UVM Theses and Dissertations
Format:
Print
Author:
Krudysz-Amblo, Jolanta Teresa
Dept./Program:
Biochemistry
Year:
2011
Degree:
PhD
Abstract:
The blood coagulation cascade and its regulation is a very complex system of events. Numerous factors contribute to the initiation and propagation state of thrombin generation. The interaction of various activators and inhibitors is in part regulated by threshold events and series of regulated and complicated processes. Regulation through posttranslational modifications is an emerging concept and adds to the already established multifaceted regulatory mechanisms of hemostasis. Despite its approbation as an important biological regulatory tool, few affirmations have been established as to the exact role of each modification in natural proteins isolated from human tissues. Most studies are conducted in vitro with recombinant proteins from various sources.
This work describes the structure-function relationship of tissue factor also known as coagulation factor III, tissue thromboplastin, and CD142. This single chain protein belongs to a Cytokine Receptor Superfamily with a Fibronectin Type III topology. Proteins of the Cytokine Receptor Superfamily group are transmembrane receptor proteins. Fibronectins have been implicated in numerous functions such as blood clotting, wound healing, metastasis, cell signaling and embryogenesis. In addition to its primary role in coagulation, tissue factor has also been an important player in many biological processes. Tissue factor is expressed by cells of arteriosclerotic lesions associated with plaque rupture in the vasculature.
In addition, calcium-and phosphorylation-dependent regulation of tissue factor makes it an important molecule in signal transduction and cell-to-cell interactions in the vasculature. In oncology, tissue factor has been described as a marker of cancer development and a prometastatic molecule. The numerous functions of tissue factor contribute to its importance in human biology. Although presently it is most prominently known for its role in coagulation, evolutionary sequence homologies and similar structural topologies with fibronectins of the CRS proteins place tissue factor in a much wider spectrum of functions.
Tissue factor is a single chain transmembrane glycoprotein that initiates the coagulation pathway. Tissue factor initiates the extrinsic pathway of blood coagulation leading to thrombin generation and clot formation. Overall it is found in very low concentrations with the exception of a few tissues including placenta, lungs and brain. Notwithstanding its low abundance, it is an extremely efficient and potent cofactor in the onset of thrombin generation. Tissue factor is essential to life. Most of the functional studies on tissue factor have been performed using recombinant proteins. Posttranslational modifications described, also were those of recombinant proteins expressed in various organisms. Numerous inconsistencies and lack of direct evidence is found regarding the influence of posttranslational modifications on tissue factor function.
Our goal was to characterize modifications, specifically glycosylation, phosphorylation and disulfide bridge formation, of the natural protein from human placenta. We showed how these modifications influence the activity of the natural tissue factor protein in vitro. Our major findings indicated that glycosylation influences TF activity and that carbohydrate composition of the natural placental tissue factor is significantly different from that of its recombinant counterpart. We have also determined that the two disulfide bonds, found in the extracellular domain, do not play a role in tissue factor function. Additionally we have identified threonine 172 as being phosphorylated in the natural placental but not in the recombinant tissue factor protein.
This work describes the structure-function relationship of tissue factor also known as coagulation factor III, tissue thromboplastin, and CD142. This single chain protein belongs to a Cytokine Receptor Superfamily with a Fibronectin Type III topology. Proteins of the Cytokine Receptor Superfamily group are transmembrane receptor proteins. Fibronectins have been implicated in numerous functions such as blood clotting, wound healing, metastasis, cell signaling and embryogenesis. In addition to its primary role in coagulation, tissue factor has also been an important player in many biological processes. Tissue factor is expressed by cells of arteriosclerotic lesions associated with plaque rupture in the vasculature.
In addition, calcium-and phosphorylation-dependent regulation of tissue factor makes it an important molecule in signal transduction and cell-to-cell interactions in the vasculature. In oncology, tissue factor has been described as a marker of cancer development and a prometastatic molecule. The numerous functions of tissue factor contribute to its importance in human biology. Although presently it is most prominently known for its role in coagulation, evolutionary sequence homologies and similar structural topologies with fibronectins of the CRS proteins place tissue factor in a much wider spectrum of functions.
Tissue factor is a single chain transmembrane glycoprotein that initiates the coagulation pathway. Tissue factor initiates the extrinsic pathway of blood coagulation leading to thrombin generation and clot formation. Overall it is found in very low concentrations with the exception of a few tissues including placenta, lungs and brain. Notwithstanding its low abundance, it is an extremely efficient and potent cofactor in the onset of thrombin generation. Tissue factor is essential to life. Most of the functional studies on tissue factor have been performed using recombinant proteins. Posttranslational modifications described, also were those of recombinant proteins expressed in various organisms. Numerous inconsistencies and lack of direct evidence is found regarding the influence of posttranslational modifications on tissue factor function.
Our goal was to characterize modifications, specifically glycosylation, phosphorylation and disulfide bridge formation, of the natural protein from human placenta. We showed how these modifications influence the activity of the natural tissue factor protein in vitro. Our major findings indicated that glycosylation influences TF activity and that carbohydrate composition of the natural placental tissue factor is significantly different from that of its recombinant counterpart. We have also determined that the two disulfide bonds, found in the extracellular domain, do not play a role in tissue factor function. Additionally we have identified threonine 172 as being phosphorylated in the natural placental but not in the recombinant tissue factor protein.