RESUMO
An important negative regulator of factor VIIIa (FVIIIa) cofactor activity is A2 subunit dissociation. FVIII molecules with stabilized activity have been generated by elimination of charged residues at the A1-A2 and A2-A3 interfaces. These molecules exhibited reduced decay rates as part of the enzymatic factor Xa generation complex and retained their activities under thermal and chemical denaturing conditions. We describe here the potency and efficacy of 1 such stability variant, D519V/E665V, derived from B domain-deleted FVIII (BDD-FVIII). The major effect of A2 stabilization was on cofactor activity. D519V/E665V potency was increased twofold by the 2-stage chromogenic assay relative to BDD-FVIII. D519V/E665V demonstrated enhanced thrombin generation responses (fivefold by peak thrombin) relative to BDD-FVIII. In vivo consequences of enhanced cofactor activity of D519V/E665V included >fourfold increased maximal platelet-fibrin deposition after laser injury and twofold increased protection from bleeding in acute and prolonged vascular injury model in hemophilia A mice. These results demonstrate that noncovalent stabilization of the FVIII A2 subunit can prolong its cofactor activity, leading to differential enhancement in clot formation over protection from blood loss in hemophilia. The FVIII molecule described here is the first molecule with clear efficacy enhancement resulting from noncovalent stabilization of the A2 domain.
Assuntos
Fator VIII/química , Fator VIII/farmacologia , Hemofilia A/genética , Animais , Arteríolas/lesões , Modelos Animais de Doenças , Fator VIII/genética , Feminino , Camundongos , Camundongos Knockout , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.
