Factor Xa (FXa) is a key protease of the coagulation pathway whose activity is known to be in part modulated by binding to factor Va (FVa) and sodium ions. Previous investigations have established that solvent-exposed, charged residues of the FXa ␣-helix 163-170 (h163-170), Arg 165 and Lys 169 , participate in its binding to FVa. In the present study we aimed to investigate the role of the other residues of h163-170 in the catalytic functions of the enzyme. FX derivatives were constructed in which point mutations were made or parts of h163-170 were substituted with the corresponding region of either FVIIa or FIXa. Purified FXa derivatives were compared with wild-type FXa. Kinetic studies in the absence of FVa revealed that, compared with wild-type FXa, key functional parameters (catalytic activity toward prothrombin and tripeptidyl substrates and non-enzymatic interaction of a probe with the S1 site) were diminished by mutations in the NH 2 -terminal portion of h163-170. The defective amidolytic activity of these FXa derivatives appears to result from their impaired interaction with Na ؉ because using a higher Na ؉ concentration partially restored normal catalytic parameters. Furthermore, kinetic measurements with tripeptidyl substrates or prothrombin indicated that assembly of these FXa derivatives with an excess of FVa in the prothrombinase complex improves their low catalytic efficiency. These data indicate that residues in the NH 2 -terminal portion of the FVa-binding h163-170 are energetically linked to the S1 site and Na ؉ -binding site of the protease and that residues Val 163 and Ser 167 play a key role in this interaction. Factor X (FX)4 is a vitamin K-dependent, two-chain glycoprotein that plays a central role in blood coagulation. During this process, FX is activated to FXa and forms a high affinity macromolecular complex with other components of the prothrombinase complex, factor Va (FVa), negatively charged phospholipid surfaces, and calcium to activate prothrombin to thrombin (1-6). These macromolecular interactions lead to an increase of 5 orders of magnitude in the catalytic efficiency of FXa toward prothrombin (2, 7). Enhancement of the k cat of the reaction is mainly due to the cofactor function of FVa. Two basic residues of h163-170 5 of the protease domain of FXa, namely Arg 165 and Lys 169 , directly interact with FVa (8, 9). All known sequences from different species in this surface-exposed helix of FXa are similar. Interestingly despite being stimulated by different cofactors, the catalytic domains of other blood coagulation proteins, such as factor IXa (FIXa) and factor VIIa (FVIIa), share the same cofactor-dependent activity binding site based on the structural equivalences with chymotrypsin (10 -12).Like other serine proteases of blood coagulation, small ligands such as calcium and sodium can allosterically modulate the activity and the specificity of FXa (13-20) by binding to several exposed surface loops near or remote from the catalytic pocket of the enzyme (21). According to the th...