title: The missense Thr211Pro mutation in the factor X activation peptide of a bleeding patient causes molecular defect in the clotting cascade.
abstract: Factor X (FX) is a vitamin K-dependent coagulation zymogen, which upon activation to factor Xa assembles into the prothrombinase complex to activate prothrombin to thrombin. FX can be activated by either factor VIIa-tissue factor or factor IXa-factor VIIIa in extrinsic and intrinsic pathways, respectively. In this study, we identified a bleeding patient with moderate FX deficiency who exhibits a clotting defect only in the intrinsic pathway. Exome sequencing revealed that the patient carries a novel homozygous missense mutation that results in substitution of Thr211 with Pro in the activation peptide of FX. Thr211 is the site of an O-linked glycosylation in the activation peptide of FX. We postulated that the lack of this post-translational modification specifically impacts the activation of FX by intrinsic Xase, thereby impairing thrombin generation in the subject. To test this hypothesis, we expressed both wild-type FX and FX containing this mutation in mammalian cells and following the purification of the zymogens to homogeneity characterized their properties in both purified and plasma-based assay systems. Analysis of the results suggests that Thr211 to Pro substitution renders the FX mutant a poor substrate for both physiological activators, however, at physiological concentration of the substrate, the clotting defect manifest itself only in the intrinsic pathway, thus explaining the bleeding phenotype for the patient carrying this mutation.
authors.affiliation: Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1100 S. Grand Blvd., St. Louis, MO 63104, USA.
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.forename: Yiping
authors.initials: Y
authors.lastname: Shen
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000293:Adolescent; D001777:Blood Coagulation / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D001779:Blood Coagulation Factors / Q000008:administration & dosage; D015949:Factor IXa / Q000378:metabolism; D005170:Factor X / Q000235:genetics* / Q000378:metabolism; D005171:Factor X Deficiency / Q000097:blood / Q000150:complications / Q000235:genetics*; D005260:Female; D006031:Glycosylation; D057809:HEK293 Cells; D006470:Hemorrhage / Q000209:etiology / Q000517:prevention & control; D006801:Humans; D008875:Middle Aged; D020125:Mutation, Missense / Q000235:genetics; D010375:Pedigree; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D013917:Thrombin / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
chemical_list: D001779:Blood Coagulation Factors; C025667:prothrombin complex concentrates; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin