scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

scholarly journals Characterization of a factor IX variant with a glycine207 to glutamic acid mutation

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1866-1873 ◽  
Author(s):  
SW Lin ◽  
CN Lin ◽  
N Hamaguchi ◽  
KJ Smith ◽  
MC Shen
Keyword(s):  
Glutamic Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intrinsic Fluorescence ◽  
Factor Ix ◽  
Active Site Pocket ◽  
Factor Ixa ◽  
Factor Xia

Abstract Factor IXTaipei9 is a factor IX variant from a hemophilia B patient with reduced levels of circulating protein molecules (cross-reacting material reduced, CRM). This variant contained a glycine (Gly) to glutamic acid (Glu) substitution at the 207th codon of mature factor IX. The functional consequences of the Gly-->Glu mutation in factor IXTaipei9 (IXG207E) were characterized in this study. Plasma-derived IXG207E exhibited a mobility similar to that of normal factor IX on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its specific activity was estimated to be 3.5% that of the purified normal factor IX in a one-stage partial thromboplastin time assay (aPTT). Cleavage of factor IXG207E by factor XIa or factor VIIa-tissue factor complex appeared to be normal. When the calcium-dependent conformational change was examined by monitoring quenching of intrinsic fluorescence, both normal factor IX and IXG207E exhibited equivalent intrinsic fluorescence quenching. Activated factor IXG207E (IXaG207E) also binds antithrombin III equally as well as normal factor IXa. However, aberrant binding of the active site probe p-aminobenzamidine was observed for factor XIa-activated factor IXG207E, indicating that the active site pocket of the heavy chain of factor IXaG207E was abnormal. Moreover, the rate of activation of factor X by factor IXaG207E, as measured in a purified system using chromogenic substrates, was estimated to be 1/40 of that of normal factor IXa. A computer-modeled heavy-chain structure of factor IXa predicts a hydrophobic environment surrounding Gly-207 and this Gly forms a hydrogen bound to the active site serine-365. The molecular mechanism of the Gly-->Glu mutation in factor IXTaipei9 might result in the alteration of the microenvironment of the active site pocket which renders the active site serine-365 inaccessible to its substrate.

In Vitro Mutagenesis Study of Two Critical Glutamic Acids in the Calcium Binding Loop of the Factor IX Heavy Chain

1994 ◽  
Vol 72 (06) ◽  
pp. 856-861 ◽  
Author(s):  
Nobuko Hamaguchi ◽  
Darrel Stafford
Keyword(s):  
Glutamic Acid ◽  
Binding Site ◽  
Heavy Chain ◽  
Active Site ◽  
Calcium Binding ◽  
Factor Ix ◽  
Calcium Binding Site ◽  
Factor Ixa ◽  
Factor Xia ◽  
Binding Loop

SummaryWe investigated the structural and functional significance of calcium binding in the factor IXa heavy chain by introducing point mutations into the probable calcium binding site (residues 235 and 245). According to factor IXa computer modelling based on trypsin x-ray structure, side chains of two glutamic acid residues, 235 and 245, together with backbone carbonyl groups of residues 237 and 240, bind a calcium ion. Factor IX clotting activity decreased approximately 25 percent on substitution of glutamic acid 235 with lysine. Activity decreased more than 90 percent on substitution of glutamic acid 245 with lysine. Activity also decreased more than 90 percent on substitution of both glutamic acids by lysines. Factor XIa cleavage of factor IXGlu235Lys and factor IXGlu245Lys appeared normal by polyacrylamide gel analysis. (Factor IXGlu235Lys: Factor IX with Lysine substituted for Glutamic acid at residue 235. Factor IXGlu245Lys: Factor IX with Lysine substituted for Glutamic acid at residue 245. Factor IXGlu235&245Lys: Factor IX with Lysine substituted for Glutamic acid at residues 235 and 245.) Activated factor IXGlu235Lys bound the fluorescent active site probe, p-aminobenzamidine, normally, while factor XIa cleaved factor IXGlu245Lys and factor IXGlu235&245Lys failed to bind p-aminobenzamidine. Plasma purified factor IX titrated with terbium showed an increase in luminescence; however, factor IXGlu235Lys and factor IXGlu245Lys had no effect on terbium luminescence. Radioimmunoassays indicate that in calcium’s absence, factor IXGlu235Lys adopts a conformation similar to normal factor IX in the presence of calcium. By contrast, factor IXGlu245LyS’s conformation in the presence of calcium is similar to that of plasma purified factor IX in the absence of calcium. We conclude that glutamic acids 235 and 245 are, as predicted, involved in the calcium binding site in the factor IX catalytic domain, and that the correct conformation of this calcium binding loop is essential for maintaining the active site pocket.

