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dc.description.abstractCollagen-stimulated generation of ROS regulates signal transduction in platelets, although the direct mechanism is unclear. The family of PTPs is one of redox-sensitive targets and PTPs have an oxidation-sensitive cysteine in catalytic site. Here I show that the SHP-2 is oxidized in platelets by ROS produced upon collagen stimulation. In addition to, I have identified Syk, Vav1, and Btk as putative substrates of SHP-2 in collagen-stimulated platelets. Oxidative inactivation of SHP-2 promotes collagen-induced phosphorylation of Syk, Vav1, and Btk in LAT signaling complex, which culminates in enhanced tyrosine phosphorylation-mediated activation of PLCγ2 and platelet aggregation. Genetic approaches using platelets obtained from mice deficient in H2O2 eliminating enzymes demonstrate the role of H2O2 for redox regulation of SHP-2. Relative to wild-type platelets, both glutathione peroxidase 1/catalase double-deficient and peroxiredoxin II-null platelets show the enhancement of cellular H2O2, oxidative inactivation of SHP-2, and specific tyrosine phosphorylation of Syk, Vav1, Btk, and PLCγ2 in respond to collagen, which subsequently leads to augmentation of integrin αIIbβ3 activation and surface expression of P-selectin. Furthermore, H2O2 eliminating enzyme deficiency accelerates thrombotic response in FeCl3-injured carotid artery in mice. Taken together, the data indicate that collagen-induced H2O2 generation leads to SHP-2 oxidation, which promotes platelet activation and pathologic thrombus formation through the tyrosine phosphorylation-based signaling pathways.;Collagen 자극에 의해 생성된 활성산소종 (reactive oxygen species, ROS)은 혈소판 활성화 신호전달을 조절한다. 그러나 이렇게 생성된 ROS에 의한 신호전달기전은 명백하게 밝혀진 바가 없다. Protein tyrosine phosphatases (PTPs) family는 redox에 민감한 signaling molecule 중 하나로 효소 활성 부위에 ROS에 의해 산화되는 cysteine 잔기를 가지고 있다. 본 논문에서는 SH2 domain-containing PTP (SHP-2)가 collagen 자극에 의해서 생성된 ROS에 의해서 산화되는 것을 밝혔다. 또한 collagen 자극에 의해 활성화된 혈소판에서 spleen tyrosine kinase (Syk), Vav1, Bruton's tyrosine kinase (Btk)가 SHP-2의 잠정적인 substrates임을 밝혔다. ROS에 의해 산화되어 활성이 저해된 SHP-2는 LAT (the linker for the activation of T cells) signaling complex 내에서 collagen 자극에 의한 Syk, Vav1, Btk의 인산화를 증가시켰고, 이것은 결국 phospholipase Cγ2 (PLCγ2)의 tyrosine 인산화 증가에 의한 활성화와 혈소판 aggregation을 증강시켰다. 유전자적 접근방법으로 H2O2를 제거하는 효소들이 결핍된 마우스로부터 혈소판을 얻어 SHP-2의 redox 조절에 관련된 H2O2의 역할을 입증하였다. 정상적인 혈소판들에 비해, glutathione peroxidase 1/catalase가 결핍된 혈소판과 peroxiredoxin II (Prx II)가 결핍된 혈소판들은collagen 자극에 따른 세포 내 H2O2 생성, SHP-2의 산화적 불활성화, 신호전달에 중요한 단백질들인 Syk, Vav1, Btk, PLCγ2의 특정 tyrosine 잔기의 인산화가 증가되었고, 이에 따른 integrin αIIbβ3 활성화와 세포막 부위로의 P-selectin 발현도 증가되었다. 또한, 마우스의 경동맥이 FeCl3에 의해 손상될 때 나타나는 혈전의 형성속도가, H2O2를 제거하는 효소들이 결핍된 경우에 정상군에 비하여 가속화되었다. 이상의 결과들을 종합하면, collagen 자극에 의해 혈소판 내에 생성이 증가된 H2O2에 의하여 SHP-2의 산화가 증가되고, 이에 따라 단백질의 tyrosine 인산화를 기반으로 하는 신호전달체계의 활성화를 통해 혈소판의 활성화와 혈관 내 혈전형성이 촉진된다는 것을 규명하였다.-
dc.description.tableofcontentsIntroduction 1 I. Role of platelets in hemostasis and thrombosis 2 A. Platelet structure and function 2 B. Platelets in hemostasis 4 C. Platelets in thrombosis 6 II. Signal transduction in platelet activation 7 A. Platelet agonists and their receptors 7 B. Collagen-GPVI signaling 9 III. Role of ROS and antioxidants in platelets 11 A. ROS in platelets 11 B. H2O2 as signaling messenger 13 C. H2O2 eliminating Enzymes 15 D. H2O2 eliminating enzymes in thrombosis or atherosclerosis 17 IV. The specific aims of the thesis 18 Materials & Methods 20 1. Antibodies and reagents 21 2. Experimental animals 21 3. Polymerase chain reaction for genotyping 22 A. Mouse tail DNA isolation 22 B. Genotyping of GPx1/Catalase double deficiency mice 22 C. Genotyping of Prx II deficiency mice 23 4. Human platelet preparation 25 5. Mouse platelet preparation 25 6. Aggregation studies 26 7. Analysis of intracellular ROS 26 8. Analysis of intracellular cytosolic calcium 26 9. Immunoblot analysis 27 10. Immunoprecipitation analysis 27 11. Detection of oxidized SHP-2 with oxPTP antibodies 28 12. Biotinylation for detection of oxidized thiols in SHP-2 30 13. Flow cytometry 32 14. Assessment of arterial thrombosis after ferric chloride exposure 32 15. Statistical analysis 33 Results 34 1. Collagen-induced ROS generation enhances a cascade of tyrosine phosphorylation events in platelets 35 2. Collagen-induced ROS generation leads to oxidation of SHP-2 in platelets 40 3. SHP-2 associates with multiple proteins in LAT signalsome upon collagen stimulation 44 4. Effects of collagen-induced ROS generation on LAT-mediated PLCγ2 activation 47 5. Deficiency of H2O2 eliminating enzymes leads to enhanced activation of PLCγ2 via oxidative inactivation of SHP-2 in collagen-stimulated platelets 50 6. Deficiency of H2O2 eliminating enzyme enhances platelet activation and aggregation in a redox-dependent manner 58 7. Deficiency of H2O2 eliminating enzyme accelerates thrombotic response in injured carotid artery 62 Discussion 64 Graphical Abstract 71 References 72 논문개요 85-
dc.format.extent3628404 bytes-
dc.publisher이화여자대학교 대학원-
dc.titleCollagen-induced H₂O₂ generation regulates platelet activation through oxidation of SHP-2-
dc.typeDoctoral Thesis-
dc.creator.othernameJang, Ji Yong-
dc.format.pageix, 86 p.-
dc.identifier.major대학원 생명·약학부약학전공- 2-
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