Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that impacts multiple diverse processes required during platelet activation. The overall goal of this project is to define the intracellular signaling pathways that Phosphatidylinositol Transfer Proteins (PITPs) contribute to the synthesis of phosphorylated phosphatidylinositols (phosphoinositides) in platelets, and to understand the role of PITPs in platelet biology. PITPs are a small protein family that has been found to bind and transfer phosphoinositide monomers from one cellular compartment to another in an energy-independent manner during vesicle trafficking and phospholipid signaling. Although there are no studies on the role of PITPs in hematopoietic cells, there is evidence in yeast cells that these proteins are essential for the biosynthesis and metabolism of phosphoinositides. Platelets have two dominant PITP family members, PITPa and PITPB. The overall hypothesis of this proposal is that the function of these PITP isoforms in platelets is not overlapping, and each is essential for the generation and spatial localization of discrete species of phosphoinositides within platelets. A secondary hypothesis is that the enzymatic activities of both PITP isoforms are necessary for normal platelet function ex vivo and in vivo. To understand the unique roles ofthe PITP isoforms in platelet biology. My laboratory has generated mice containing conditional null mutations in the PITPa and PITPP genes. The mature megakaryocytes and platelets of these mice lack PITPa, PiTPB, or both PITP isoforms, but the proteins are expressed normally in all other tissues. Loss of either isoform results in thrombocytopenia. Preliminary data also indicate that platelets lacking PITPa have a complete loss in the second messenger, lns(3,4,5)P3 (also known as 1P3) following stimulation by maximal doses of thrombin.
In Aim 1 of this proposal, we will determine the link between PITP isoforms and phosphoinositide synthesis, as well as analyze the contribution of PITPs to platelet signaling.
In Aim 2, i propose experiments designed to understand the distinct biochemical functions ofthe individual PITP isoforms.
In Aim 3, we will study the role of PITP isoforms in platelet activation ex vivo and in vivo.

Public Health Relevance

Activated (sticky) platelets that form at sites of atherosclerosis are often the precipitating event for heart attacks and strokes. Our work demonstrates that two platelet proteins, PITPa and PITPa are important regulators of platelet activation. We believe that a better understanding ofthe events that regulate platelet activation will lead to new therapeutic approaches to prevent vascular occlusion, as well as lead to a better understanding of platelet biology .

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
2P01HL040387-26A1
Application #
8694334
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2014-05-16
Budget End
2015-04-30
Support Year
26
Fiscal Year
2014
Total Cost
$451,038
Indirect Cost
$169,139
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Tomaiuolo, Maurizio; Stalker, Timothy J; Welsh, John D et al. (2014) A systems approach to hemostasis: 2. Computational analysis of molecular transport in the thrombus microenvironment. Blood 124:1816-23
Welsh, John D; Stalker, Timothy J; Voronov, Roman et al. (2014) A systems approach to hemostasis: 1. The interdependence of thrombus architecture and agonist movements in the gaps between platelets. Blood 124:1808-15
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
Stalker, Timothy J; Welsh, John D; Tomaiuolo, Maurizio et al. (2014) A systems approach to hemostasis: 3. Thrombus consolidation regulates intrathrombus solute transport and local thrombin activity. Blood 124:1824-31
Stalker, Timothy J; Welsh, John D; Brass, Lawrence F (2014) Shaping the platelet response to vascular injury. Curr Opin Hematol 21:410-7
Kowalska, M Anna; Zhao, Guohua; Zhai, Li et al. (2014) Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation. Arterioscler Thromb Vasc Biol 34:120-6
Min, Sang H; Suzuki, Aae; Stalker, Timothy J et al. (2014) Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice. Nat Commun 5:4691
Brass, Lawrence F; Tomaiuolo, Maurizio; Stalker, Timothy J (2013) Harnessing the platelet signaling network to produce an optimal hemostatic response. Hematol Oncol Clin North Am 27:381-409
Lu, Qiongyu; Dong, Ningzheng; Wang, Qi et al. (2013) Increased levels of plasma soluble Sema4D in patients with heart failure. PLoS One 8:e64265
Stalker, Timothy J; Traxler, Elizabeth A; Wu, Jie et al. (2013) Hierarchical organization in the hemostatic response and its relationship to the platelet-signaling network. Blood 121:1875-85

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