Abstract
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 alpha-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.
Highlights
Factor X (FX)4 is a vitamin K-dependent, two-chain glycoprotein that plays a central role in blood coagulation
We extended the finding of previous studies that have implicated the basic residues of h163– 170 in the regulation of Factor Xa (FXa) by cofactors [8, 9]
Our data show (Fig. 3) that FXa derivatives with a mutation at position Arg165 exhibited a severe defect in thrombin formation in the presence of small amount of factor Va (FVa)
Summary
Factor X (FX) is a vitamin K-dependent, two-chain glycoprotein that plays a central role in blood coagulation. According to the three-dimensional structure of FXa [22], the FXa Naϩ-binding site is close to the catalytic pocket of the enzyme and to the FVa-binding h163– 170 (Fig. 1). There is some evidence that both the FVa- and Naϩ-binding sites of FXa are energetically linked [20, 23]. The relationship between h163–170, which is a crucial FVa-binding site, and the Naϩ-binding site were investigated To this end, FXa derivatives were designed in which h163–170 was either substituted by the corresponding region of FVIIa and FIXa or mutated (Fig. 2) at residues known, based on the three-dimensional structure of FXa, to be solventexposed (Arg165) or to be proximate to the Naϩ-binding site (Val163 and Ser167). Lalanyl-L-pipecolyl-L-arginine-para-nitroanilide dihydrochloride (H-D-Phe-PipArg-pNA1⁄72HCl); S-2765, benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-paranitroanilide dihydrochloride (Z-D-Arg-Gly-Arg-pNA); SpeFXa, methoxycarbonyl-cyclohexylglycyl-glycyl-arginine-para-nitroanilide dihydrochloride (CH3CO2-D-CHGly-Gly-Arg-pNA), product name Spectrozyme FXa; PAB, paraaminobenzamidine; D-FFR-CK, methanesulfonyl-D-phenyl-phenyl-arginylchloromethyl ketone; ATIII, antithrombin III. 5 Amino acid sequence numbering of FXa and of all the proteases mentioned in this study is based on the three-dimensional topological equivalences with chymotrypsin
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