scholarly journals Cleavage and activation of human factor IX by serine proteases

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 821-831
Author(s):  
DL Enfield ◽  
AR Thompson
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Immunoradiometric Assay ◽  
Single Chain ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia

Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

Activation of Bovine Factor IX By Bovine Factor XIa . Active Site Titration Of Factor IXa and Development of A spectro-Photometric assay for Factor IX

1981 ◽  
Author(s):  
G Tans ◽  
T Janssen-Claessen ◽  
G v Dieijen ◽  
J Rosing ◽  
H C Hemker
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Factor Ix ◽  
Plasma Samples ◽  
Factor X ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Bovine Factor

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate.Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.

scholarly journals Cleavage and activation of human factor IX by serine proteases

Blood ◽  
1984 ◽  
Vol 64 (4) ◽  
pp. 821-831 ◽  
Author(s):  
DL Enfield ◽  
AR Thompson
Keyword(s):  
Active Site ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Factor Ix ◽  
Immunoradiometric Assay ◽  
Single Chain ◽  
Heavy Chains ◽  
Factor Ixa ◽  
Human Factor Ix ◽  
Factor Xia

Abstract Human factor IX circulates as a single-chain glycoprotein. Upon activation in vitro, it is cleaved into disulfide-linked light and heavy chains and an activation peptide. After reduction of activated 125I-factor IX, the heavy and light chains are readily identified by gel electrophoresis. A direct, immunoradiometric assay for factor IXa was developed to assess activation of factor IX for proteases that cleaved it. The assay utilized radiolabeled antithrombin III with heparin to identify the active site and antibodies to distinguish factor IX. After cleavage of factor IX by factor XIa, factor VIIa- tissue thromboplastin complex, or the factor X-activating enzyme from Russell's viper venom, antithrombin III bound readily to factor IXa. Cleavage of 125I-factor IX by trypsin, chymotrypsin, and granulocyte elastase in the presence of calcium yielded major polypeptide fragments of the sizes of the factor XIa-generated light and heavy chains. Kallikrein did not cleave the zymogen. Nonactivation cleavage was noted by thrombin, but only in the absence of calcium. When the immunoradiometric assay was used to assess trypsin-cleaved factor IX, the product bound antithrombin III, but not maximally. After digesting with insolubilized trypsin, clotting activity confirmed activation. In contrast, incubation of factor IX with elastase (Takaki A et al, J Clin Invest 71:1706, 1983) or chymotrypsin did not lead to generation of an antithrombin III-binding site, despite their digestion of 125I-factor IX into heavy and light chain-sized fragments. In evaluating activation of factor IX, physical evidence of activation cleavages does not necessarily correlate with generation of an active site.

scholarly journals Isolation and Properties of the Abnormal Factor IX Molecule of Hemophilia BM

1977 ◽  
Author(s):  
B. Østerud ◽  
K. Lavine ◽  
C. K. Kasper ◽  
S. I. Rapaport
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Heavy Chain ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Factor Ix ◽  
Procoagulant Activity ◽  
Ca Ions

