Kenneth M. Lerea - US grants

The role of platelets in wound healing and inflammation processes has been well established. In performing these functions, platelets undergo massive biochemical and morphological changes. The molecular mechanism(s), however, by which platelet reactivity is controlled is poorly understood. Thus, why platelets fail to function normally in certain disease states remains an open question. Recognized as being important reactions in stimulusresponse coupling, the role of phosphorylation of platelet proteins has been studied by many laboratories including ours; but the involvement of specific phosphorylation reactions in regulating platelet responses remains unclear. The broad long-term objective of this application is to understand the role of these reactions in platelets. The phosphorylation status of proteins reflects a balance between protein kinase and phosphatase activities. Thus, there are several paths one can pursue to address this problem. The objectives of this research program are to characterize basic properties of platelet phosphatases and their roles. The proposed studies will concentrate on the protein serine/threonine phosphatase, PP1, focussing attention primarily on: (i) The physiochemical properties of this enzyme, which includes an understanding of its structure, subcellular localization, and substrate specificity making use of exogenous phosphoproteins. (ii) Identifying which of the biochemical event that occur in response to a platelet agonist depends on PP1 activity. These studies will make use of okadaic acid, a membrane permeable inhibitor of PP1, in attempt to define the intracellular pathway that requires PP1 to proceed normally. Finally, (iii) attempts will be made to identify the platelet phosphoproteins that are endogenous substrates for PP1. Characterizing PPl-sensitive phosphorylations may help identify proteins that regulated platelet reactivity.

The role of platelets in wound healing and inflammation processes has been well established. In performing these functions, platelets undergo massive biochemical and morphological changes. The molecular mechanism(s), however, by which platelet reactivity is controlled is poorly understood. Thus, why platelets fail to function normally in certain disease states remains an open question. Recognized as being important reactions in stimulusresponse coupling, the role of phosphorylation of platelet proteins has been studied by many laboratories including ours; but the involvement of specific phosphorylation reactions in regulating platelet responses remains unclear. The broad long-term objective of this application is to understand the role of these reactions in platelets. The phosphorylation status of proteins reflects a balance between protein kinase and phosphatase activities. Thus, there are several paths one can pursue to address this problem. The objectives of this research program are to characterize basic properties of platelet phosphatases and their roles. The proposed studies will concentrate on the protein serine/threonine phosphatase, PP1, focussing attention primarily on: (i) The physiochemical properties of this enzyme, which includes an understanding of its structure, subcellular localization, and substrate specificity making use of exogenous phosphoproteins. (ii) Identifying which of the biochemical event that occur in response to a platelet agonist depends on PP1 activity. These studies will make use of okadaic acid, a membrane permeable inhibitor of PP1, in attempt to define the intracellular pathway that requires PP1 to proceed normally. Finally, (iii) attempts will be made to identify the platelet phosphoproteins that are endogenous substrates for PP1. Characterizing PPl-sensitive phosphorylations may help identify proteins that regulated platelet reactivity.

Integrins on the plasma membrane of cells are central recognition elements for conveying signals from the extracellular matrix to the cell interior. One of the best studied model systems for integrin function is the platelet, which undergoes complex biochemical and structural changes in response to integrin activation. The overall goal of this project is to define the molecular mechanism by which protein serine/threonine (Ser/Thr) phosphatases control platelet responses associated with integrins. The first objective is to examine Thr phosphorylation of the beta subunit of the platelet-specific integrin alphaIIb/beta3. Platelet spreading and other responses induced by platelet aggregation are critical steps in the activation by this integrin. Inhibitors of protein Ser/Thr phosphatases block spreading on fibrinogen matrices and aggregation-induced signaling. Thus, these responses are apparently regulated by phosphatase activity. The carboxy-terminal segment of the beta3 subunit of the integrin has a sequence that includes sites for both tyrosine (Tyr) and Thr phosphorylation. Under normal conditions, activation of alphaIIb/beta3 integrin by fibrinogen causes phosphorylation of Tyr residues, which in turn promotes the assembly of signaling complexes on the carboxy-terminus of beta3. In contrast, inhibition of platelet Ser/Thr phosphatases promotes phosphorylation of Thr residues within the carboxy-terminus. The hypothesis is that this phosphorylation modulates alphaIIb/beta3 activity. The first specific aim is to test whether Thr phosphorylation of beta3 affects the Tyr phosphorylation or the ability of this subunit to form signaling complexes. This will be accomplished using intact beta3 and peptides derived from its cytoplasmic domain, with and without target Thr residues, as Tyr kinase substrates. The second specific aim is to characterize the protein kinase that phosphorylates the beta3 subunit, again using an appropriate synthetic peptide to assay kinase activity. The third specific aim is to assess whether the phosphatase inhibitor calyculin A affects just beta3 integrins or, in addition, integrins with other beta subunits. Specifically, the ability of calyculin A to alter the localization of beta1 integrins to focal adhesions will be tested. The studies are significant for understanding both specific integrin signaling in platelets as well as more general mechanisms of integrin regulation in other cell types.