Functional analysis of two abnormal antithrombin proteins with different intracellular kinetics.

INTRODUCTION: Hereditary antithrombin (AT) deficiency type I causes venous thrombosis due to decreased levels of AT antigen in the blood. We identified one novel and one known abnormal variant in two unrelated Japanese families with venous thrombosis. In this study, we analyzed the mechanism by which these abnormal variants cause type I AT deficiency. MATERIALS AND METHODS: Wild-type and variant AT expression vectors were constructed and transiently expressed in human embryonic kidney 293 cells, and AT antigen levels and N-glycosylation of cell lysates and culture medium were evaluated by western blot analysis. Subcellular co-localization of AT was also examined using confocal microscopy, and chase experiments with cycloheximide and MG132 were performed to investigate the degradation pathway of AT variants. RESULTS: Genetic analysis identified a novel variant, c.613delC (p.Leu205Trpfs⁎79), and the known variant c.283T>C (p.Tyr95His). These AT variants exhibited significantly reduced extracellular secretion compared with the wild-type; N-glycosylation of the AT protein was normal. Co-localization analysis suggested that the transport of these abnormal AT proteins to the Golgi apparatus was impaired. The c.613delC variant was degraded early by the proteasome, suggesting that the c.283T>C variant is stored in the endoplasmic reticulum (ER). CONCLUSIONS: The AT variants identified here synthesize abnormal AT proteins that exhibit suppressed secretion and impaired transport from the ER to the Golgi apparatus. These results provide clues that could help elucidate the mechanism of type I AT deficiency and facilitate therapy development.

Identification and functional analysis of three novel genetic variants resulting in premature termination codons in three unrelated patients with hereditary antithrombin deficiency.

Hereditary antithrombin (AT) deficiency is an autosomal dominant inherited thrombophilia. In three pedigrees of hereditary type I AT deficiency, we identified novel variants c.126delC (p.Lys43Serfs*7), c.165C > G (p.Tyr55*), and c.546delA (p.Lys182Asnfs*102) in the open reading frame encoding AT in each patient. Each of these aberrant variants leads to premature termination of AT protein synthesis. To investigate whether these abnormal variants are involved in the pathogenesis of type I AT deficiency, we analyzed the function of these variants in HEK293 cells. Results of western blot analysis and immunofluorescence microscopy showed that all abnormal variants were expressed intracellularly, but p.Lys43Serfs*7 and p.Tyr55* protein were aggregated in the cells. These three variants were not detected in the spent culture medium, indicating that these novel variants affect protein secretion. In summary, we suggest that these variants in the AT-encoding gene are translated in the cell, but form abnormal proteins that form aggregates and/or inhibit secretion. These results provide insight into novel mechanisms of type I AT deficiency and potential therapies for the condition.

A case of venous thromboembolism caused by protein C deficiency due to a novel gene mutation.

Hereditary protein C (PC) deficiency is a quantitative or qualitative abnormality of the coagulation regulator PC resulting in a decreased PC activity. It is caused by mutations in the PC gene (PROC) located on chromosome 2q13-q14. Although hereditary PC deficiency is an important risk factor for venous thromboembolism (VTE), it is often overlooked because of difficulties in genetic examination. The low prevalence of this disease has led to a lack of evidence for its treatment. We report the case of a 21-year-old male with VTE caused by hereditary PC deficiency due to a novel PROC gene mutation, c.566G>A, p.Arg 189 Gln. The patient was refractory to treatment with direct oral anticoagulants, but responded to catheter-directed thrombolysis. Further intrafamilial genetic survey revealed the presence of the same mutation in five of the six family members. Learning objectives: Venous thromboembolism (VTE) caused by hereditary protein C deficiency that is refractory to direct oral anticoagulants may respond to catheter-directed thrombolysis. Furthermore, the first VTE in young patients with a strong family history and female family members of childbearing age should be considered for genetic testing. In addition, genetic examination will help establish evidence for the treatment of such patients.

Effect on Plasma Protein S Activity in Patients Receiving the Factor Xa Inhibitors.

