Thrombin generation at the human platelet surface is effected through the assembly and function of the enzymatic complex, Prothrombinase, consisting of a 1:1 stoichiometric, Ca/2+ complex of the cofactor factor Va and the serine protease factor Xa. Subsequent to platelet activation, released platelet factor V(a) and/or plasma-derived factor Va bind to the platelet membrane surface and in so doing form at least part of the receptor for factor Xa. Thus, several protein/protein and protein/membrane interactions dictate functional complex assembly. A goal of this research project is to define how platelets actively participate in and regulate complex assembly. Because platelets have been shown to express the factor Xa membrane receptor Effector Protease Receptor-1 (EPR-1) following agonist-induced activation, substantial effort will be placed on defining its role in, and regulation of, a functional Prothrombinase complex. The platelet's ability to regulate events critical for enzyme complex assembly subsequent to agonist-induced activation will be assessed as well, specifically as related to 1) functional EPR-1 expression and 2) expression of a platelet factor Va molecule resistant to activated protein C inactivation, and perhaps other proteases as well. Because thrombin once formed positively regulates Prothrombinase complex assembly and function through platelet activation, a second goal is to understand how thrombin interactions with platelet membrane proteins affect its function. Effort will be placed on defining and isolating the thrombin high affinity binding site which appears to render the thrombin molecule transiently inactive and delineating the relationship of glycoprotein Ib/IX to this site. Finally, the observation that both a thrombomodulin-like molecule constitutively expressed on the platelet membrane and platelet-derived factor Va inhibit fibrinolysis through a thrombin-dependent mechanism will be explored, especially as related to the recently identified Thrombin Activatable Fibrinolysis Inhibitor (TAFI). The ultimate goal of this research project is to define the mechanisms by which platelets actively regulate both the generation and function of thrombin at their membrane surface. Defining the cellular and molecular events which regulate thrombin production is fundamental to our overall understanding of the hemostatic and thrombotic processes.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL046703-08
Application #
6110091
Study Section
Project Start
1998-09-01
Project End
1999-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Vermont & St Agric College
Department
Type
DUNS #
066811191
City
Burlington
State
VT
Country
United States
Zip Code
05405
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
Brummel-Ziedins, Kathleen E; Everse, Stephen J; Mann, Kenneth G et al. (2014) Modeling thrombin generation: plasma composition based approach. J Thromb Thrombolysis 37:32-44
Bouchard, Beth A; Gissel, Matthew T; Whelihan, Matthew F et al. (2014) Platelets do not express the oxidized or reduced forms of tissue factor. Biochim Biophys Acta 1840:1188-93
de Haan, Hugoline G; Bezemer, Irene D; Vossen, Carla Y et al. (2014) Genetic variants in Cell Adhesion Molecule 1 (CADM1): a validation study of a novel endothelial cell venous thrombosis risk factor. Thromb Res 134:1186-92
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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