Abnormal factor IX from a hemophilia Bm patient (F. IX-Bm) has been isolated to homogeneity on SDS Polyacrylamide gel electrophoresis by the same technique utilized for purifying normal F. IX. F. IX-Bm generated no measurable procoagulant activity when incubated with F. XIa in a two-stage F. IXa assay (normal F. IX, 55 sec; F. IX-Bm, >30 min).F. IX-Bm inhibited the activation of F. X by F. VII and ox brain thromboplastin as measured in an amidolytic assay for factor Xa. Normal F. IX also inhibited this reaction but to a five times lesser degree. F. IX-Bm has the same molecular weight on SDS gel electrophoresis as normal F. IX (55,000) and does not differ from normal F. IX in its amino acid composition. F. XIa cleaves F. IX-Bm in the presence of Ca ions at the same rate as it cleaves normal F. IX, yielding a heavy chain of 27,000 molecular weight and a light chain of 16,000 molecular weight. However, the cleavage does not give rise to procoagulant activity. Like normal F. IXa, the cleaved forms of F. IX-Bm appear to bind phospholipid since F. IX-Bm protein was precipitated with phospholipid in the presence of Ca ions. These data support an hypothesis that the abnormality in the F. IX-Bm molecule stems from a defect at the active site.

scholarly journals The Role of Factor IX (Christmas Factor) in Blood Coagulation

1977 ◽  
Author(s):  
Earl W. Davie ◽  
Kazuo Fujikawa ◽  
Patricia Lindquist ◽  
Richard Di Scipio ◽  
Kotoku Kurachi ◽  
...  
Keyword(s):  
Blood Coagulation ◽  
Disulfide Bond ◽  
Heavy Chain ◽  
Peptide Bond ◽  
Factor Ix ◽  
Activation Mechanism ◽  
Amino Terminal ◽  
Factor Ixa ◽  
Factor Xia ◽  
Russell’S Viper

Factor IX participates in the middle phase of the intrinsic pathway of blood coagulation. The reactions leading to the activation of factor IX involve prekallikrein, high molecular weight kininogen, and factor XII. These proteins interact in the presence of a surface such as kaolin and give rise to the activation of factor XI. Factor XIa then converts factor IX to factor IXa in the presence of calcium ions. In this reaction, factor IX (a single-chain glycoprotein of mol. wt.-~55,000) is converted to factor IXa in a two-step reaction. In the first step, an internal peptide bond is cleaved leading to the formation of an intermediate lacking enzymatic activity. This intermediate contains two polypeptide chains held together by a disulfide bond(s). In the second step, an activation peptide is split from the heavy chain of the intermediate giving rise to factor IXa (mol. wt. ~45,000). Factor IXa is composed of a heavy chain (mol. wt.~27,000) and a light chain (mol. wt. ~16,000) held together by a disulfide bond(s). The activation mechanism is essentially identical for human and bovine factor IX. Factor IXa is a serine protease with esterase activity and is sensitive to protease inhibitors such as antithrombin III. Factor IX is also activated by the protease from Russell’s viper venom, but this reaction involves only a single cleavage in the precursor molecule. The critical step in the activation of factor IX by factor XIa or the protease from Russell’s viper venom is the cleavage of the same internal Arg-Val peptide bond and the formation of a new amino-terminal sequence of Val-Val-Gly-Gly- in the heavy chain of the enzyme.

Factor IX Substrate Binding Exosite on the Light Chain of Factor Xia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1959-1959 ◽  
Author(s):  
Dipali Sinha ◽  
Mariola Marcinkiewicz ◽  
Duraiswamy Navaneetham ◽  
Peter N. Walsh
Keyword(s):  
Heavy Chain ◽  
Light Chain ◽  
Active Site ◽  
Substrate Binding ◽  
Factor Ix ◽  
Reversible Inhibitor ◽  
Factor Xia ◽  
Presence And Absence ◽  
Kinetics Of ◽  
Hydrolysis Of

