Structural-functional studies of the non-enzymatic domains of prothrombin

• Main Author: Hung, Vo Cong
• Language: English
• Published: 2009
• Online Access: http://hdl.handle.net/2429/9988

To study the role of the non-enzymatic domains of the clotting protein prothrombin, human prothrombin and several variants were produced by using recombinant DNA techniques and in vitro tissue culture expression. The prothrombin variants included the first kringle domain deleted (rΔKl), the second kringle domain deleted (rΔK2), both kringle domains deleted (rΔKl/ΔK2), the second kringle domain substituted with the bovine counterpart (rhBK2), and the second kringle domain substituted with the first kringle (rKl/Kl). The recombinant proteins were expressed by using the pNUT-baby hamster kidney cell system under methotrexate selection. The expressed proteins were purified from the media using barium citrate precipitation followed by fast performance liquid chromatography (FPLC). Different gamma-carboxylated recombinant proteins were resolved by pseudo-affinity FPLC using a calcium gradient. The expressed recombinant proteins subjected to several characterization assays including Gla content analysis by capillary electrophoresis-laser induced fluorescence (CE-LIF), calcium and phospholipid binding assays, and activation assays by factor Xa and by the prothrombinase complex using purified systems. Results from the studies have indicated that a reduced Gla content in the protein, by as little as three residues, led to a substantial loss of calcium dependent phospholipid binding, and a reduced clotting ability. Results from the activation assays of the prothrombin variants have demonstrated that the second kringle domain was necessary for the activation of prothrombin by both the protease factor Xa alone and by the prothrombinase complex, implying important interactions of the second kringle with factor Xa and the cofactor Va. In addition, peptides derived from the loops of the second kringle domain were demonstrated to have potent inhibitory activity in the activation of prothrombin by the prothrombinase complex. Taken together the study has demonstrated that the nonenzymatic domains of prothrombin play important roles in the ability of prothrombin to be activated physiologically. The Gla domain is responsible for calcium and phospholipid binding, the kringle domains mediate protein-protein interactions with the protease factor Xa and cofactor Va of the prothrombinase complex.