[보고서]혈소판 응집시 증가되는 활성산소종이 세포신호전달을 증폭시키는 기전에 관한 연구

창동신

보고서 정보

주관연구기관

이화여자대학교
Ewha Womans University

연구책임자

창동신

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2010-02

과제시작연도

2007

주관부처

과학기술부

사업 관리 기관

한국과학재단
Korea Science and Engineering Foundtion

등록번호

TRKO201100005161

과제고유번호

1355050332

사업명

특정기초연구지원사업

DB 구축일자

2013-04-18

키워드

혈관계 질환.혈소판 응집.단백질 타이로신 인산화.활성산소종.산화환원 조절.타이로신 탈인산화 효소.vascular disease.platelet aggregation.protein tyrosine phosphorylation.reactive oxygen species.redox regulation.protein tyrosine phosphatase.

표 NAD(P)H oxidase inhibitor and ROS scavenger reduce platelet aggregation. Mice platelets were preincubated with inhibitor of NAD(P)H oxidase diphenylene iodonium (DPI, 50 uM) and potent antioxidant compound N-acetylcysteine (NAC, 5 mM) for 5 minutes and then stimulated with collagen (10 ug/ml) and allowed to aggregate during indicated time, with stirring at 1,200 rpm. Platelet aggregation was demonstrated by the change in light transmission. A representative aggregation tracing from 3 independent experiments is shown.
표 DPI and NAC inhibit collagen-induced increase in ROS level in platelets. Mice platelets were preincubated with CM-H2DCFDA (10 uM) for 15 minutes at 37℃ in phosphate-buffered saline (PBS), excessive dye was washed out, and platelets were resuspended in PBS. Platelets were preincubated with inhibitors of DPI (50 uM) and NAC (5 mM) for 5 minutes. Then, after activation by collagen (10 ug/ml), ROS production was measured during indicated time by fluorospectrophotometer. A representative ROS detection tracing from 3 independent experiments is shown.
표 NADPH oxidase inhibitor and ROS scavenger inhibit aggregation and an elevation of intracellular ROS level in collagen-stimulated human platelets. Washed human platelets were preincubated with diphenylene iodonium (DPI, 50 μM), an inhibitor of NAD(P)H oxidase or N-acetylcysteine (NAC, 5 mM), a potent antioxidant for 5 min and then stimulated with collagen (10 μg/ml). Platelet aggregation (A) and intracellular ROS level (B) were measured as in Figure 1 and 2
표 Collagen-induced tyrosine phosphorylation in platelets. Tyrosine phosphorylation in platelets was detected using the anti-phosphotyrosine antibody 4G10. Collagen (10 ug/ml) induced tyrosine phosphorylation during indicated time in mice platelets.
표 Effect of DPI and NAC on tyrosine phosphorylation induced by collagen in platelets. Mice platelets were preincubated with 50 uM DPI (A) or 5 mM NAC (B) for 5 minutes and then stimulated with collagen (10 ug/ml) for indicated times. Cell lysates were then subjected to immunoblot analysis with the anti-phosphotyrosine antibody 4G10. A representative data from 3 independent experiments is shown.
표 Collagen induces oxidative modification of SHP-2 in platelets. (A) Reduced thiols (PTP-SH) are labeled with PEO-iodoacetyl biotin but oxidized thiols (PTP-S-OH; sulfenic acid) are not. (B) After collagen stimulation for the indicated times, platelets were lysed and labeled with PEO-iodoacetyl biotin as described in Materials and methods. SHP2 was immunoprecipitated and biotinylation detected with HRP-conjugated streptavidin and ECL. Equal application of protein was confirmed by immunoblot analysis. The level of biotin incorporation (by densitometry) was corrected for total protein levels. Loss of biotin incorporation into immunoprecipitated SHP2 indicates oxidation of reactive thiols. The graph represent the average of three separate experiments±S.E.M.
