Structural and mutagenesis data have indicated that the 220-loop of thrombin is stabilized by a saltbridge between Glu-217 and Lys-224, thereby facilitating the octahedral coordination of Na + with contributions from two carbonyl O atoms of Arg-221a and Lys-224. All three residues are also conserved in fXa and the x-ray crystal structure of fXa indicates that both Glu-217 and Lys-224 are within hydrogen-bonding distance from one another. To investigate the role of these three residues in the catalytic function of fXa and their contribution to interaction with Na + , we substituted them with Ala and characterized their properties in both amidolytic and proteolytic activity assays. The results indicate that the affinity of all three mutants for interaction with Na + has been impaired. The mutant with the greatest loss of affinity for Na + (E217A or E217Q) also exhibited a dramatic impairment (~3-4 orders of magnitude) in its activity toward both synthetic and natural substrates. Interestingly, factor Va (fVa) restored most of the catalytic defect with prothrombin, but not with the synthetic substrate. Both Glu-217 mutants exhibited a near normal affinity for fVa in the prothrombinase assay, but a markedly lower affinity for the cofactor in a direct-binding assay. These results suggest that, similar to thrombin, an ionic interaction between Glu-217 and Lys-224 stabilizes the 220-loop of fXa for binding Na + . They further support the hypothesis that the Na + and fVabinding sites of fXa are energetically linked and that a cofactor function for fVa in the prothrombinase complex involves inducing a conformational change in the 220-loop of fXa that appears to stabilize this loop in the Na + -bound active conformation.Factor Xa (fXa) 1 is a multi-domain vitamin K-dependent trypsin-like serine protease in plasma that, upon complex formation with factor Va (fVa) on the membrane surfaces expressing negatively charged phospholipids (prothrombinase complex), converts prothrombin to thrombin at the final stage of the clotting cascade (1-5). The assembly of fXa into the prothrombinase complex enhances the catalytic efficiency of fXa by greater than five orders of magnitude by improving both the K m and k cat of the substrate activation reaction (6,7). The improvement in K m (~two orders of magnitude) has been demonstrated to arise from the γ-carboxyglutamic acid (Gla) domain-dependent binding of both fXa and prothrombin on the same membrane surfaces (1,6,7). The remaining ~three orders of magnitude improvement in the rate of prothrombin activation by fXa has been attributed to the cofactor effect of fVa which increases the k cat of the reaction (2,7). The mechanism by which fVa promotes the k cat of *Address of Corresponding Author: Alireza R. Rezaie, Ph.D., Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1100 S. Grand Blvd., St. Louis, MO 63104, Phone: (314) Fax: (314) 977-9205, E-mail: rezaiear@slu.edu. † Both authors contributed equally to this study.1 Abbreviations -FX...