Coagulation Factor VII Gln100 → Arg

Abstract

We have used recombinant mammalian expression and purification of the factor VII (FVII) variant Gln 100 3 Arg (Q100RFVII) to study FVII deficiency in subjects with this mutation. Q100RFVII was secreted poorly in comparison with wild-type FVII (WTFVII) in a stable mammalian expression system, and purified variant protein was found to have undetectable clotting activity. Following activation by immobilized factor Xa, Q100RFVIIa had amidolytic activity similar to WTFVIIa in the absence of soluble tissue factor (sTF); however, unlike WTFVIIa no typical increase in activity was seen after addition of sTF. In a factor X activation assay using relipidated transmembrane truncated tissue factor (residues 1-243), Q100RFVIIa showed less than 5% of the ability of WTFVIIa to activate factor X.We performed direct binding analysis of WT and Q100RFVII/FVIIa to immobilized sTF using surface plasmon resonance, and severely reduced binding of both non-activated and activated Q100RFVII to sTF was seen, indicating a pronounced defect in tissue factor (TF) interaction with this variant.In the sTF-FVIIa crystal structure the candidate residue Gln 100 is not in contact with TF but is at the epidermal growth factor 2-protease domain interface. We suggest that the mutation results in a global fold change severely reducing tissue factor interaction; mutation of FVII residues not directly involved in the interaction with TF may still result in variant FVII unable to take part in the initiation of coagulation.

The effect of the chemical chaperone 4-phenylbutyrate on secretion and activity of the p.Q160R missense variant of coagulation factor FVII

“…Before treatment, FXaG was barely detectable in conditioned medium from cells expressing rFVII-160R. This is in agreement with data published by Kemball-Cook et al [10], who showed that the activated form of the mutant recombinant protein had reduced affinity for TF and, in the presence of soluble TF, had less than 5% of the ability of rFVIIwt to activate FX. Accordingly, evaluation of the FXa generation parameters before 4-PBA treatment was not feasible because mutant curves were not suitable for quantitative analysis.…”

“…On the other hand the specific activity of the 4-PBA-induced rFVII-160R was still considerably reduced compared to rFVIIwt, as expected for FVII molecules bearing important structural changes. Particularly, the replacement of the glutamine side chain by the larger, positively charged arginine side chain in FVII-160R would disrupt hydrogen bonding networks and destabilize protein inter-domain areas [10]. It is tempting to speculate that folding disturbances in the EGF-2 protease region of FVII-160R, probably responsible both for barely detectable affinity for TF and for the secretion defect, would be partially overcome by the 4-PBA.…”

“…This variant, reported in the FVII variant database [8], is present in all the Norwegian FVII deficient patients and is associated with a bleeding phenotype due to reduced FVII antigen and activity in plasma [9]. Recombinant (r) FVII Q160R (FVII-160R) has been shown to be poorly secreted and, in the presence of relipidated TF, the activated rFVII-160R showed less than 5% of the ability of wild type FVIIa to activate factor X [10].…”

Predicted solution structure of zymogen human coagulation FVII

“…Retention of the overall fold of this domain on solvation is not surprising, because it has been shown that the EGF-like domains of FVIIa remains stable under high guanidine hydrochloride concentrations and at elevated temperatures. 59 The mutation Gln100 → Arg in the EGF2 domain has been detected 60 -62 61 using surface plasmon resonance. The outcome was severely reduced binding of the mutant of both FVII and FVIIa to TF.…”

Generation of a polyclonal rabbit anti-mouse tissue factor antibody by nucleic acid immunisation

“…1A). We thenattemptedtoexpress recom-binant mouse TF 1-225 using the same system as we had used for recombinant hTF (1),h owever no mTF wasp roduced. We thenimmunised rabbits with synthetic peptidesderivedfrom the mTF extracellulard omains (including one previouslyr eported to generate an effective anti-mTF antibody(2)).All the peptides induced an immune response and the antisera specifically recognised the linear peptide sequence, howevertheyreacted poorly withmouse TF expressed by murine fibroblasts in Westernblot, immunohistochemical and flow cytometric analyses (datan ot shown).W ehavenow circumvented these problems by using a simple nucleic acidi mmunisation method to generate ar abbit polyclonalanti-mTF antibody that is effective in detecting both recombinanta nd native mTF in av arietyo fe xperimental settings.…”