title: Molecular basis of the clotting defect in a bleeding patient missing the Asp-185 codon in the factor X gene.
abstract: Factor X (FX) is a vitamin K-dependent plasma zymogen, which following activation to factor Xa (FXa), converts prothrombin to thrombin in the blood clotting cascade. It was recently demonstrated that a natural variant of FX carrying the Asp-185 deletion (FX-D185del, chymotrypsinogen numbering) was associated with mild bleeding in a patient with severe FX deficiency. In this study, we expressed FX-D185del in mammalian cells and characterized its properties in appropriate kinetic assays in purified systems. We discovered that while the FX variant can be normally activated by physiological activators; both amidolytic and proteolytic activities of the mutant are dramatically impaired. Interestingly, factor Va (FVa) significantly improved the proteolytic defect when the mutant protease was assembled into the prothrombinase complex. Thus, in contrast to >50-fold catalytic defect in the absence of FVa, the variant activated prothrombin with only ~2.5-fold decreased catalytic efficiency in the presence of the cofactor. The FXa variant dramatically lost its susceptibility to inhibition by antithrombin and tissue factor pathway inhibitor, thus exhibiting ~2-3 orders of magnitude lower reactivity with the plasma inhibitors. Further studies revealed that Na(+) no longer activates the variant protease, suggesting that the functionally important allosteric linkage between the Na(+)-binding and the P1-binding sites of the protease has been eliminated. These results suggest that the lower catalytic efficiency of FXa-D185del in the bleeding patient may be partially compensated by the loss of its reactivity with plasma inhibitors, possibly explaining the basis for the paradoxical severe FX deficiency with only mild bleeding tendency for this mutation.
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China.
authors.forename: Qiuya
authors.initials: Q
authors.lastname: Lu
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO, USA.
authors.forename: Likui
authors.initials: L
authors.lastname: Yang
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO, USA.
authors.forename: Chandrashekhara
authors.initials: C
authors.lastname: Manithody
authors.affiliation: Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China. Electronic address: wangxuefeng6336@hotmail.com.
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.affiliation: Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO, USA. Electronic address: rezaiear@slu.edu.
authors.forename: Alireza R
authors.initials: AR
authors.lastname: Rezaie
mesh_terms: D000990:Antithrombin III / Q000378:metabolism; D001777:Blood Coagulation; D003062:Codon; D015943:Factor Va / Q000378:metabolism; D005170:Factor X / Q000235:genetics* / Q000378:metabolism; D005171:Factor X Deficiency / Q000150:complications* / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D006470:Hemorrhage / Q000209:etiology* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008074:Lipoproteins / Q000378:metabolism; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D003062:Codon; D008074:Lipoproteins; D011994:Recombinant Proteins; C051928:lipoprotein-associated coagulation inhibitor; D015943:Factor Va; D000990:Antithrombin III; D011516:Prothrombin; D005170:Factor X
keywords: Bleeding; FVa; FX deficiency; Prthrombinase; Sodium