 unit/mL thrombin was added, but there was no corresponding redistribution of actin binding protein, talin, vinculin, alpha-actinin, GPIIb-IIIa, PECAM, PI3-kinase, and pp60c-src to the cytoskeleton. Treatment of platelets with calyculin A resulted in an increase in the phosphorylation state of a membrane skeletal protein of 50 kDa. These data strongly suggest that platelet aggregation is dissociable from aggregation-induced signaling, which is dependent on type 1 and 2A phosphatase activities.
authors.affiliation: Department of Cell Biology and Anatomy, New York Medical College, Valhalla 10595, USA.
authors.forename: C H
authors.initials: CH
authors.lastname: Hoyt
authors.forename: K M
authors.initials: KM
authors.lastname: Lerea
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D003598:Cytoskeletal Proteins / Q000097:blood* / Q000302:isolation & purification; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008387:Marine Toxins; D008565:Membrane Proteins / Q000097:blood / Q000302:isolation & purification; D019319:Okadaic Acid; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010750:Phosphoproteins / Q000097:blood / Q000302:isolation & purification; D010766:Phosphorylation; D010974:Platelet Aggregation / Q000187:drug effects* / Q000502:physiology; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*
chemical_list: D003598:Cytoskeletal Proteins; D004988:Ethers, Cyclic; D008387:Marine Toxins; D008565:Membrane Proteins; D010080:Oxazoles; D010750:Phosphoproteins; D010980:Platelet Membrane Glycoproteins; D019319:Okadaic Acid; C059041:calyculin A; D010749:Phosphoprotein Phosphatases; D013917:Thrombin