UVM Theses and Dissertations
Format:
Print
Author:
Bravo, Maria Cristina
Dept./Program:
Cell and Molecular Biology Program
Year:
2012
Degree:
Ed. D.
Abstract:
Maintenance of the human vascular system is critical for maintaining proper blood flow. Upon injury to the vascular system an appropriate measured response is initiated to stem the loss of blood, repair the damage, and eventually restore proper blood flow. This complex coordinated response involves circulating plasma proteins, circulating cells such as platelets, and the endothelial cell lining of vascular vessel walls. Following injury there is a coordinated amplification of the procoagulant response that is regulated via antithrombotic proteins and the initiation of negative feedback regulatory mechanisms. Due to the complex nature of the multiple interactions involved in coagulation, the development of mathematical models to describe these interactions help the biochemist to understand the reaction network. These models can also serve as a tool for prediction ofclinical conditions.
The prothrombinase complex is the final procoagulant enzyme·cofactor complex formed following initiation of the coagulation cascade. This complex, composed of the enzyme factor Xa (FXa) and cofactor factor Va (FVa), is responsible for the large burst of thrombin generation that participates in the formation ofthe vessel plug that stems blood loss. Thrombin also initiates the protein C (PC) pathway whose major role is the inhibition of the prothrombinase complex. Activation of the PC pathway leads to the formation of activated protein C (APC) which is responsible for inactivating the cofactor, FVa, of the prothrombinase complex. Due to the critical involvement of the PC pathway, we pursued the development and validation of a mathematical model demonstrating its effects on the procoagulant response. In order to address the intricacies of the PC pathway we set out to engineer recombinant versions of FVa and its relevant mutants to assist in the development and validation of this mathematical model.
We successfully optimized a recombinant expression system of recombinant human FVa which were capable of activation profiles similar to plasma derived FVa. However, inactivation studies with these recombinant species were different from plasma derived FVa and precluded us from utilizing these proteins in further studies. Therefore we developed and validated a mathematical model of the human APC inactivation of human FVa using plasma derived proteins. Validation studies were conducted by comparing multiple in vitro analytes of the inactivation reaction conducted in stepwise complexity. The integrated studies of in vitro and in silico experiments served as a useful tool to identify the contribution of accessibility to membrane binding sites to the inactivation reaction, and to develop the hypothesis that the FXa protection of FVa against APC inactivation does not extend to all partially proteolyzed species of FVa. Our studies on the complexity of the PC pathway were extended to include experiments with dynamic activation of this pathway upon cells. Mathematical models of these systems were able to qualitatively recapitulate behavior of experiments ofthe dynamically activated PC pathway.
The prothrombinase complex is the final procoagulant enzyme·cofactor complex formed following initiation of the coagulation cascade. This complex, composed of the enzyme factor Xa (FXa) and cofactor factor Va (FVa), is responsible for the large burst of thrombin generation that participates in the formation ofthe vessel plug that stems blood loss. Thrombin also initiates the protein C (PC) pathway whose major role is the inhibition of the prothrombinase complex. Activation of the PC pathway leads to the formation of activated protein C (APC) which is responsible for inactivating the cofactor, FVa, of the prothrombinase complex. Due to the critical involvement of the PC pathway, we pursued the development and validation of a mathematical model demonstrating its effects on the procoagulant response. In order to address the intricacies of the PC pathway we set out to engineer recombinant versions of FVa and its relevant mutants to assist in the development and validation of this mathematical model.
We successfully optimized a recombinant expression system of recombinant human FVa which were capable of activation profiles similar to plasma derived FVa. However, inactivation studies with these recombinant species were different from plasma derived FVa and precluded us from utilizing these proteins in further studies. Therefore we developed and validated a mathematical model of the human APC inactivation of human FVa using plasma derived proteins. Validation studies were conducted by comparing multiple in vitro analytes of the inactivation reaction conducted in stepwise complexity. The integrated studies of in vitro and in silico experiments served as a useful tool to identify the contribution of accessibility to membrane binding sites to the inactivation reaction, and to develop the hypothesis that the FXa protection of FVa against APC inactivation does not extend to all partially proteolyzed species of FVa. Our studies on the complexity of the PC pathway were extended to include experiments with dynamic activation of this pathway upon cells. Mathematical models of these systems were able to qualitatively recapitulate behavior of experiments ofthe dynamically activated PC pathway.