The ability of platelets to regulate thrombin generation at their membrane surfaceis central to their role in hemostasis, thrombosis, and atherosclerosis. Thrombin generation is effectedthrough the assembly and function of the enzymatic complex, Prothrombinase, consisting of a Ca^-dependent, membrane-bound complex of the cofactorfactorVa and the serine protease factorXa. Subsequent to platelet activation, platelet-derived factor Va and/or plasma-derived factor Va are expressed on or bind to the platelet membrane surfaceand in so doing form at least part of the receptorfor factorXa. Thus, several protein/protein and protein/membrane interactions participate in and regulate complex assembly. The major goal of this project is to define how platelets actively participate in and regulate Prothrombinase assembly and function. The following hypotheses have been formulated regarding unique mechanisms by which platelets regulate thrombin formation, catalyzed by Prothrombinase, and will be tested. 1) Platelets regulate thrombin generation through the expression of three discrete platelet subpopulations. One binds both factors Va and Xa to effect Prothrombinase assembly and function;one binds only factor Va, whereas one subpopulation is incapable of binding either protein. 2) Unique membrane proteins regulate Prothrombinase assembly and function on the activated platelet surface.3) Platelets release a cofactor molecule, factor Va, which is functionally and physically unique when compared to plasma-derived factor Va. 4) Physical and functional characteristics of Prothrombinase assembled on defined phospholipid vesicles are not mimicked by complex assembly on the activated platelet membrane. State of the art arterial and venous flow models will be used to characterizethe recruitment of the procoagulant platelet subpopulations to thrombogenic surfaces. Hematopoietic regulation will be determined using CD34+""""""""derived megakaryocytes in flow cytometric analyses. Factor Xa will be labeled with a trrfunctionalcross-linking reagent that will result inthe transfer of a biotin handle to activated platelet membrane proteins, other than factorVa, with which it interacts to effect its function. State of the art mass spectrometric analyses will be used to identify the isolated biotinylated proteins. Functional and physical differences between plasma and platelet-derived factor Va will be assessed using analyses of kinetic of prothrombin activation coupled to mass spectrometric techniques. Definition of important factor Va interactions with factor Xa and prothrombin, which regulate Prothrombinase assembly and function on the activated platelet surface will require the synthesis of select peptides mimicking important regions in both proteins for use in both competitive binding and functional analyses. Successful completion of these goals will demonstrate the mechanisms by which platelets actively and uniquely regulate the generation of thrombin attheir membranesurface. These studies will identify several different mechanisms essential for thrombin generation at the activated platelet membrane, all of which will be potential therapeutic targets in hypercoagulable and thrombotic states.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
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Heart, Lung, and Blood Initial Review Group (HLBP)
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University of Vermont & St Agric College
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Kusak, Piotr; Czarnecka, Danuta; Gissel, Matthew et al. (2016) Activated factor IX, factor XI and tissue factor identify patients with permanent atrial fibrillation treated with warfarin who are at risk of ischemic stroke. Arch Med Sci 12:1000-1007
Bouchard, Beth A; Chapin, John; Brummel-Ziedins, Kathleen E et al. (2015) Platelets and platelet-derived factor Va confer hemostatic competence in complete factor V deficiency. Blood 125:3647-50
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Undas, A; Brummel-Ziedins, K E; Mann, K G (2014) Anticoagulant effects of statins and their clinical implications. Thromb Haemost 111:392-400
Whelihan, Matthew F; Kiankhooy, Armin; Brummel-Ziedins, Kathleen E (2014) Thrombin generation and fibrin clot formation under hypothermic conditions: an in vitro evaluation of tissue factor initiated whole blood coagulation. J Crit Care 29:24-30
Krudysz-Amblo, Jolanta; Jennings 2nd, Mark E; Knight, Tyler et al. (2013) Disulfide reduction abolishes tissue factor cofactor function. Biochim Biophys Acta 1830:3489-96
Ayombil, F; Abdalla, S; Tracy, P B et al. (2013) Proteolysis of plasma-derived factor V following its endocytosis by megakaryocytes forms the platelet-derived factor V/Va pool. J Thromb Haemost 11:1532-9
Baker, Jason V; Brummel-Ziedins, Kathleen; Neuhaus, Jacqueline et al. (2013) HIV replication alters the composition of extrinsic pathway coagulation factors and increases thrombin generation. J Am Heart Assoc 2:e000264

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