The Clinical Aspects And Molecule Machanisms Of A Hereditary Resistance To Activated Protein C Family

Background The phenomenon of activated protein C resistance (APC-R) was first reported by Dahlbanck et al in 1993. It has been documented that the main mechanism of APC-R is a single point mutation in FV gene which results in the substitution of Arginine at position 506 by Glutamine (FV Leiden) and is one of the most frequent risk factors for thrombosis (hereditary thrombophilia) in Caucasians. However, there is predominant regional difference in the distribution of FV Leiden mutation. In China, Korea and Japan, although the APC-R are present in some of the healthy individuals and thrombotic patients, FV Leiden is almost undetectale. Therefore, it is noteworthy whether other gene defects are associated with APC-R and thrombotic tendency in Asian population.Objective To investigate the clinical aspects and molecular pathogenesis of hereditary APC-R with thrombotic history in a Chinese family, and identify the novel genetic defects related to APC-R and functional abnormality of the novel mutation coded coagulation factor.Methods1. Collecting clinical data and blood samples from the proband and four of her available family members, and analyzing inherited trait.2. APC-R and protein S (PS) activity were measured by a clotting assay; protein C (PC) activity and antithrombin (AT-â…¢) activity were measured by the synthetic chromogenic substrate method, and plasma level of FV and FVâ…¢were measured by a one-stage coagulation assay.3. FV Leiden and FV Cambridge/Hong Kong mutations were determined by PCR-RELP and direct sequencing.4.25 exons of FV gene were amplificated by PCR. The DNA sequence of PCR products were analysed by direct sequencing and then compared with cDNA sequence of FV gene published in GeneBank. 5. The novel FV gene mutations we identified were screened in the healthy individuals and thrombotic patients, and allele frequency of the mutations were compared.6. Mixing the plasma from the proband or standard plasma with FV-deficient plasma in various proportions. Then APC-sr values were determined using Coatest APC Resistance kit (Chromogenix, Molndal, Sweden) to explore the mechanism of novel mutation related to APC-R in this family.Results1. The proband was a 53-year-old woman, and she was admitted to our hospital because of swelling and pain on her left leg and progessing chest pain. The suspicion of deep vein thrombosis (DVT) on left lower extremity and pulmonary embolism (PE) was confirmed by Color Doppler ultrasound and computed tomography angiography of the pulmonary artery. No hereditary and acquired thrombophilia was detected with the expection of a positive APC-R. She received warfarin as long-term oral anticoagulation following the initial low molecular heparin therapy. There was no recurrence of VTE after a follow-up for 16 months.2. The parents of the proband were not consanguineous marriage. One of her elder sisters died of VTE two years ago. The other family members had no thrombotic history. APC-R were positive in four out of five available members including the proband. All other risk factors for VTE, including anti-phospholipid antibodies, PS, PC, AT-â…¢, FVâ…¢C, were normal. The hereditary APC-R family was identified, and it appeared to manifest autosomal dominant inheritance.3. FV Leiden and FV Hong Kong/Cambridge mutations were absent among the family.4. Two FV gene mutations were identified in the proband:1) FV R485K, a homozygous G to A substitution at base 1628 in exon10, changing the codon for amino acid 485 from AGA (Arg) to AAA (Lys). It has been reported that FV R485K is a single nucleotide polymorphism (SNP), poor response to APC and might be associated with VTE. FV R485K was dectected in all of available family members. Its allele frequency was shown to be 60%and 64% in the healthy individuals and the thrombotic patients, respectively.2) FV E666D, a heterozygous G to C substitution at base 2172 in exonl3, changing the codon for amino acid 666 from GAG (Glu) to GAC (Asp). This mutation is a novel FV gene mutation, and also detectable in the family members with positive APC-R and absent in her niece whose APC-R was negative and all of the healthy individuals and the thrombotic patients. The mutation is at the position near the third APC cleavage site-Arg679, which might interfere with cleaving of FVa at this site and result in APC-R.5. There was little difference in APC-sr values between normal plasma and FV E666D-heterozygous plasma when the concentration of FV were very low, suggesting that there was no difference in the susceptibility to APC between normal and mutant FVa. The initial slope of the APC-sr plot was lower for mutant FV than for normal FV, suggesting that the mutant FV E666D reduced APC cofactor activity.Conclusions1. In present study, we have identified the first Chinese family of hereditary APC-R with thrombotic history.2. A novel FV mutation (FV E666D) in exon 13 was identified and detectable in all the available members who had positive APC-R. The mutation was undetectable in the family member without APC-R and all of the detected healthy individuals and the thrombotic patients. We inferred that FV E666D mutation was responsible for the observed APC-R phenotype in this family.3. There was little difference in allele frequency of FV R485 between the healthy individuals and thrombotic patients, suggesting that FV R485K was not a risk factor for thrombosis.4. It has been showed that FV E666D mutant protein had lower APC cofactor activity in comparison with normal FV, which might be one of the reasons for the APC-R. Further studies are needed to determine the importance of our observations.