Purification and Characterization of Factor VII Inhibitor Found in a Patient with Life Threatening Bleeding

2000 ◽  
Vol 83 (01) ◽  
pp. 60-64 ◽  
Author(s):  
Seiji Miyamoto ◽  
Atsushi Iwasa ◽  
Masao Ishii ◽  
Kenji Okajima ◽  
Yu-ichi Kamikubo
Keyword(s):  
Light Chain ◽  
Calcium Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Vii ◽  
Immunoblotting Analysis ◽  
Calcium Dependent ◽  
Sds Page ◽  
Carboxyglutamic Acid ◽  
Life Threatening ◽  
Gla Domain

SummaryWe recently observed a patient with acquired inhibitor-induced F.VII deficiency whose plasma level of F.VII was < 1.0%. However, the biochemical nature of the inhibitor has not yet been clarified. In the present study, we purified the F.VII inhibitor from the patient’s plasma by using activated F.VII (F.VIIa)-conjugated gel and characterized the inhibitor. The results showed that the inhibitor comprised two kinds of antibodies: one was eluted with EDTA (antibody 1) and the other with glycine-HCl buffer (pH 2.3) (antibody 2) from the F.VIIa affinity gel. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of these inhibitors demonstrated that both antibodies had features of immunoglobulin G1 (IgG1) with κ and λ-light chains. Antibody 1 bound to the immobilized F.VIIa with a high affinity in the presence of calcium ion, while antibody 2 bound to the F.VIIa very weakly and the binding was independent of calcium ion. Immunoblotting analysis demonstrated that antibody 1 bound to the light chain of F.VIIa after reduction with 2-mercaptoethanol, while it did not react with either the γ carboxyglutamic acid (Gla)-domainless light chain of F.VIIa or the heavy chain with the protease domain. Antibody 1 markedly inhibited the activity of tissue factor-F.VIIa complex. Based on these observations, it is suggested that F.VIIa autoantibody (antibody 1) recognizes the calcium-dependent conformation within or near the Gla domain and inhibits F.VIIa activity by interacting with the light chain.

Kinetic Description Of Conversion Of Bovine Prethrombin 2 To Thrombin

1981 ◽  
Author(s):  
Thomas L Carlisle ◽  
Craig M Jackson
Keyword(s):  
Calcium Ion ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Maximum Velocity ◽  
Catalytic Efficiency ◽  
Kinetic Description ◽  
Factor Va ◽  
Carboxyglutamic Acid ◽  
Thrombin Formation

Bovine Factor Xa slowly converts Prethrombin 1 to thrombin plus Fragment 2. Inclusion of Ca2+ increased the rates of Prethrombin 1 consumption, Prethrombin 2 production and thrombin formation detected by SDS polyacrylamide gel electrophoresis or by thrombin assay. Ca2+ also increased the rate of thrombin formation from equimolar mixtures of Prethrombin 2 and Fragment 2 (Prethrombin 2/Fragment 2) by approximately 1.8 fold. Calcium ion thus increases the rate of both proteolyses required to generate thrombin from Prethrombin 1. Studies using Factor Xa (des light chain residues 1-44) indicated that this effect of Ca2+ required the region of Factor Xa containing gamma-carboxyglutamic acid.Factor Va markedly lowered the apparent Km of Factor Xa for Prethrombin 2/Fragment 2, with decreases greater than 20 fold observed under some conditions. The apparent maximum velocity also increased by up to 50 fold. The extent of increase was greater at higher concentrations of Factor Va, and was about 6 fold greater in the presence of Ca2+ than in its absence. Factor Va binding to Factor Xa (forming XaVa with enhanced substrate binding and/or catalytic efficiency), and Factor Va binding to Prethrombin 2/Fragment 2 (forming a substrate more readily bound and/or cleaved) must be considered among the possible explanations for these effects. Previous qualitative observations suggest that these effects of Factor Va on activation of Prethrombin 2/ Fragment 2 are important in understanding the activation of prothrombin.