AIMS: Measurement of protein S (PS) activity in patients taking direct oral anticoagulants (DOACs) using reagents based on a clotting assay results in falsely high PS activity, thus masking inherited PS deficiency, which is most frequently seen in the Japanese population. In this study, we investigated the effect of factor Xa (FXa) inhibitors on PS activity using the reagent on the basis of the chromogenic assay, which was recently developed in Japan. METHODS: The study enrolled 152 patients (82 males and 70 females; the average age: 68.5±14.0 years) receiving three FXa inhibitors (rivaroxaban, edoxaban, and apixaban). PS activity was measured using the reagents on the basis of the clotting and chromogenic assays. RESULTS: PS activity measured by the clotting assay reagents exhibited falsely high values depending on the plasma concentrations of FXa inhibitors in patients taking either rivaroxaban or edoxaban. However, none of the three FXa inhibitors affected PS activity when measured using the chromogenic assay. CONCLUSION: In patients taking rivaroxaban or edoxaban, inherited PS deficiency is likely missed because the levels of PS activity measured using the reagents based on the clotting assay are falsely high. However, we report that three FXa inhibitors do not affect PS activity measured by the chromogenic assay. When measuring the levels of PS activity in patients undergoing DOACs, the principles of each reagent should be understood. Furthermore, plasma samples must be collected at the time when plasma concentrations of DOACs are lowest or the DOAC-Stop reagent should be used.

A Case of Treatment With Dabigatran for Cerebral Venous Thrombosis Caused by Hereditary Protein C Deficiency.

A 37-year-old woman was admitted to our hospital with involuntary movements. She had no medical or family history of thromboembolism, nor was she on any medication. She showed no impaired consciousness, cranial nerve abnormalities, abnormal breathing, stiff neck or paralysis. Magnetic resonance venography exhibited poor visualization of intracranial vein. The protein C activity level reduced but the protein C antigen level was normal. Genetic analysis revealed a heterozygous mutation in exon 7 c.577-579delAAG, p.Lys193del on protein C gene. She was diagnosed with cerebral venous thrombosis and hereditary protein C deficiency type II. She received heparin in acute phase, and switched to dabigatran in chronic phase. Consequently, she had no recurrence of cerebral venous thrombosis and other complications. Dabigatran might be one of the alternative choices for patients with cerebral venous thrombosis and protein C deficiency.

Protein C Gene Mutation in an Older Adult Patient with Clostridium perfringens Septicemia-Related Visceral Vein Thrombosis.

A 78-year-old Japanese male with Clostridium perfringens septicemia and cholecystitis was found to have thrombosis in the left branch of intrahepatic portal vein as well as superior mesenteric vein. Visceral vein thrombosis (VVT) in this case was associated with protein C deficiency, due to a heterozygous mutation, p. Arg185Met. Our experience emphasizes that VVT, or other thromboembolic events, may occur in later life, triggered by environmental thrombosis risk factors, together with underlying hereditary protein C gene mutation.

Evaluation of Optimal Sample Processing Conditions for Accurate Measurement of Protein S Activity.

OBJECTIVE: Protein S(PS) activity, especially PS-specific activity calculated by total PS activity (tPSAct) divided by total PS antigen (tPSAg), is important in the diagnosis of hereditary PS deficiency (PSD). The cleavage of PS at a thrombin-sensitive region (TSR) by proteases reduces the anticoagulant activity of PS. Therefore, we investigated the effect of sample processing and storage on tPSAct and PS cleavage. METHODS: Blood samples were collected from ten healthy subjects, and tPSAg and tPSAct were measured in whole blood or plasma stored at room temperature (RT) or 4°C. The cleaved PS was detected by western blotting, and the relationship between decreases in PS-specific activity and increase rates of cleaved PS was evaluated. Furthermore, the stability of tPSAg and tPSAct on the long-term storage of plasma was also evaluated. RESULTS: Both whole blood and plasma stored at RT and whole blood stored at 4°C showed decreased tPSAct (50-80%) after 24 hours (P<0.05). PS-specific activity levels negatively correlated with the increase rate of cleaved PS (r =-0.84, P<0.001). The tPSAct was decreased to 60% after three days in plasma stored at 4°C (P<0.05) but was stable for about one month when stored at -20°C or below. CONCLUSION: Inappropriate processing and storage result in falsely low PS-specific activity due to the cleavage of PS in the blood collection tubes, which may lead to misdiagnosis of PSD. Samples should be centrifuged immediately after collection, and the plasma should be frozen.

Genetic analysis of a compound heterozygous patient with congenital factor X deficiency and regular replacement therapy with a prothrombin complex concentrate.

