Development of a Sensitive and Rapid Chromogenic Factor IX Assay for Clinical Use

Wagenvoord, Rob; Hendrix, H; Tran, Thuy; Hemker, H.C.

doi:10.1159/000216139

Cited by 11 publications

(10 citation statements)

References 15 publications

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“…Haemophilia type B is displayed by severe and often spontaneous haemorrhagic disease (Christmas disease), similar to that observed in some patients with severe DENV disease (DHF grades II to IV) [72,73]. These findings, therefore, confirmed the essential role of: i) factor IXa in maintaining normal haemostasis, despite its very low blood concentration (44–88 nM: mean 67 nM = 0.038 μg/ml) [76], and ii) the 2-N residue binding to a Ca 2+ ion thereby causing its rotation, and which would result in increased MAb 1G5.3 binding. Reduced functions of factor IXa were however only found in 17% of patients with DHF/DSS, while 50% and 100% showed reduced factor VIIa and factor I (fibrinogen) functions/concentrations, respectively [77].…”

Section: Discussionsupporting

confidence: 66%

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies

Falconar

2012

BMC Res Notes

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BackgroundEpitope-mapping of infectious agents is essential for pathogenesis studies. Since polyclonal antibodies (PAbs) and monoclonal antibodies (MAbs) are always polyspecific and can react with multiple epitopes, it is important to distinguish between specific and non-specific reactions. Relative antibody discriminating specificity (RADS) values, obtained from their relative ELISA reactions with L-amino acid peptides prepared in the natural versus reverse orientations (x-fold absorbance natural/absorbance reverse = RADS value) may be valuable for this purpose.PAbs generated against the dengue type-2 virus (DENV-2) nonstructural-1 (NS1) glycoprotein candidate vaccine also reacted with both DENV envelope (E) glycoproteins and blood-clotting proteins. New xKGSx/xSGKx amino acid motifs were identified on DENV-2 glycoproteins, HIV-1 gp41 and factor IXa. Their potential roles in DENV and HIV-1 antibody-enhanced replication (AER) and auto-immunity were assessed.In this study, a) RADS values were determined for MAbs and PAbs, generated in congeneic (H2: class II) mice against DENV NS1 glycoprotein epitopes, to account for their cross-reaction patterns, and b) MAb 1G5.3 reactions with xKGSx/xSGKx motifs present in the DENV-4 NS1, E and HIV-1 glycoproteins and factor IXa were assessed after the introduction of amino acid substitutions, deletions, or intra-/inter-cysteine (C-C) bridges.ResultsMAbs 1H7.4, 5H4.3, 3D1.4 and 1G5.3 had high (4.23- to 16.83-fold) RADS values against single epitopes on the DENV-2 NS1 glycoprotein, and MAb 3D1.4 defined the DENV complex-conserved LX1 epitope. In contrast, MAbs 1G5.4-A1-C3 and 1C6.3 had low (0.47- to 1.67-fold) RADS values against multiple epitopes. PAb DENV complex-reactions occurred through moderately-high (2.77- and 3.11-fold) RADS values against the LX1 epitope. MAb 1G5.3 reacted with xSGKx motifs present in DENV-4 NS1 and E glycoproteins, HIV-1 gp41 and factor IXa, while natural C-C bridge formations or certain amino acid substitutions increased its binding activity.ConclusionsThese results: i) were readily obtained using a standard 96-well ELISA format, ii) showed the LX1 epitope to be the immuno-dominant DENV complex determinant in the NS1 glycoprotein, iii) supported an antigenic co-evolution of the DENV NS1 and E glycoproteins, and iv) identified methods that made it possible to determine the role of anti-DENV PAb reactions in viral pathogenesis.

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Section: Discussionsupporting

confidence: 66%

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies

Falconar

2012

BMC Res Notes

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“…Activation of FIX was assessed by a modification of the method of Wagenvoord [16]. FIX (0.05–10.0 μ m ) in TBSA with 5 m m CaCl 2 was incubated at 37 °C with 0.5 n m FXIa for 60 s. Reactions were stopped with EDTA (25 m m ) and chilled to 4 °C.…”