Dicoumarol-induced prothrombins containing 6, 7, and 8 γ-carboxyglutamic acid residues: isolation and characterization

1989 ◽  
Vol 67 (8) ◽  
pp. 411-421 ◽  
Author(s):  
Om P. Malhotra
Keyword(s):  
Gel Electrophoresis ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Calcium Dependent ◽  
Isolation And Characterization ◽  
Carboxyglutamic Acid ◽  
K Deficiency ◽  
Agar Gel Electrophoresis

Isolation and characterization of γ-carboxyglutamic acid (Gla) deficient prothrombins induced by Warfarin or dicoumarol are useful for studying the role of specific Gla residues in prothrombin. In addition to 7-Gla prothrombin, we have isolated two more atypical prothrombins from the barium citrate eluate, one containing 6.11, and the other, 7.85 Gla residues, presumably 6- and 8-Gla prothrombins. The actual Gla content of the 7-Gla isomer was 7.05. Each of the 6-, 7-, and 8-Gla variants showed a single component by agar or dodecyl sulfate Polyacrylamide gel electrophoresis. When agar gel electrophoresis was performed in calcium, each of the variants moved more rapidly than normal (10-Gla) prothrombin. In the presence of EDTA, the 8-Gla isomer exhibited the fastest mobility, equivalent to that of normal prothrombin, followed by 7-, and then 6-Gla variants. The physiological activities of the isomers were found to be 18 to 23% for 8-, 6 to 8% for 7-, and 2 to 3% of normal prothrombin for 6-Gla variant. Prothrombin fragment 1, derived from 8-Gla prothrombin, exhibited 23% of calcium-induced fluorescence quenching, compared with 40% for 10-Gla and 8% or less for 7- and 6-Gla fragments 1. Competition radioimmunoassay data show that calcium-dependent anti (normal) prothrombin polyclonal antibodies are not specific for 10-Gla prothrombin, since the 7- and 8-Gla isomers were able to displace radiolabeled (125I) normal prothrombin.Key words: prothrombin, blood clotting, dicoumarol, Warfarin, γ-carboxyglutamic acid, vitamin K deficiency.

Role of Gamma-Carboxyglutamic Acid (GLA) Domain in Phosphatidylserine (PS)-Regulated Activity of Factor IXa.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2696-2696
Author(s):  
Rinku Majumder ◽  
Judson Craft ◽  
Alireza Rezaie ◽  
R. Barry ◽  
Dougald Monroe
Keyword(s):  
Equilibrium Dialysis ◽  
Factor Xa ◽  
Regulatory Site ◽  
Calcium Dependent ◽  
Catalytic Domains ◽  
Factor Ixa ◽  
Carboxyglutamic Acid ◽  
Proteolytic Activities ◽  
Gla Domain

