Characterization of molecular defect of 13387-9delG mutated antithrombin in inherited type I antithrombin deficiency

Abstract

As a major physiological inhibitor of thrombin and other coagulation proteases, antithrombin (AT) plays an important role in the maintenance of normal hemostasis and its deficiency is associated with a predisposition for familial venous thromboembolic disease. Recently, we found a novel mutation (13387-9delG) in the antithrombin gene that is associated with type I AT deficiency. To examine the molecular pathologic mechanism of this mutation causing type I AT deficiency, the wild-type and the mutant AT constructs were expressed in COS-7 cells or Chinese Hamster Ovary cells. No AT antigen could be detected by enzyme-linked immunosorbent assay in the conditioned media of cells expressing the mutant protein, and the AT antigen level was reduced in cell lysates. The mutant AT-expressing cells did not have less intracellular mRNA levels than the wild-type transfectants as estimated by quantitative reverse transcriptase-polymerase chain reaction. Metabolic and pulse-chase experiments showed the newly synthesized wild-type AT protein was gradually secreted into the media, whereas no labeled mutant AT protein was detected in the media and the total amount of radioactivity was significantly reduced in the cells during the chase periods. By immunofluorescence analysis, the staining of the mutant AT was weaker than that of the wild type, and was predominantly diffuse without perinuclear enhancement. These results indicate that the 13387-9delG mutation, which disrupts the disulfide bridge Cys247-Cys430, impairs the secretion and stability of the truncated AT protein associated with intracellular degradation.

Clinical Manifestation and Mutation Spectrum of 53 Unrelated Pedigrees with Protein S Deficiency in China

“…Mutations in the PROC and SERPINC1 were identified as previously described. 29,30 PROS1 Messenger Ribonucleic Acid Analysis and In Silico Analysis PROS1 messenger ribonucleic acid (mRNA) analysis was performed in proband 28 with a novel heterozygote splice site mutation c.1871-2A > G to identify its effect on normal transcription. The following primers: forward 5′-GTGCCCTTT-GCTGTGTCCT-3′, reverse 5′-CACCATCTCTTCTGCCTTC-3′, were used to amplify the sequence from exon 13 to 15 of PROS1.…”

Identification and characterization of two SERPINC1 mutations causing congenital antithrombin deficiency

“…For example, p.Cys127Arg causes the rupture of disulfide bonds between Cys53-Cys127, significantly slows the rate of AT secretion, and persists in the intracellular reticulum in the form of oligosaccharides, which cause AT deficiency [ 57 ]. Mutations 13,387-9delG and c.964A > T (p.Lys322*) cause breakage of the disulfide bond between Cys279 and Cys462, impairing secretion and structural stability of AT-truncated proteins degraded intracellularly [ 58 , 59 ]. In addition, AT protein truncation caused by c.964A > T (p.Lys322*) led to the loss of the P1-P1′(Arg393-Ser394) bond, which does not act as an inactivated protease [ 58 ].…”

Genetic Analysis of a Pedigree With Antithrombin and Prothrombin Compound Mutations and Antithrombin Heterozygotes

“…The study by Michiels JJ et al pointed out that the absence of cross-reactive substances in the patients’ plasma indicated that p.Arg197stop either prevented the formation of stable mRNA or the translated peptide was rapidly degraded ( Michiels et al, 1995 ). It has been reported that the 13387-9delG mutation resulted in the loss of the disulfide bond between Cys247 and Cys430, impairing the secretion and stability of the truncated AT protein associated with intracellular degradation ( Wang et al, 2005 ). Since both c.964A > T (p.Lys322stop) and 13387-9delG mutations will cause the loss of the disulfide bond between Cys247 and Cys430, we assumed that c.964A > T (p.Lys322stop) caused the reduction of AT:A and AT:Ag by the same mechanism as 13387-9delG.…”