Congenital factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million. The proband, a 2-year-old girl, exhibited easy bruising and a history of umbilical cord bleeding at birth. Prothrombin time (> 40 s) and activated partial thromboplastin time (65.0 s) were prolonged. Marked declines in FX activity (< 1%) and FX antigen levels (5%) were also observed. Genetic analysis of the proband identified two types of single-base substitutions, c.353G>A (p.Gly118Asp) and c.1303G>A (p.Gly435Ser), indicating compound heterozygous congenital FX deficiency. Genetic analysis of family members revealed that her father and older sister (5-year-old) were also heterozygous for p.Gly118Asp, and that her mother was heterozygous for p.Gly435Ser. To improve the bleeding tendency, the proband received regular replacement of 500 units of PPSB-HT, a prothrombin complex concentrate (PCC). Following continued regular replacement of 500 units of PPSB-HT once per week, the proband has exhibited no bleeding tendencies and no new bruises have been observed. There are no previous report of the use of PPSB-HT for regular FX replacement. Regular replacement therapy with PPSB-HT may be an effective method for preventative control of bleeding tendencies in FX deficiency.

Congenital coagulation factor X deficiency: Genetic analysis of five patients and functional characterization of mutant factor X proteins.

Congenital factor X (FX) deficiency is a rare bleeding disorder that is inherited as an autosomal recessive trait. In this study, a genetic analysis of the FX gene was performed in five families with this disorder. Four heterozygous mutations [p.Gly154Arg, p.Val236Met, p.Gly263Val and p.Arg387Cys] and one pair of compound heterozygous FX gene mutations consisting of p.Gly406Ser and p.Val424Phe were identified. Mutant FX proteins containing the identified amino acid substitutions were also expressed in cultured cells. These proteins were analysed by enzyme-linked immunosorbent assay and pulse-chase experiments. The results demonstrated normal intracellular synthesis and extracellular secretion of mutant FX proteins carrying the p.Val236Met, p.Arg387Cys and p.Gly406Ser amino acid substitutions. However, the results also showed that the p.Gly154Arg, p.Gly263Val and p.Val424Phe proteins were secreted less efficiently than the wild-type protein, although they were synthesized normally in the cell. Collectively, these observations suggest that the amino acid substitutions p.Gly154Arg, p.Gly263Val and p.Val424Phe induce protein misfolding, leading to the intracellular degradation of many FX proteins containing any of these mutations, and ultimately to the development of FX deficiency. On the other hand, for the p.Val236Met, p.Arg387Cys and p.Gly406Ser mutant proteins, we hypothesize that secreted FX proteins have impaired coagulant activities due to functional defects caused by these amino acid substitutions.

[An elderly case of congenital prekallikrein deficiency].

The proband is a 69-year-old woman with purpura and subcutaneous hematoma.We investigated this patient with prekallikrein (PK) deficiency, using both standard coagulation study and molecular genetic analysis of the PK gene. In a coagulation study, the prothrombin time (PT) was normal but the activated partial thromboplastin time (APTT) was prolonged. Preincubation of normal plasma with APTT reagent caused shortening of abnormal clotting time. Plasma PK activity was <1%. Her parents were cousins. Molecular genetic analysis showed a homozygous Gly401Glu substitution in exon 11 in the PK gene. This mutation has already been reported in a Japanese patient as PK Tokushima. Gly401 is positioned in PK light chain, which encodes the serine protease domain. The disulfide binding is formed between Cys400 and Cys416, thus Gly401 is located next to His415, which is one of the activation peptides and is important in supporting the correct conformation of proteins. Therefore, we suggest that this mutation may prevent formation of disulfide binding and reduce enzyme activity. In conclusion, in the elderly case with prolonged APTT, we should consider the contact factor deficiency and determine PK activity apart from the abnormality of coagulation factor VIII, IX, XI and XII activities.

A case of factor X (FX) deficiency due to novel mutation V196M, FX Hofu.

The patient, a 20-year-old male, was found to have a slightly prolonged prothrombin time (PT). No episodes of bleeding were noted. The measurement of coagulation factors revealed that the level of factor X (FX) activity was solely deficient, 51% (normal range: 70-130% ), and that of FX antigen was 100%. Analysis of the entire FX gene revealed the novel missense mutation of GTG to ATG, resulting in the substitution of the 196th amino acid valine --> methionine. The mother and younger brother had a normal PT time and expressed no episode of bleeding. The mother exhibited a normal level of FX activity and antigen; however the younger brother showed a slight decrease in both the parameters. This mutation was not observed in the mother and younger brother. Polymorphism is not observed at this point in healthy persons. The present novel FX mutation was named FX Hofu.