Factors XWenatchee I and II: compound heterozygosity involving two variant proteins

Abstract

Variant factor X in an individual with a mild bleeding tendency was suspected based on deficient procoagulant activity (10-20% of normal) and antigen (30-35% of normal) levels of plasma factor X. Heteroduplex analysis of factor X gene exons indicated heterozygosity for mutations in both exons 6 and 4, confirmed by direct sequencing of the amplified exons. Substitution of C by T at nucleotide position 13,984 (Arg-139 to Cys) was found in the factor X gene exon 6 of the propositus. This mutation creates a BsmI site and the patient tested heterozygous for the BsmI cleavage involved, as did one of his two daughters. In addition, exon 4 was found to have the normal A and a novel C (Asn-57 to Thr) at nucleotide position 9338. The exon 4 mutation creates a BsaJI site, detectable after amplification mismatch to remove an existing BsaJI site. Both the patient and the second of his two daughters were heterozygous for this cleavage. The two variant proteins are called factors XWenatchee I (Arg-139 to Cys) and II (Asn-57 to Thr). A mixed variant isolate derived from the plasma of the propositus exhibited heavy/light chains of normal size, as well as an apparent single-chain molecule not dissociable by reducing agent. A single-chain molecule would be predicted for form I, if the mutation blocks processing cleavages that normally remove a tripeptide interposed between the heavy and light chains. A Western blot of partially purified factor X from the daughter who inherited the form I defect revealed a component migrating the same as the putative single-chain species. Based upon the factor X activity vs. antigen ratios for the propositus and both daughters, both forms I and II are probably dysfunctional molecules.

Structure and Dynamics of Zymogen Human Blood Coagulation Factor X

“…For instance, mutational studies suggest that Arg36 of VIIa plays a key role in substrate interactions (Ruf et al, 1999). Similarly, the GLA domain of FX has been implicated in the interaction with the TF-VIIa complex based on the study of several mutants in the GLA domain of FX (Kim et al, 1995;Rudolph et al, 1996). TF residues Lys165 and Lys166 have been shown to contribute to protein-protein interactions with FX in the ternary TF-VIIa-FX complex (Ruf et al, 1992).…”

A candidate activation pathway for coagulation factor VII

“…Sheep anti-human factor XI polyclonal was from Enzyme Research Laboratories (South Bend, IN). 10H10 anti-human tissue factor monoclonal antibody [18], 1150 anti-human factor VII calcium-dependent monoclonal antibody [19], and 4G3 anti-human factor X calcium-dependent monoclonal antibody [20] were purified from hybridoma cells generously provided by James Morrissey (University of Michigan). The HPC4 calcium-dependent monoclonal antibody was purified from hybridoma cells generously provided by Charles Esmon (Oklahoma Medical Research Foundation) [21].…”

“…Recombinant mutant S419A factor X (FA10, UniProtKB P00742) was purified from conditioned cell culture media with modifications of a previously described serial chromatography method [27]. Anion exchange chromatography using Q Sepharose was done first, followed by immunoaffinity chromatography using the calcium-dependent anti-factor X antibody 4G3 [20], followed by anion exchange on monoQ (GE Healthcare Life Sciences, Pittsburgh, PA). An amount of 10 mM benzamidine was maintained in all buffers.…”

The activation peptide of coagulation factor IX and X serves as a high affinity receptor to cationic ligands

“…Starting with recombinant single-chain protein (rFX SC ), we produced rFXα and rFXaα by addition of the proteases furin or RVV-X, respectively. rFXaα represents pathological forms such as FX Kurayoshi (10,11). The furincleaved rFXα is proteolytically inactive and lacks the tripeptide Arg 140 -Lys-Arg 142 while rFXaα is cleaved at the Arg 194 -Ile 195 peptide bond.…”