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...

Factor X (FX) has high structure homology with other proteins of blood coagulation such as factor IX (FIX) and factor VII (FVII). These proteins present at their aminoterminal extremity a ␥-carboxyglutamic acid containing domain (Gla domain), followed by two epidermal growth factor-like (EGF1 and EGF2) domains, an activation peptide, and a serine protease domain. After vascular damage, the tissue factor-FVIIa (TF-FVIIa) complex activates both FX and FIX. FXa interacts stoichiometrically with tissue pathway inhibitor (TFPI), regulating TF-FVIIa activity by forming the TF-FVIIa-TFPI-FXa quaternary complex. Conversely, FXa boosts coagulation by its association with its cofactor, factor Va (FVa). To investigate the contribution of the Gla and EGF1 domains of FX in these complexes, FX chimeras were produced in which FIX Gla and EGF1 domains substituted the corresponding domains of FX. The affinity of the two chimeras, FX/FIX(Gla) and FX/FIX(EGF1), for the TF-FVIIa complex was markedly reduced compared with that of wild-type-FX (wt-FX) independently of the presence of phospholipids. Furthermore, the association rate constants of preformed FX/FIX(Gla)-TFPI and FX/FIX(EGF1)-TFPI complexes with TF-FVIIa were, respectively, 10-and 5-fold slower than that of wt-FXa-TFPI complex. Finally, the apparent affinity of FVa was 2-fold higher for the chimeras than for wt-FX in the presence of phospholipids and equal in their absence. These data demonstrate that FX Gla and EGF1 domains contain residues, which interact with TF-FVIIa exosites contributing to the formation of the TF-FVIIa-FX and TF-FVIIa-TFPI-FXa complexes. On the opposite, FXa Gla and EGF1 domains are not directly involved in FVa binding.The blood coagulation cascade consists of a series of enzymatic conversions driven by the formation of complexes between serine proteases and cell membrane-bound cofactors. Human factor X (FX) 1 is one of the serine protease zymogens playing a central role in coagulation processes leading to the formation of a fibrin clot. This is illustrated by the behavior of FX as a substrate or as an enzyme in three essential blood coagulation complexes. First, FX is a natural substrate, as well as factor IX (FIX), of the tissue factor-factor VIIa (TF-FVIIa) complex (1) considered as the initial enzyme complex in the cascade following vascular damage. FX activation by TF-FVIIa results from specific cleavage and release of a 52-residue activation peptide. Activated FX (FXa) can generate a tiny amount of thrombin from prothrombin in an extremely inefficient reaction (2). Tissue factor pathway inhibitor (TFPI) binds to TF-FVIIa-FXa to limit the production of FXa and FIXa by TF-FVIIa (3, 4). Nevertheless, once produced, thrombin and the initially formed FXa activate small quantities of factor V (FV) to FVa and factor VIII (FVIII) to FVIIIa (5-8). The activation of these two cofactors leads to the formation of two other essential procoagulant complexes, both involving FX, at the surface of procoagulant phospholipids in the presence of calcium ions (9),...

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