The N-terminal Epidermal Growth Factor-like Domain of Coagulation Factor IX

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Binding of short chain phosphatidylserine (C6PS) enhances the proteolytic activity of factor X a by 60-fold (Koppaka, V., Wang, J., Banerjee, M., and Lentz, B. R. (1996) Biochemistry 35, 7482-7491). In the present study, we locate three C6PS binding sites to different domains of factor X a using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements on proteolytic and biosynthetic fragments of factor X a . Our results demonstrate that the structural responses of human and bovine factor X a to C6PS binding are somewhat different. Despite this difference, data obtained with fragments from both human and bovine factor X a are consistent with a common hypothesis for the location of C6PS binding sites to different structural domains. First, the ␥-carboxyglutamic acid (Gla) domain binds C6PS only in the absence of Ca 2؉ (k d ϳ 1 mM), although this PS site does not influence the functional response of factor X a . Second, a Ca 2؉ -dependent binding site is in the epidermal growth factor domains (EGF NC ) that are linked by Ca 2؉ and C6PS binding to the Gla domain. This site appears to be the lipid regulatory site of factor X a . Third, a Ca 2؉ -requiring site seems to be in the EGF C -catalytic domain. This site appears not to be a lipid regulatory site but rather to share residues with the substrate recognition site. Finally, the full functional response to C6PS requires linkage of the Gla, EGF NC , and catalytic domains in the presence of Ca 2؉ , meaning that PS regulation of factor X a involves linkage between widely separated parts of the protein.The substantial effects of soluble phosphatidylserine (C6PS 1 ) on the kinetics of prothrombin activation by factor X a(1) and on the structure of factor X a , as documented here, indicate that phosphatidylserine (PS) may act as an allosteric regulator of prothrombin activation. PS located on the cytoplasmic face of resting platelet plasma membranes is exposed on the surface of activated platelet vesicles (2, 3). The implication of this PS exposure and of the effect of PS on factor X a and on its ability to catalyze activation of prothrombin is that PS may act as a second messenger in regulating thrombin formation. Because of the crucial role of thrombin in hemostasis, the exposure of PS may be a crucial regulatory step in blood coagulation. To better define this regulatory process, it is important to know the locations of the PS binding sites on factor X a . The organization of factor X into structural domains is illustrated below in Fig. 1. Factor X consists of two peptides. The light chain consists of an N terminus ␥-carboxyglutamic acidrich region (Gla module) and two Cys-rich cassette modules. The heavy chain consists of the serine protease catalytic domain. The two cassette modules of the light chain show strong sequence and structural homology to epidermal growth factor (EGF) (4) and are thus referred to as EGF N and EGF C , where N and C indicate the domain nearer to the N and C termini, respectively. Crystal structures o...

Section: Effect Of Soluble C6ps On the CD Spectra Of Expressed Human mentioning

confidence: 84%

mentioning

confidence: 99%

Localization of Phosphatidylserine Binding Sites to Structural Domains of Factor Xa

Srivastava¹,

Wang²,

Majumder³

et al. 2002

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“…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)(14)(15)(16)(17)(18)(19)(20) by binding to several exposed surface loops near or remote from the catalytic pocket of the enzyme (21). According to the three-dimensional structure of FXa (22), the FXa Na ϩ -binding site is close to the catalytic pocket of the enzyme and to the FVa-binding h163-170 (Fig.…”

mentioning

confidence: 99%

Role of the α-Helix 163-170 in Factor Xa Catalytic Activity

Levigne

Thiec

Cherel

et al. 2007

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...

“…The Gla domain of FIXa appears to interact directly with FVIIIa in the FX activating complex as suggested by cross-linking experiments, implicating Phe 26 and perhaps Val 46 but not Phe 9 (34). The salt bridge between Glu 78 within the EGF1 domain and Arg 94 within the EGF2 domain is also shown to contribute to stimulation by FVIIIa in FX activation (24) by maintaining FIXa structure, although an FIX antibody directed to the C terminus of EGF1 domain had a marginally inhibitory effect on FX activation (35). The connecting segment between the two EGF domains (Leu 84 -Thr 87 ), but not segment Asn 89 -Lys 91 in the EGF2 domain, appeared to mediate stimulation of FX activation by …”

Section: Fixa Plateletsmentioning

confidence: 96%

Identification of Residues Asn89, Ile90, and Val107 of the Factor IXa Second Epidermal Growth Factor Domain That Are Essential for the Assembly of the Factor X-activating Complex on Activated Platelets

Yang

Chang

Lin

et al. 2004

Activated platelets promote intrinsic factor X-activating complex assembly by presenting high affinity, saturable binding sites for factor IXa mediated by two disulfide-constrained loop structures (loop 1, Cys 88 -Cys 99 ; loop 2, Cys 95 -Cys 109 ) within the second epidermal growth factor (EGF2) domain. To identify amino acids essential for factor X activation complex assembly, recombinant factor IXa point mutants in loop 1 (N89A, I90A, K91A, and R94A) and loop 2 (D104A, N105A, and V107A) were prepared. All seven mutants were similar to the native factor IXa by SDS-PAGE, active site titration, and content of ␥-carboxyglutamic acid residues. Kinetic constants obtained by either titrating factor X or factor VIIIa on SFLLRN-activated platelets or phospholipid vesicles revealed near normal values of K m(app) and K d(app)FVIIIa for all mutants, indicating normal substrate and cofactor binding. In a factor Xa generation assay in the presence of activated platelets and cofactor factor VIIIa, compared with native factor IXa (K d(app)FIXa ϳ 1.1 nM, V max ϳ 12 nM min ؊1 ), N89A displayed an increase of ϳ20-fold in K d(app)FIXa and a decrease of ϳ20-fold in V max ; I90A had an increase of ϳ5-fold in K d(app)FIXa and ϳ10-fold decrease in V max ; and V107A had an increase of ϳ3-fold in K d(app)FIXa and ϳ4-fold decrease in V max . We conclude that residues Asn 89 , Ile 90 , and Val 107 within loops 1 and 2 (Cys 88 -Cys 109 ) of the EGF2 domain of factor IXa are essential for normal interactions with the platelet surface and for the assembly of the factor X-activating complex on activated platelets.