The goal of this proposal is to understand the roles of pleckstrin and pleckstrin-2 in platelet adhesion and thrombosis. Pleckstrin is a prominent PKC substrate that makes up about 1 percent of total cellular protein in platelets. It consists of amino- and carboxy-terminal Pleckstrin Homology (PH) domains and an intervening DEP domain. Although pleckstrin was first described in platelets, its role in platelet activation is still not completely understood. We have found in overexpression studies that pleckstrin, once it was phosphorylated by PKC, regulates phospholipid second messengers generated by both phospholipase C and phosphatidylinositol 3- kinase, and enhances integrin mediated cytoskeletal changes and adhesion. We have also cloned a cDNA for pleckstrin-2, which is a widely expressed paralog that is also present in platelets. Pleckstrin-2 is not phosphorylated by PKC, but instead is regulated by binding to specific phospholipid products of phosphatidylinositol 3-kinase (PI3K). Although overexpression studies have been useful for gaining insight into the potential role of these two proteins, I believe that studies of platelets lacking these enzymes are critical for a complete understanding of their contributions to platelet biology. Consequently, we have introduced a null mutation into the murine pleckstrin gene, and have generated chimeric mice that are currently being bred to produce pleckstrin knockout animals. We have also targeted the pleckstrin-2 gene, and have recently generated mice that are homozygous for a pleckstrin-2 null mutation. Pleckstrin-2 knockout platelets have impaired aggregation in response to thrombin and collagen, have defective dense granule secretion, and have impaired the spreading of immobilized fibrinogen. I hypothesize that in platelets, pleckstrin, and pleckstrin-2 moderate phospholipid second messengers, regulate platelet exocytosis, and function in concert with integrins to induce actin reorganization and stable platelet adhesion. Based upon this hypothesis, I propose three specific aims.
In Aim 1, we will determine the molecular link between platelet actin dynamics and pleckstrin isoforms.
In Aim 2, by using several ex vivo and in vivo models we will determine the contribution of pleckstrin and pleckstrin-2 to stable platelet adhesion, and test the hypothesis that both of these isoforms are required for thrombus formation.
In Aim 3, we will use in vitro approaches, including structural studies, to further investigate phosphoinositide binding by the two pleckstrin isoforms ? ?

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL083392-02
Application #
7169204
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Sarkar, Rita
Project Start
2006-02-01
Project End
2010-01-31
Budget Start
2007-02-01
Budget End
2008-01-31
Support Year
2
Fiscal Year
2007
Total Cost
$381,825
Indirect Cost
Name
University of Pennsylvania
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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Lian, Lurong; Suzuki, Aae; Hayes, Vincent et al. (2014) Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation. Haematologica 99:554-60
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