Reference number : GC-2017-Q1-02596, amendment number : , agreement type : G, recipient legal name : Kim, Paul (McMaster University), recipient country : CA, recipient province : nan, recipient city : Ontario, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Upon vascular injury, the coagulation system is activated in order to stop the blood loss and begin the process of wound healing and damage repair. The clotting system consists of enzyme complexes that sequentially activate downstream enzyme complexes. One characteristic in common is that these complexes all contain 1) enzyme, 2) cofactor, 3) calcium, and 4) negatively charged cell surface. In all instances, even though the enzyme is able to act on the substrate alone, the presence of its cofactor enhances the reaction by 10 3 - to 10 5 -fold. Therefore, understanding the parallel mechanism by which these cofactors exert their effect is critical in our understanding of the coagulation system and potentially developing treatment options. 
Of these complexes, the central clotting enzyme thrombin is generated when its precursor prothrombin (PT) is activated by the prothrombinase complex (Pase). Pase consists of the enzyme factor (F) Xa, cofactor FVa, calcium and cell surface. While PT can be activated by FXa alone, this reaction is enhanced by 300,000-fold when FXa is in Pase. Despite the importance of these interactions, and decades of research work behind this topic, the exact mechanism of how FVa exerts its cofactor function remains elusive. This is partly due to the complexity in the underlying mechanism of PT activation by Pase. Furthermore, how FVa binds PT and is presented to FXa remains unanswered. This is particularly of importance since the abundance of FV(a) relative to FXa dictates that PT-FVa interaction may be crucial in presenting PT to FXa. Therefore, we wish to investigate the region of PT that is responsible for interacting with FVa, and thus allowing FVa to exert its cofactor activity during PT activation by Pase. 
We have preliminary data that demonstrate that the outer face of kringle 2 domain of PT is involved in binding the heavy chain of FVa. More specifically, we have identified six residues on PT that directly bind FVa using NMR. We then generated a PT variant with these six residues substituted with inert residues. Activation of this PT variant led to a substantial decrease in the formation of a major intermediate as well as a significant decrease in thrombin generation, suggesting that there has been a major change in the mechanism of PT activation. In this proposal, we aim to further investigate our initial findings by directly characterizing the FVa-PT interaction. Furthermore, in place of the negatively charged synthetic lipid vesicles that are typically used to mimic the activated cell surface to which Pase is assembled, we will also use activated platelets that are obtained fresh. 
Therefore, the potential impact our research will have is a better understanding of how these non-enzymatic protein cofactors work to enhance blood clot formation. By answering these long standing questions, we aim to break exciting new ground in understanding the biochemical processes that modulate coagulation., program purpose french : Upon vascular injury, the coagulation system is activated in order to stop the blood loss and begin the process of wound healing and damage repair. The clotting system consists of enzyme complexes that sequentially activate downstream enzyme complexes. One characteristic in common is that these complexes all contain 1) enzyme, 2) cofactor, 3) calcium, and 4) negatively charged cell surface. In all instances, even though the enzyme is able to act on the substrate alone, the presence of its cofactor enhances the reaction by 10 3 - to 10 5 -fold. Therefore, understanding the parallel mechanism by which these cofactors exert their effect is critical in our understanding of the coagulation system and potentially developing treatment options. 
Of these complexes, the central clotting enzyme thrombin is generated when its precursor prothrombin (PT) is activated by the prothrombinase complex (Pase). Pase consists of the enzyme factor (F) Xa, cofactor FVa, calcium and cell surface. While PT can be activated by FXa alone, this reaction is enhanced by 300,000-fold when FXa is in Pase. Despite the importance of these interactions, and decades of research work behind this topic, the exact mechanism of how FVa exerts its cofactor function remains elusive. This is partly due to the complexity in the underlying mechanism of PT activation by Pase. Furthermore, how FVa binds PT and is presented to FXa remains unanswered. This is particularly of importance since the abundance of FV(a) relative to FXa dictates that PT-FVa interaction may be crucial in presenting PT to FXa. Therefore, we wish to investigate the region of PT that is responsible for interacting with FVa, and thus allowing FVa to exert its cofactor activity during PT activation by Pase. 
We have preliminary data that demonstrate that the outer face of kringle 2 domain of PT is involved in binding the heavy chain of FVa. More specifically, we have identified six residues on PT that directly bind FVa using NMR. We then generated a PT variant with these six residues substituted with inert residues. Activation of this PT variant led to a substantial decrease in the formation of a major intermediate as well as a significant decrease in thrombin generation, suggesting that there has been a major change in the mechanism of PT activation. In this proposal, we aim to further investigate our initial findings by directly characterizing the FVa-PT interaction. Furthermore, in place of the negatively charged synthetic lipid vesicles that are typically used to mimic the activated cell surface to which Pase is assembled, we will also use activated platelets that are obtained fresh. 
Therefore, the potential impact our research will have is a better understanding of how these non-enzymatic protein cofactors work to enhance blood clot formation. By answering these long standing questions, we aim to break exciting new ground in understanding the biochemical processes that modulate coagulation., agreement title english : Identification and characterization of the factor Va binding site on prothrombin., agreement title french : Identification and characterization of the factor Va binding site on prothrombin., agreement number : RGPIN, agreement value : 130000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada