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Mutation of individual protein C cleavage sites in Human coagulation factor Va
김성욱(Suhng Wook Kim),이승관(Suhng Wook Kim),이창규(Suhng Wook Kim) 고려대학교 보건과학연구소 2004 보건과학논집 Vol.30 No.1
Resistance to activated protein C (APC) is the most common inherited risk factor for venous thrombosis. In the present study we have investigated the effect of mutations of the APC cleavage sites at Arg<SUB>306</SUB>, Arg<SUB>506</SUB> and Arg<SUB>679</SUB> in the factor V heavy chain on the inactivation of factor Va by APC. Mutants R306A, R506Q and R679A were expressed using B-domain deleted factor V constructs in COS-7 cells. The specific activity of the purified mutant proteins was identical to the wild-type protein (rHFVa) and APC cleavage at the mutated sites was blocked. Characterization of these mutants using a clinical assay for APC resistance demonstrated that R506Q was resistant to APC, but that R306A and R679A were sensitive. In clotting assays, R506Q showed delayed inactivation by APC, whereas R306A showed a rapid but incomplete loss of activity. APC inactivation of R679A was rapid and indistinguishable from rHFVa. These results indicate that mutation of individual APC cleavage sites in factor Va leads to variable degrees of APC. These findings have significant implications for the identification of patients with APC resistance and the understanding of the importance of individual APC cleavage sites in regulating the activity of the prothrombinase complex.
Rapid and direct detection of apolipoprotein E genotypes using whole blood from humans.
Kim, Suhng Wook,Heo, Ji Hye,Kim, Chun Huem,Yoo, Dong Chul,Won, Dong Hwan,Lee, Seung Gwan,Cho, Kyoung Jin,Song, Jung Han,Park, Su Jeong,Yang, Young Geun,Choi, Dal Woong Taylor Francis 2010 Journal of toxicology and environmental health. Pa Vol.73 No.21
<P>Polymerase chain reaction (PCR) is a powerful molecular biological tool in the field of toxicity testing and diagnostics. The use of PCR for large-scale genetic testing requires an effective method of sample processing. Unfortunately, isolation of PCR-quality DNA is time-consuming. PCR performed directly on whole blood is preferred because of time efficiency, cost of the procedure, and possible automation for large-scale toxicity evaluation and diagnosis. The apolipoprotein E (APOE) gene contains two single-nucleotide polymorphisms (SNP) located at codons 112 and 158, producing three APOE protein isoforms known to be associated with the risks of developing cardiovascular disease and susceptibility to Alzheimer's disease. In the present study, an attempt was made to use the AnyDirect solution for APOE genotyping by PCR using whole blood directly without DNA purification. Results for two PCR methods, (1) conventional PCR using purified DNA and conventional buffer and (2) direct PCR using whole blood and AnyDirect solution, were compared in four different PCR-based APOE genotyping methods including PCR restriction-fragment-length polymorphism (PCR-RFLP), allele-specific PCR, SNaPshot mini-sequencing, and multiplex tetra-primer amplification refractory mutation system (T-ARMS) PCR. There was complete concordance in the APOE genotypes between conventional PCR and direct PCR, in all four different PCR-based APOE genotyping methods. Data demonstrated that the four different PCR-based APOE genotyping methods are able to determine the APOE genotypes successfully using whole blood directly with the use of AnyDirect solution. The direct multiplex T-ARMS PCR using whole blood may be the most rapid, simple, and inexpensive method for detecting APOE genotypes among four different APOE genotyping methods.</P>
Kim, Suhng-Wook Korean Society of Life Science 2003 생명과학회지 Vol.13 No.4
제5인자와 지질막 phosphatidylserine과의 상호작용은 prothrombinase 복합체의 활성을 조절하는데 중요하다. 본 연구에서 제5인자의 지질 결합부위에 위치한 Trp2063과 Trp2064를 동시에 돌연변이 시킨 재조합 제5인자를 과발현 시키고 정제하였다. 돌연변이된 제5인자는 1-10%의 phosphatidylserine을 포함하는 지질막에서 아주낮은 활성을 보였다. surface plasmon resonance에 의해서 지질막과의 결합을 측정한 결과 돌연변이된 제5인자가 본래의 제5인자보다 고정된 지질막에의 결합이 현저하게 떨어지는 것을 관찰하였다. 제5인자가 phosphatidylserine을 포함하는 지질막에 높은 친화력으로 결합하기 위해서는 Trp2063과 Trp2064가 필수적이고 이러한 상호작용은 생리적인 phosphatidylserine 농도를 포함하는 지질막 위에서 prothrombinase 복합체의 형성에 필요하다는 결론을 내렸다. Interactions between factor Va (HFVa) and membrane phosphatidylserine (PS) regulate the activity of the prothrombinase complex. I have previously shown that two solvent exposed hydrophobic residues located in the C2-domain, Trp2063 and Trp2064, are required for binding to immobilized PS and for expression of procoagulant activity on membranes containing 5% PS. In order to fully define the functional importance of these two residues I have expressed and isolated recombinant factor Va (rHFVa) W2063A/W2064A double mutant. In contrast to the native protein the two glycoforms resulting from alternative glycosylation of Asn2181 eluted as a single peak with rHFVa1 W2063A/W2064A eluting on the leading edge and rHFVa2 W2063A/W2064A eluting on the trailing edge. The double mutant rHFVa2 W2063A/W2064A expressed little or no procoagulant activity on membranes containing 1-10% mol % PS. In contrast, the procoagulant activity of this mutant was slightly greater than the native protein on membranes containing>18 mol % PS. The binding of rHFVa2 W2063A/W2064A to immobilized phospholipid vesicles was markedly reduced compared to the native protein in a surface plasmon resonance binding assay. I conclude that Trp2063 and Trp2064 are required for high affinity binding of factor Va to PS membranes and that this interaction is necessary for assembly of the prothrombinase complex on membranes containing physiological concentrations of PS.