Abstract Both factors Xa and IXa are vitamin-K-dependent serine proteases that consist of disulfide-linked heavy and light chains. The heavy chain contains the serine protease active site, while the light chain contains the GLA (gamma-carboxyglutamic acid) domain, EGF-I (epidermal growth factor-like region) and EGF-II domains. Effect of PS on FXa: We have extensively studied the interaction of factor Xa with 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS). C6PS is a soluble analogue of phosphatidylserine, which is present in platelet membranes; it serves as a model for the effect of membrane-bound PS on factor Xa activity and structure. We located three C6PS binding sites to different domains of factor Xa using a combination of activity, circular dichroism, fluorescence, and equilibrium dialysis measurements, showing that : the Gla domain binds C6PS only in the absence of calcium (kd ∼ 1 mM); a calcium-dependent, regulatory, PS-binding site exists in the EGFN domain when linked by calcium to the Gla domain; and a second calcium-requiring site exists in the EGFC-catalytic domains and shares residues with the substrate recognition site. We have shown that full functional response to C6PS requires linkage of the Gla, EGFNC, and catalytic domains in the presence of calcium. Recently we have observed that the proteolytic activity of des EGFN factor Xa is not affected in the presence of C6PS, locating more precisely a significant component of the PS-triggered regulatory site to the EGFN domain. Efect of PS on FIXa: We have previously reported that C6PS induces a calcium-dependent conformational change in factor IXa that regulates the amidolytic and proteolytic activities. We have also shown that factor IXa binds 2 molecules of C6PS. Here we have examined the role of the GLA domain in this C6PS regulation of factor IXa using a GLA-domainless variant of factor IXa (GD-IXa). In the absence of the GLA domain, binding to C6PS has no effect on the rate of proteolytic or amidolytic activity of GD-IXa toward both factor X and synthetic substrates. The binding of C6PS to GD-IXa studied using intrinsic tryptophan fluorescence (Kd = ∼50μM) was different from that for native factor IXa (Kd = ∼2μM). The critical micelle concentration (CMC) of C6PS under the conditions of these experiments (∼300μM) was much greater than the C6PS concentrations used in our experiments. We conclude that: C6PS does bind to GD-IXa, but at a reduced affinity compared to factor IXa; C6PS does not regulate the amidolytic or proteolytic activities of factor IXa in the absence of GLA domain. This could be due to two C6PS sites existing in FIXa, with the regulatory site requiring the Gla domain (as observed for FXa), or to a requirement for the Gla domain for the regulatory activity of a single site. Future studies looking at the stoichiometry of binding of C6PS with GD-IXa will help distinguish between these possibilities. Supported by Supported by NHLBI (HL 072827).

Role of the Direct Interaction between Factor VIII C2 and Factor IXa Gla Domain in the Factor Xase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2687-2687
Author(s):  
Tetsuhiro Soeda ◽  
Keiji Nogami ◽  
Masahiro Takeyama ◽  
Kenichi Ogiwara ◽  
Kazuhiko Tomokiyo ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Dependent Manner ◽  
C2 Domain ◽  
Factor X ◽  
Calcium Dependent ◽  
Factor Ixa ◽  
Factor Viiia ◽  
Gla Domain

Abstract Factor VIII functions as a cofactor for factor IXa in the anionic phospholipid surface-dependent conversion of factor X to Xa. It is well-known that the A2 and A3 domains of factor VIII interact with the catalytic domain and EGF2 domain of factor IXa, respectively. Recently, Furie et al. have reported that the Gla domain of factor IXa (factor IXa-GD) interacts with the light chain of factor VIII. However, the factor IXa-GD-interactive site on the light chain remained to be investigated. In the current study, the recombinant C2 (rC2) domain of factor VIII was prepared using a yeast secretion system. ELISA-based assay in the absence of phospholipid showed the Glu-Gly-Arg-active site modified factor IXa (EGR-factor IXa) bound to the immobilized rC2 domain dose-dependently, and the binding ability was maximum under the condition of 150 mM NaCl/1 mM CaCl2. This binding was competitively inhibited by the addition of excess of factor VIII or rC2 domain, supporting the specificity of this interaction. Furthermore, the presence of high ionic strength and the metal-ion chelator EDTA blocked this binding by ∼95 and ∼75%, respectively. Surface plasmon resonance-based assay showed that the binding affinity (Kd) of rC2 domain for EGR-factor IXa was 108 ± 15.5 nM. GD less-factor IXa, deleting the GD completely, failed to bind to rC2 domain. A monoclonal antibody against factor IXa-GD specific for calcium-dependent conformation (mAbIXa-GD) also inhibited (∼ 95%) the rC2 domain binding to EGR-factor IXa in a dose-dependent manner (IC50; 758 nM), suggesting the authentic of the C2 domain and factor IXa-GD interaction. The addition of rC2 domain or mAbIXa-GD inhibited the factor IXa-catalyzed factor X activation with factor VIIIa in the absence of phospholipid (IC50; 15.7 μM or 43.2 nM, respectively), whilst both any little affected in the absence of factor VIIIa. In addition, the ∼8-kDa C2 fragment obtained by V8 protease digestion (residues 2182–2259) bound directly to EGR-factor IXa. Taken together, these results indicate that factor VIII C2 domain directly interacts with factor IXa-GD via both the electrostatic- and calcium-dependent interactions. Furthermore, our results provide the first evidence for an essential role of the C2 domain in the association between factor VIII and factor IXa in the factor Xase complex.

