Role of the Residues of the 39-Loop in Determining the Substrate and Inhibitor Specificity of Factor IXa

Abstract

The activation of antithrombin (AT) by heparin facilitates the exosite-dependent interaction of the serpin with factors IXa (FIXa) and Xa (FXa), thereby improving the rate of reactions by 300-to 500-fold. Relative to FXa, AT inhibits FIXa with ϳ40-fold slower rate constant. Structural data suggest that differences in the residues of the 39-loop (residues 31-41) may partly be responsible for the differential reactivity of the two proteases with AT. This loop is highly acidic in FXa, containing three Glu residues at positions 36, 37, and 39. By contrast, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. To determine whether differences in the residues of this loop contribute to the slower reactivity of FIXa with AT, we prepared an FIXa/FXa chimera in which the 39-loop of the protease was replaced with the corresponding loop of FXa. The chimeric mutant cleaved a FIXa-specific chromogenic substrate with normal catalytic efficiency, however, the mutant exhibited ϳ5-fold enhanced reactivity with AT specifically in the absence of the cofactor, heparin. Further studies revealed that the FIXa mutant activates factor X with ϳ4-fold decreased k cat and ϳ2-fold decreased K m , although the mutant interacted normally with factor VIIIa. Based on these results we conclude that residues of the 39-loop regulate the cofactor-independent interaction of FIXa with its physiological inhibitor AT and substrate factor X.Factor IXa (FIXa) 2 is a vitamin K-dependent plasma serine protease that, upon complex formation with factor VIIIa (FVIIIa), on negatively charged membrane surfaces in the presence of Ca 2ϩ (intrinsic Tenase) activates factor X (FX) to factor Xa (FXa) during the blood coagulation process (1-5). FIXa plays an important role in the clotting cascade, because its deficiency is associated with the life-threatening disease, hemophilia B (6). The activity of FIXa toward its physiological substrate FX is very poor in the absence of FVIIIa, however, complex formation with the cofactor improves the catalytic efficiency of FIXa by 4 -5 orders of magnitude in the intrinsic Tenase complex (1-5). The proteolytic activity of FIXa in plasma is regulated by the serpin inhibitor, antithrombin (AT) (7)(8)(9)(10)(11). Surprisingly, in contrast to a dramatic cofactor-mediated improvement in the activity of FIXa toward FX, FVIIIa has a minimal cofactor effect on the reactivity of the protease with AT or its activity toward small synthetic substrates (12, 13), suggesting that FVIIIa-mediated exosite interactions with FX, involving sites remote from the active-site pocket, play dominant roles in the catalytic reaction (14, 15). In the case of the FIXa reaction with AT, the cofactor function of heparin-like glycosaminoglycans, similar to those lining the surface of the endothelium (16), is required to facilitate the protease recognition of the serpin (17-19).Heparin accelerates the reactivity of AT with FIXa by two distinct mechanisms: a conformational ac...

Residues of the 39-Loop Restrict the Plasma Inhibitor Specificity of Factor IXa

“…However, although the FIXa mutant exhibited a normal affinity for FVIIIa, its catalytic activity (k cat ) toward the natural substrate FX in the presence of FVIIIa was decreased ϳ4-fold, suggesting that residues of the 39-loop contribute to the recognition specificity of the substrate in the intrinsic tenase complex (10). Further studies revealed that the reactivity of the FIXa mutant with AT has been improved ϳ5-fold specifically in the absence of pentasaccharide, suggesting that the 39-loop of FIXa contributes to the slower reactivity of the protease with the serpin in the absence of a heparin cofactor (10). In light of the latter observation that the 39-loop of FXa improves the recognition specificity of the FIXa chimera with the circulating native conformer of AT, we wondered whether the 39-loop of FIXa plays any role in rendering FIXa resistant to inhibition by other plasma inhibitors, which readily inhibit FXa in circulation.…”

“…The expression and purification of the FIX mutant in which the residues of the 39-loop from residues Val 31 to Phe 41 ( 31 Val-Val-Leu-Asn-GlyLys-Val-Asp-Ala-Phe 41 ) in the chymotrypsin numbering (12) were replaced with the same 39-loop residues of FXa ( ) using the same expression/purification vector system have been described (10). The activation of FIX derivatives by the FXactivating enzyme from Russell's viper venom and determination of their active site concentration by an amidolytic activity assay and active site titrations using known concentrations of AT in the presence of heparin have been described (13).…”

“…Briefly, FIXa (5-10 nM) was incubated with increasing concentration of ZPI (100 -1000 nM) in 0.1 M NaCl, 0.02 M Tris-HCl, pH 7.5, and 5 mM Ca 2ϩ (TBS/Ca 2ϩ ) containing 0.1 mg/ml BSA and 0.1% PEG 8000 at room temperature for 0.5-2 h. In the presence of PZ (5-160 nM), the inhibition of the FIXa derivatives (3-5 nM) by ZPI (200 nM) was monitored for 0.5-5 min on 50 M PC/PS vesicles in the same TBS buffer system. All of the reactions were carried out in 50-l volumes in 96-well plates and at different time points, 50 l of the chromogenic substrate CBS 31.39 in TBS was added to each reaction, and the remaining enzyme activity was measured at 405 nm using a V max kinetic microplate reader (Molecular Devices, Menlo Park, CA) as described (10,16). The observed pseudo-first order rate constants (k obs ) were determined by fitting the time course data to a single exponential decay function with a non-zero end point, and the second order association rate constants (k 2 ) for uncatalyzed and PZ-catalyzed reactions were obtained from the slopes of the plots of k obs versus concentrations of ZPI as described (10,16).…”

“…All of the reactions were carried out in 50-l volumes in 96-well plates and at different time points, 50 l of the chromogenic substrate CBS 31.39 in TBS was added to each reaction, and the remaining enzyme activity was measured at 405 nm using a V max kinetic microplate reader (Molecular Devices, Menlo Park, CA) as described (10,16). The observed pseudo-first order rate constants (k obs ) were determined by fitting the time course data to a single exponential decay function with a non-zero end point, and the second order association rate constants (k 2 ) for uncatalyzed and PZ-catalyzed reactions were obtained from the slopes of the plots of k obs versus concentrations of ZPI as described (10,16). All of the values are presented as the average of at least three independent measurements Ϯ S.D.…”

“…One of these surface loops, known as the variable region 1 or 39-loop (also referred to as the 37-loop), is known to participate in restricting the substrate and inhibitor specificity of all coagulation proteases (11,12). In a recent study, we prepared a FIXa mutant in which the residues of this loop (residues 31-41) were replaced with the corresponding residues of FX (FIXa-FX 39-loop ), and then we demonstrated that this loop plays a critical role in the reactivity of FIXa with AT and its catalytic activity in intrinsic tenase (10). In this study, we demonstrate that the 39-loop of FIXa also plays a critical role in restricting the ZPI and TFPI specificity of the protease.…”