The goal of this proposal is to define the intracellular signaling pathways that contribute to the synthesis of phosphorylated phosphatidylinositols (phosphoinositides) in platelets, and to understand the role of phosphoinositides in platelet biology. Our recent research has focused on the synthesis of two phosphatidylinositol bisphosphates, PtdIns(4,5)P2 and PtdIns(3,5)P2. The hypotheses underlying this proposal are that the synthesis of PtdIns(4,5)P2 and PtdIns(3,5)P2 occurs within unique microdomains in platelets, and that PtdIns(4,5)P2 and PtdIns(3,5)P2 are each responsible for discrete aspects of platelet signaling and in vivo thrombus formation. We have studied the synthesis of PtdIns(4,5)P2 by the ?, ?, and ? isoforms of phosphatidylinositol phosphate 5-kinase I (PIP5KI), and found clear evidence that spatially distinct pools of PtdIns(4,5)P2 exist in platelets and in megakaryocytes. Moreover, the loss of either PIP5KI? or PIP5KI? leads to impaired in vivo thrombosis, but for different reasons. The pool of PtdIns(4,5)P2 that is synthesized by PIP5KI? is required to form second messengers and to extend lamellipodia. In contrast, PtdIns(4,5)P2 synthesized by PIP5KI? is vital for the membranecytoskeletal dynamics and allows for stable adhesion that is resistant to shear forces. We have also found that PtdIns(3,5)P2, which is synthesized by PIKFyve, regulates the biogenesis of platelet lysosomes. Deletion of PIKFyve specifically in murine platelets leads to a platelet lysosome disorder, which remarkably is associated with inflammation and accelerated thrombus formation in vivo. Taken together, these results support the hypothesis that the synthesis of particular phosphoinositides within discrete platelet subcellular microdomains regulates specific spatially distinct intracellular signaling pathways. The proposed experiments are designed to comprehensively and systematically study how PtdIns(4,5)P2 and PtdIns(3,5)P2 production regulates the platelet cytoskeleton and lysosomes. We will also study their effect on stable platelet adhesion in vivo.
In Aim 1, we will determine how PtdIns(4,5)P2 regulates platelet membrane-cytoskeletal dynamics.
In Aim 2, we will examine the spatial regulation of PtdIns(4,5)P2 synthesis. In the final Aim, we will formally analyze how PtdIns(3,5)P2 regulates the biology of the platelet lysosome and its contribution to in vivo thrombus formation.

Public Health Relevance

PROJECT 5 - NARRATIVE Platelets and macrophages both play key roles in the pathogenesis of atherosclerosis as well as arterial thrombosis that lead to acute myocardial infarctions and strokes. This proposal will analyze a previously unrecognized link between platelet secretion and macrophage activation. A better understanding of this process will identify novel therapeutic targets that could be used to treat arterial thrombosis, the most common cause of morbidity and mortality in the US.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL120846-01A1
Application #
8742313
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2014-08-08
Budget End
2015-06-30
Support Year
1
Fiscal Year
2014
Total Cost
$451,429
Indirect Cost
$169,286
Name
University of Pennsylvania
Department
Type
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Paul, David S; Casari, Caterina; Wu, Congying et al. (2017) Deletion of the Arp2/3 complex in megakaryocytes leads to microthrombocytopenia in mice. Blood Adv 1:1398-1408
Tomaiuolo, Maurizio; Brass, Lawrence F; Stalker, Timothy J (2017) Regulation of Platelet Activation and Coagulation and Its Role in Vascular Injury and Arterial Thrombosis. Interv Cardiol Clin 6:1-12
Zhao, Liang; Thorsheim, Chelsea L; Suzuki, Aae et al. (2017) Phosphatidylinositol transfer protein-? in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis. Nat Commun 8:1216
Lee, Robert H; Bergmeier, Wolfgang (2017) Sugar makes neutrophils RAGE: linking diabetes-associated hyperglycemia to thrombocytosis and platelet reactivity. J Clin Invest 127:2040-2043
Shen, Jian; Sampietro, Sara; Wu, Jie et al. (2017) Coordination of platelet agonist signaling during the hemostatic response in vivo. Blood Adv 1:2767-2775
Kononova, Olga; Litvinov, Rustem I; Blokhin, Dmitry S et al. (2017) Mechanistic Basis for the Binding of RGD- and AGDV-Peptides to the Platelet Integrin ?IIb?3. Biochemistry 56:1932-1942
Welsh, J D; Poventud-Fuentes, I; Sampietro, S et al. (2017) Hierarchical organization of the hemostatic response to penetrating injuries in the mouse macrovasculature. J Thromb Haemost 15:526-537
Bennett, Joel S (2016) Shedding New Light on the Platelet Storage Lesion. Arterioscler Thromb Vasc Biol 36:1715-6
Zhu, S; Welsh, J D; Brass, L F et al. (2016) Platelet-targeting thiol reduction sensor detects thiol isomerase activity on activated platelets in mouse and human blood under flow. J Thromb Haemost 14:1070-81
Zhou, Zinan; Tang, Alan T; Wong, Weng-Yew et al. (2016) Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling. Nature 532:122-6

Showing the most recent 10 out of 42 publications