Abstract Binding of factor IX (FIX) to an exosite on the heavy chain of factor XIa (FXIa) is essential for the optimal activation of FIX (Biochemistry26:3768–3775, 1987). We now provide evidence that a second substrate binding exosite resides on the light chain (LC) of FXIa. To understand the mechanism of activation of FIX by LC we have investigated 1) the kinetics of S-2366 hydrolysis by FXIa and its isolated LC in the presence and absence of a reversible inhibitor of serine proteases (p-aminobenzamidine, PAB); 2) the kinetics of activation of the macromolecular substrate FIX by FXIa and its isolated light chain in the presence and absence of either PAB or the alternative substrate S-2366; and 3) the effect of active site inhibited FXIa LC (LCi, i.e., FXIa LC treated with phenylmethylsulfonyl fluoride) on the activation of FIX by FXIa. Kinetic parameters for the hydrolysis of S-2366 by FXIa and its isolated LC are comparable. However kcat for the activation of the macromolecular substrate FIX by LC compared to full-length FXIa is reduced by ~300-fold whereas the Km was only slightly higher (2- to 3-fold). PAB inhibited the hydrolysis of S-2366 both by FXIa and LC in a classical competitive fashion. If an exosite on the heavy chain of FXIa is solely responsible for formation of the Michaelis complex then PAB should have inhibited the activation of FIX by LC in a competitive fashion. However, PAB and S-2366 were found to be noncompetitive inhibitors of FIX activation by FXIa or LC demonstrating the presence of an exosite for FIX binding on the LC remote from its active site. The presence of the second exosite on the light chain of FXIa was further confirmed by the fact that the V vs. S as well as the V vs. I plots of the activation of FIX by FXIa in the presence of LCi were sigmoidal suggesting formation of a nonproductive SI complex. We conclude that for FIX-activation, two macromolecular substrate-binding exosites, one on the heavy chain and one on the light chain of FXIa are required to mediate the formation of the Michaelis complex.

The Activation of Human Factor IX

1975 ◽  
Vol 33 (03) ◽  
pp. 553-563 ◽  
Author(s):  
B Østerud ◽  
K Laake ◽  
H Prydz
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Tissue Thromboplastin ◽  
Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

scholarly journals Functional consequences of an arginine180 to glutamine mutation in factor IX Hilo

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1540-1544 ◽  
Author(s):  
DM Monroe ◽  
DM McCord ◽  
MN Huang ◽  
KA High ◽  
RL Lundblad ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Active Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Ix ◽  
Affinity Column ◽  
Extrinsic Pathway ◽  
Control Value ◽  
Amino Terminal ◽  
Active Site Probe ◽  
Covalently Linked

Abstract Factor IX Hilo is a variant factor IX molecule that has no detectable coagulant activity. The defect in factor IX Hilo arises from a point mutation in the gene such that in the protein Arg180 is converted to a Gln. Activation of factor IX Hilo by factor Xla was monitored using the fluorescent active site probe p-aminobenzamidine. Normal factor IX showed complete activation in one hour as determined by measuring the increase in fluorescence when p-aminobenzamidine bound to activated factor IX. Factor IX Hilo showed no increase in fluorescence even after 24 hours, indicating that the active site was not exposed. Polyacrylamide gel electrophoresis showed that factor IX Hilo was cleaved to a light chain plus a larger peptide with a molecular weight equivalent to a heavy chain covalently linked to an activation peptide. Amino terminal amino acid sequencing of factor IX Hilo cleaved by factor Xla showed cleavage only at Arg145-Ala146, indicating that the Gln180-Val181 bond was not cleaved and that the active site was thus not exposed. The presence of factor IX Hilo in patient plasma was responsible for the patient having a very long ox brain prothrombin time characteristic of severe hemophilia Bm. Patient plasma had an ox brain prothrombin time of 100 seconds using a Thrombotest kit, significantly prolonged over the normal control value of 45 seconds. When factor IX Hilo was depleted from patient plasma using an immunoaffinity column, the ox brain prothrombin time decreased to 41 seconds. When factor IX Hilo was added back to depleted patient plasma, to normal plasma depleted of factor IX by the same affinity column, or to plasma from a CRM- hemophilia B patient, the ox brain prothrombin time was significantly prolonged. We conclude that the Arg180 to Gln mutation in factor IX Hilo results in a molecule that cannot be activated by factor Xla. Further, our data suggest that the mutation results in a molecule that interacts with components of the extrinsic pathway to give a prolonged ox brain prothrombin time.

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