| Summary: | 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. === Medicine, Faculty of === Biochemistry and Molecular Biology, Department of === Graduate
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