Platelet-rich, arterial thrombi mediate tissue infarction in stroke, peripheral vascular disease, and myocardial infarction. During thrombus formation, platelets secrete their granule contents. The most abundant platelet granule, the alpha-granule, contains adhesion molecules, coagulation factors, and vasoactive factors that contribute to thrombus propagation. We have used a permeabilized platelet secretory model to define a role for SNARE proteins in membrane fusion events leading to platelet alpha-granule secretion. The mechanisms by which agonist-induced stimulation of the platelet results in SNARE protein-mediated membrane fusion, however, remain largely unknown. We have found that phosphatidylinositol (4,5)-bisphosphate synthesis is required for alpha-granule secretion. These studies demonstrated a role for type II phosphatidylinositol 5-phosphate 4-kinase in alpha-granule secretion. The synthetic pathway responsible for the synthesis of phosphatidylinositol (4,5)-bisphosphate required for alpha-granule secretion, however, is poorly characterized. Experiments described in Specific Aim 1 of this proposal will determine the relative contributions of type I phosphatidylinositol 4-phosphate 5-kinase and type II phosphatidylinositol 5-phosphate 4-kinase in platelet alpha-granule secretion. These studies will also determine whether type I phosphatidylinositol 5-phosphate 4-kinase serves as a downstream effector of protein kinase C during platelet alpha-granule secretion. Phosphatidylinositol (4,5)-bisphosphate mediates both remodeling of the platelet actin cytoskeleton and secretion of alpha-granules. Experiments described in Specific Aim 2 will determine whether the actin cytoskeleton mediates the effects of phosphatidylinositol (4,5)-bisphosphate in stimulating platelet alpha-granule secretion. Experiments described in Specific Aim 3 will define the role of the actin cytoskeleton in directing SNARE protein complex formation during alpha-granule secretion. These studies will define a critical activation pathway required for alpha-granule secretion and reveal interactions between the actin cytoskeleton and platelet secretory machinery that are important for platelet alpha-granule secretion.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL063250-05
Application #
6773113
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Ganguly, Pankaj
Project Start
2000-05-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
5
Fiscal Year
2004
Total Cost
$340,000
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
071723621
City
Boston
State
MA
Country
United States
Zip Code
02215
Flaumenhaft, Robert (2015) A new story ARC for ?-granule formation. Blood 126:123-4
Dowal, Louisa; Sim, Derek S; Dilks, James R et al. (2011) Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1. Proc Natl Acad Sci U S A 108:2951-6
Woronowicz, Kamil; Dilks, James R; Rozenvayn, Nataliya et al. (2010) The platelet actin cytoskeleton associates with SNAREs and participates in alpha-granule secretion. Biochemistry 49:4533-42
Flaumenhaft, Robert; Dilks, James R; Richardson, Jennifer et al. (2009) Megakaryocyte-derived microparticles: direct visualization and distinction from platelet-derived microparticles. Blood 113:1112-21
Tanaka, Eiichi; Chen, Frederick Y; Flaumenhaft, Robert et al. (2009) Real-time assessment of cardiac perfusion, coronary angiography, and acute intravascular thrombi using dual-channel near-infrared fluorescence imaging. J Thorac Cardiovasc Surg 138:133-40
Graham, Gwenda J; Ren, Qiansheng; Dilks, James R et al. (2009) Endobrevin/VAMP-8-dependent dense granule release mediates thrombus formation in vivo. Blood 114:1083-90
Blair, Price; Flaumenhaft, Robert (2009) Platelet alpha-granules: basic biology and clinical correlates. Blood Rev 23:177-89
Flaumenhaft, R; Dilks, J R (2008) Discovery-based strategies for studying platelet function. Mini Rev Med Chem 8:350-7
Flaumenhaft, Robert; Rozenvayn, Nataliya; Feng, Dian et al. (2007) SNAP-23 and syntaxin-2 localize to the extracellular surface of the platelet plasma membrane. Blood 110:1492-501
Sim, Derek S; Dilks, James R; Flaumenhaft, Robert (2007) Platelets possess and require an active protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus formation. Arterioscler Thromb Vasc Biol 27:1478-85

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