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock

2002 ◽  
Vol 76 (3) ◽  
pp. 217-223 ◽  
Author(s):  
J. Martinez ◽  
J. Perez-Serrano ◽  
W.E. Bernadina ◽  
F. Rodriguez-Caabeiro
Keyword(s):  
Alkaline Phosphatase ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Differential Survival ◽  
Immunoblotting Analysis ◽  
Sds Page ◽  
Shock Proteins ◽  
Dose Dependent ◽  
Trichinella Nativa

AbstractStress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS–PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS–PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS–PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.

Molecular characterization and immunodiagnostics of Dicrocoelium dendriticum species isolated from sheep of north-west Himalayan region

2020 ◽  
Vol 94 ◽  
Author(s):  
J.S. Dar ◽  
U. Shabir ◽  
S.A. Dar ◽  
B.A. Ganai
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Ovis Aries ◽  
Diagnostic Specificity ◽  
Immunoblotting Analysis ◽  
Accurate Identification ◽  
North West ◽  
Dicrocoelium Dendriticum ◽  
Specificity And Sensitivity ◽  
Sds Page

Abstract Despite its extensive presence among grazing ruminants, dicrocoeliosis, also known as ‘small liver fluke’ disease, is poorly known and often underestimated by researchers and practitioners in many countries. The accurate identification and prepatent diagnosis of Dicrocoelium dendriticum infection is an essential prerequisite for its prevention and control. In the present study, the morphologically identified specimens isolated from the bile ducts of sheep (Ovis aries) were validated through molecular data. The sequence analysis of the second internal transcribed spacer (ITS-2) of our isolates showed a high degree of similarity with D. dendriticum using the BLAST function of the National Center for Biotechnology Information (NCBI). The phylogenetic analysis of our isolates showed a close relationship with previously described D. dendriticum isolates from different countries. The antigenic profiles of somatic and excretory/secretory (E/S) antigens of D. dendriticum were revealed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting using sera from sheep naturally infected with D. dendriticum. By SDS–PAGE, 16 distinct bands were revealed from crude somatic fraction. Immunoblotting analysis of these proteins with positive sera exhibited six seroreactive bands ranging from 27 to 130 kDa. Among these, the 84 and 130 kDa bands were quite specific, with high diagnostic specificity and sensitivity. The E/S fraction comprised nine distinct bands, as revealed by SDS–PAGE analysis. Immunoblotting analysis of these proteins with positive sera exhibited five antigenic bands ranging from 27 to 130 kDa. Among these, the 130 kDa band was found to be quite specific, with high diagnostic specificity and sensitivity. The present study concludes that the protein bands of 84 and 130 kDa in somatic fraction and 130 kDa in E/S fraction can be used for the immunodiagnostic purpose for this economically important parasite, which may also encourage further studies regarding their vaccine potential.

In Vitro Characterization of High Purity Factor IX Concentrates for the Treatment of Hemophilia B

1995 ◽  
Vol 73 (04) ◽  
pp. 584-591 ◽  
Author(s):  
Steven A Limentani ◽  
Kerry P Gowell ◽  
Steven R Deitcher
Keyword(s):  
High Purity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Factor Ix ◽  
Factor Vii ◽  
Dominant Factor ◽  
Factor X ◽  
Reducing Conditions ◽  
Sds Page ◽  
Activation Products

SummaryThis study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor Vila could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected. Prothrombin contaminated all of the products except Mononine. Activation products of prothrombin were identified for three of four lots of Immunine and for one lot of Factor IX VHP. These results thus demonstrate that high purity factor IX concentrates differ substantially in the degree to which they are contaminated by potentially thrombogenic materials.

scholarly journals Two functionally distinct pools of vitronectin (Vn) in the blood circulation: identification of a heparin-binding competent population of Vn within platelet alpha-granules

