Investigating Protein-Membrane Interactions Mediated by Factor X That Regulate the Blood Clotting Cascade

Vascular trauma triggers the blood clotting cascade to maintain hemostasis by sequentially converting several zymogens to active enzymes via limited proteolysis. Coagulation factor X (FX) is activated by the extrinsic and intrinsic pathway to initiate and propagate the clotting cascade respectively. Further, activated FX (FXa) also participates in downstream reactions in the common pathway that leads to the formation of a blood clot, thereby, positioning FX centrally in the clotting cascade. Anionic membrane surfaces accelerate the activation of FX by thousands of times, underscoring the regulatory function of protein-membrane interactions in the clotting cascade. The N-terminal, γ-carboxyglutamate rich domain (Gla-domain) enables FX to reversibly bind phosphatidylserine (PS), in a calcium dependent manner. Previous studies showed that removing the Gla-domain diminished the rates of FX activation by the initiation complex even in solution, which indicates the participation of Gla-domain in protein-protein interactions as well. This demonstrates that FX Gla-domain plays a crucial role in the blood clotting cascade. However, these interactions of FX Gla-domain are poorly characterized at the molecular level. Therefore, here we investigated these interactions from the perspectives of the membrane phospholipids and clotting proteins that directly interact with FX Gla-domain. Quantitative binding studies were utilized to identify the stoichiometry of membrane lipids binding to FX Gla-domain in physiologically comparable membrane surfaces. Here for the first time, we present the evidence for a unique PS binding site in the FX Gla-domain. Next, we employed mutagenesis studies of FX Gla-domain residues to identify their impact on the rates of FX activation by the initiation complex of the clotting cascade. Along with additional membrane binding studies we identified the critical amino acid residues in the FX Gla-domain that participate in either protein-protein or membrane-protein interactions in the extrinsic pathway. A potent external activator of FX from Russell’s Viper Venom (also known as RVV-X) causes thrombotic complications in snake-bite victims, ultimately resulting in their death or disability. However, due to the high potency of RVV-X it has also been used to treat minor injuries and as a diagnostic tool in research. Biochemical studies showed that in absence of the Gla-domain, RVV-X poorly activates FX. Therefore, we extended our study to identify the residues mediating molecular interactions between FX Gla-domain and RVV-X. FX Gla-domain residues predicted to interact with RVV-X by in silico structural alignment studies were mutated to test their influence on the rates of FX activation by RVV-X. FX activated by RVV-X was utilized to investigate the effects of the mutated residues on the thrombin generation. Thus, we identified the key residues in FX Gla-domain essential for mediating the molecular interactions between FX and RVV-X. Surprisingly, we observed that the FX Gla-domain residues crucial to the extrinsic pathway, had a minimal impact on thrombin generation in the common pathway. Since several clotting reactions occur on membrane surfaces, we diversified our study beyond the molecular interactions of FX Gla-domain. We found that an anticoagulant protein, lactadherin, exhibited membrane binding in a calcium independent manner. These findings will aid in the development of therapeutic efforts against pathological conditions such as thrombosis and bleeding disorders in FX deficient patients.PHDBiological ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/172654/1/divyani_1.pd