표 Effects of ROS elimination on protein tyrosine phosphorylation and SHP-2 oxidation in collagen-stimulated human platelets. (A) Human platelets were preincubated with 5 mM NAC for 5 min and then stimulated with collagen (10 μg/ml) for indicated times. Platelets lysates were then subjected to immunoblot analysis with antibodies specific to phosphotyrosine (4G10) and α-tubulin, as indicated. (B) After collagen stimulation for the indicated times, washed human platelets were lysed and labeled with PEO-iodoacetyl biotin. SHP-2 was immunoprecipitated and its biotinylation was analyzed by immunoblot analysis with HRP-conjugated streptavidin. Equal application of protein was confirmed. Loss of biotin incorporation into immunoprecipitated SHP-2 indicates oxidation of reactive thiols. (C) Washed human platelets were preincubated with NAC (5 mM) for 5 min and then stimulated with collagen (10 μ g/ml) for indicated times and SHP-2 oxidation was measured as in (B).
표 Effects of NAC treatment on oxidative modification of SHP-1 in collagen-stimulated platelets. Human platelets were preincubated with 5 mM NAC for 5 minutes and then stimulated with collagen (10 ug/ml) for 2 minute. Activated platelets were lysed and labeled with PEO-iodoacetyl biotin as described in Materials and methods. SHP-1 was immunoprecipitated and biotinylation was detected with HRP-conjugated streptavidin and ECL. Equal application of SHP-1 was confirmed by immunoblot analysis. The level of biotin incorporation was corrected for total protein levels. Loss of biotin incorporation into immunoprecipitated SHP-1 indicates oxidation of reactive thiols.
표 Tyrosine phosphorylation level was increased in collagen stimulated Meg-01 cells. Meg-01 cells were stimulated for indicated times with collagen (10 μg/ml). Cell lysates were subjected to immunoblot analysis with anti-phosphotyrosine antibody (4G10) and anti-tubulin antibody.
표 Elevation of intracellular ROS level in collagen stimulated Meg-01 cell was inhibited by NAC. Meg-01 cells were preincubated with CM-H2DCFDA(5uM)for 15min at37℃ in phosphate-buffered saline (PBS). Excessive dye was washed out and platelets were resuspended in PBS. Meg-01 cells were incubated with NAC (0.5 mM) for 5 min and then stimulated with collagen (10 ug/ml). ROS production was measured for 5 min using fluorospectrophotometer.
표 Meg-01 cells are transefected with SHP-2 WT/CS. Meg-01 cells (1×106 cells) were transfected with pcDNA3.1/pEBG/pCGN-SHP-2 WT or CS and then incubation for 24 hrs. Cell lysates were subjected to immunoblot analysis with anti-SHP-2 monoclonal antibody.
표 Redox regulation of collagen-induced association of proteins with SHP-1 and SHP-2 in human platelets. Human platelets were preincubated with 5 mM NAC for 5 minutes and then stimulated with collagen (10 ug/ml) for 2 minute. Lysates were immunoprecipitated with anti-SHP-1 (A) or anti-SHP-2 (B) antibody. Immunoprecipitates were separated by SDSPAGE, immunoblotted with the indicated proteins. A representative data from 3 independent experiments is shown.
표 Effect of NAC on phosphorylation of specific tyrosine residue on PLCγ2, Syk, Vav and Btk in collagen-stimulated human platelets. Human platelets were preincubated with 5 mM NAC for 5 minutes and then stimulated with collagen (10 ug/ml) for 1 minute. Cell lysates were immunobloted with the anti-PLCγ2-pY753, anti-Syk-pY525/526, anti-Vav-pY174 or anti-Btk-pY551 antibody and were stripped and reprobed for the indicated proteins. A representative data from 3 independent experiments is shown.
표 Catalase/GPxI knockout mice was confirmed by genotyping and western blot analysis of platelet lysates. (A) Genotype of wild type and Cat/GPxI DKO mice was confirmed by PCR. (B) Lysates obtained from platelets of wild type or Cat/GPxI DKO mice were subjected to immunoblot analysis with the anti-Catalase and anti-GPxI antibody. A representative data from 3 independent experiments is shown.