Section: Methodsmentioning

confidence: 99%

A cross‐reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect

Gailani

Schmidt

Sun

et al. 2007

Journal of Thrombosis and Haemostasis

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Summary. Inherited deficiency of the trypsin-like protease factor (F) XI is associated with a mild to moderate bleeding diathesis. In most cases, FXI protein is reduced in plasma, and examples of dysfunctional circulating FXI variants are rare. We characterized the defect in one such variant with a proline to leucine substitution at residue 520. FXI Pro520 corresponds to chymotrypsin Pro161, and is conserved in most members of the chymotrypsin protease family. Recombinant FXI containing this substitution will be referred to as FXI P161L . k cat for cleavage of chromogenic substrates and for activation of the natural FXIa substrate FIX is 3-fold lower for activated FXI P161L (FXIa P161L ) than for wild-type FXIa (FXIa WT ), consistent with an abnormal protease active site. Inhibition of FXIa P161L by diisopropyl fluorophosphate is 2.4-fold slower than for FXIa WT , suggesting distortion of the protease oxyanion hole. Binding to p-aminobenzamidine, a probe for the integrity of the S1 substrate-binding site, was similar for FXIa WT and FXIa P161L . Rates of carbamylation of Ile16 were also similar for FXIa WT and FXIa P161L , indicating that the critical salt bridge between Ile16 and Asp194 forms normally during protease activation. Cumulatively, the data demonstrate that Pro161 is required for normal active site oxyanion hole conformation in FXIa. Examination of the FXIa crystal structure and modeling studies indicate that Pro161 forms several hydrophobic contacts with adjacent amino acids that stabilize active site conformation. Leucine can be incorporated at position 161 in FXIa, but would not form the extensive stabilizing network of hydrophobic interactions formed by Pro161.

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“…Activation of factor IX by factor XIa was assessed by a modification of the method of Wagenvoord et al (18). All reagents are diluted in TBSA.…”

Section: Determination Of the K M For Factor Ix Activation By Activatmentioning

confidence: 99%

Identification of Amino Acids in the Factor XI Apple 3 Domain Required for Activation of Factor IX

Sun

Zhao

Gailani

1999

Journal of Biological Chemistry

114

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Activated coagulation factor XI (factor XIa)Coagulation factor XI is the zymogen of a plasma serine protease that contributes to blood coagulation by proteolytically activating factor IX in a calcium-dependent manner (1-3). Human factor XI is a 160-kDa dimeric glycoprotein consisting of two identical 80-kDa polypeptides connected by a single disulfide bond (4, 5). Conversion of zymogen factor XI to the active form, factor XIa, is accomplished by a single cleavage in each polypeptide, resulting in a pair of 35-kDa C-terminal light chains with homology to the pancreatic digestive enzyme trypsin and two 45-kDa N-terminal heavy chains (4, 6). Although catalytic activity resides in the light chains, it is clear from several lines of evidence that the heavy chain is required for optimal activation of factor IX. The capacity of isolated factor XIa light chain to activate factor IX is Ͻ1% that of the intact molecule, even though both the light chain and whole molecule cleave a chromogenic substrate similarly (6 -8). In addition, antibodies directed against the heavy chain interfere with factor IX activation (6, 9)The factor XI heavy chain is composed of four 90 -91-amino acid repeats called apple domains (designated A1-A4), with A1 located at the amino terminus of the molecule (10). These domains have been implicated in binding interactions with molecules and surfaces involved in blood coagulation (9,(11)(12)(13). Previously, we prepared recombinant chimeric factor XI molecules in which individual apple domains were replaced with the corresponding domain from the related plasma protease prekallikrein (PK) 1 (9). PK, the zymogen of the kininogenase plasma kallikrein, shares 58% amino acid homology and similar domain structure with factor XI (14, 15). The two proteases are functionally distinct, however, with PK being a poor activator of factor IX (2, 9). A chimeric molecule in which the factor XI A3 domain is replaced with that of PK (FXI/PKA3) is a poor activator of factor IX, whereas chimeras with PK substitutions for A1, A2, or A4 activate factor IX normally. These data demonstrate that the A3 domain is required for interactions with factor IX (9). In this report, we describe mapping studies of the factor XI A3 domain that identify amino acids at the N and C termini of the domain required for normal activation of factor IX.

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Development of a Sensitive and Rapid Chromogenic Factor IX Assay for Clinical Use

Cited by 11 publications

References 15 publications

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies

Epitope reactions can be gauged by relative antibody discriminating specificity (RADS) values supported by deletion, substitution and cysteine bridge formation analyses: potential uses in pathogenesis studies

A cross‐reactive material positive variant of coagulation factor XI (FXIP520L) with a catalytic defect

Identification of Amino Acids in the Factor XI Apple 3 Domain Required for Activation of Factor IX

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