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 552-560 ◽  
Author(s):  
D Seiffert ◽  
RR Schleef
Keyword(s):  
Blood Circulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Plasminogen Activator Inhibitor ◽  
Heparin Binding ◽  
Immunoblotting Analysis ◽  
Sds Page ◽  
Altered Form ◽  
Activator Inhibitor

The biological functions of vitronectin (Vn) are dependent on its conformation. Whereas plasma Vn is present in a conformation that does not bind to heparin, platelet Vn has been recognized to be in a multimeric, conformationally altered form. To further understand the characteristics of platelet Vn, the molecules present in plasma and total and size-fractionated platelet releasates were compared (1) immunologically using three conformationally sensitive epitope-defined monoclonal antibodies, (2) functionally for their ability to interact with heparin, and (3) structurally using denaturing and nondenaturing polyacrylamide gel electrophoresis (PAGE). Our data indicate that Vn is present in platelet releasates in two molecular weight (M(r) forms. The high M(r) fractions contain conformationally and structurally altered Vn capable of interacting with heparin, and this form is distinct from plasma Vn and purified denatured Vn. In contrast, the lower M(r) forms of Vn are similar to plasma Vn. To determine if the presence of multimeric Vn requires platelet activation, platelets were disintegrated by sonication and fractionated by density gradients. Combined sodium dodecyl sulfate-PAGE (SDS-PAGE) and immunoblotting analysis showed a codistribution of multimeric Vn and type 1 plasminogen activator inhibitor in alpha-granule-rich fractions. Thus, platelet Vn is stored in a structurally and functionally distinct form from the molecule in plasma, raising the possibility that platelet- derived heparin-binding competent Vn will accumulate in areas of vascular injury.

THROMBIN-ACTIVATED FACTOR VIIIa IS COMPOSED OF A NONCOVALENT 73/51 kD DIMER

1987 ◽  
Author(s):  
P J Fay
Keyword(s):  
Factor Viii ◽  
Heavy Chain ◽  
Light Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Similar Extent ◽  
Factor Viiia ◽  
Polypeptide Patterns ◽  
Human Factor Viii ◽  
Sds Page

Human factor VIII purified from plasma concentrates consists of a series of heterodimers composed of a light chain of 83 kD noncovalently bound to a heavy chain which varies in size from 93 to 170 kD. Previously, we showed that each of the purified heterodimers wasactivated by thrombin to a similar extent. Activation to factor VIIIa was correlated with proteolysis of the light chain generating a73 kD polypeptide and cleavage of the heavy chain(s) generating polypeptides of 51 and 43 kD, whereas subsequent inactivation of factor VIIIa occurred in the absence of further proteolysis (Biochim Biophys Acta 871:268-278, 1986). SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of reduced or nonreduced samples showed similar polypeptide patterns indicating that there were no covalent linkages between the 51 and 43 kD chains. However, prior data does not distinguish between a factor VIIIa complex of the 73, 51 and 43 kD polypeptides and a subset of these chains. To identify factor VIIIa, thrombin- treated factor VIII at peak activity was subjected to rapid gel filtration on Superose 12. Factor VIII activity eluted as a single peak representing about 30% of the applied activity after correction for spontaneous inactivation. SDS-PAGE followedby silver staining showed that activity was correlated to fractionscontaining the 73 and 51 kD polypeptides, which co-eluted and which were separated from both the 43 kD fragment and thrombin. Densitometric scans of the stained gel indicated the stoichiometry of the 73:51 kD polypeptides in eachactive fraction to be 1:1. Addition of EDTA(50 mM) to a similar thrombin-factor VIII mixture resulted in rapid inactivation of factor VIIIa. Gel filtration followed by SDS-PAGE analysis of this sample showed that the 73 and 51 kD polypeptides eluted separately and were more included, while the elution position of the 43 kD polypeptide was unchanged. These results suggest that factor VIIIa is represented by a noncovalent dimer consisting of a 73 kD polypeptide derivedfrom the light chain plus a 51 kD polypeptide derived from the heavy chain.

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