표 Catalase/Glutathione peroxidase I deficiency enhances platelet aggregation. Platelets obtained from wild type and Cat/GPxI DKO mice were stimulated with collagen (10 ug/ml) and allowed to aggregate with stirring at 1,000 rpm. Platelet aggregation was determined by the change in light transmission. Data are means S.E.M. of triplicates.
표 Catalase/Glutathione peroxidase I deficiency enhances cellular ROS level in platelets. Platelets obtained from wild type and Cat/GPxI DKO mice were preincubated with CM-H2DCFDA (10 uM) for 15 min at 37℃ in phosphate-buffered saline (PBS). Excessive dye was washed out and platelets were resuspended in PBS. Platelets were stimulated with collagen (10 ug/ml), then ROS production was measured for 5 minutes using fluorospectrophotometer. Data are means S.E.M. of triplicates.
표 Effects of Catalase/Glutathione peroxidase I deficiency on tyrosine phosphorylation level in collagen-stimulated platelets. Platelets obtained from wild type and Cat/GPxI DKO mice were stimulated with collagen (10 ug/ml). Platelet lysates were then subjected to immunoblot analysis with the anti-phosphotyrosine antibody 4G10. A representative data from 3 independent experiments is shown.
표 Effects of Catalase/Glutathione peroxidase I deficiency on oxidative modification of SHP-2 in collagen-stimulated platelets. After collagen stimulation for 2 minutes, platelets were lysed and labeled with PEO-iodoacetyl biotin as described in Materials and methods. SHP2 was immunoprecipitated and biotinylation was detected with HRP-conjugated streptavidin and ECL. Equal application of SHP2 was confirmed by immunoblot analysis. The level of biotin incorporation was corrected for total protein levels. Loss of biotin incorporation into immunoprecipitated SHP2 indicates oxidation of reactive thiols. The graph represent the average of three separate experiments.
표 Effects of Glutathione peroxidase I and Catalase deficiency on phosphorylation of specific tyrosine residue on PLCγ2, Syk, Vav and Btk in collagen-stimulated platelets. Platelets obtained from wild type and Cat/GpxI DKO mice were stimulated with collagen (10 ug/ml) for 1 minute. Cell lysates were immunobloted with the anti-PLCr2-pY753, anti-Syk-pY525/526, anti-Vav-pY174 or anti-Btk-pY551 antibody and were stripped and reprobed for the indicated proteins. A representative data from 3 independent experiments is shown.
표 Loss of GPxI and Catalase enhances surface expression of P-selectin (CD62P-FITC) and active-Integrin αIIbβ3 (JON/A-PE) in convulxin-stimulatedplatelets. Washed platelets from WT (black line) and GPxI/Catalase double knockout (red line) mice were incubated with FITC conjugated anti-P-selectin (CD62P-FITC, 0.5 mg/ml) or PE conjugated anti-active-Integrin αIIbβ3 (JON/A-PE, 0.5 mg/ml) and convulxin (25 ng/ml) for 5min at37℃ and then stopped with PBS. Surface expression of P-selectin (A) or active-Integrin αIIbβ3 (B) on platelets were analyzed by flowcytometry.
표 Loss of GPxI and Catalase reduces blood flow in FeCl3-injured carotid artery. Photo-plethysmography was used to monitor carotid artery blood flow in WT and GPxI/Catalase double knockout mice. Flow probe was presented on exposed left carotid artery. A smaller strip of filter paper (1x1mm) saturated with 25% FeCl3 was applied proximal to the flow probe and then obtained data were analyzed with National Instruments LabVIEW software.
표 A schematic model for ROS-mediated signaling pathways in collagen-stimulated platelets.

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혈소판 응집시 증가되는 활성산소종이 세포신호전달을 증폭시키는 기전에 관한 연구
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혈소판 응집시 증가되는 활성산소종이 세포신호전달을 증폭시키는 기전에 관한 연구 Studies on the mechanism by which reactive oxygen species upregulate the cellular signaling pathways in platelet activation 원문보기

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혈소판 응집시 증가되는 활성산소종이 세포신호전달을 증폭시키는 기전에 관한 연구
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