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一种新的五步蛇蛇毒类凝血酶Cdna的克隆和序列分析 总被引:2,自引:0,他引:2
目的:克隆出五步蛇的类凝血酶cDNA,为研究其结构和功能的关系。并为下一步基因表达开发抗血栓药物提供依据和基础。方法:从五步蛇蛇腺中提取了总RNA,通过反转录合成第一链cDNA,经PCR扩增出五步蛇毒腺中类凝血酶TLE1的cDNA片段,并将其连接到质粒载体扩增,然后进行DNA测序。结果:成功克隆出一种新的五步蛇类凝血酶的cDNA片段。此片段全长794bp,包括编码部分的全长为777bp。推导其编码的蛋白质序列为258个氨基酸,其中包括18个氨基酸的信号肽,6个氨基权的前肽和234个氨基酸的成熟氨基酸顺序。TLE1成熟肽与其它蛇毒类凝血酶相比,蛋白质一级结构具有较高的同源性。结论:从五步蛇中成功克隆出一 种新的类凝血酶cDNA,并确定了它的DNA顺序。 相似文献
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Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of α-thrombin, an essential protease in the coagulation cascade. Two α-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain α-helical coiled coils at pH 7 and 25 °C. The control peptides were only partially α-helical. E14C remained folded at 90 °C but E8C was half unfolded at 49 °C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of α-thrombin. None of these compounds accelerated antithrombin inhibition of α-thrombin, and neither control peptide accelerated HC inhibition of α-thrombin. Acceleration of the HC inhibition of α-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic α-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biologcal activity. © Munksgaard 1995. 相似文献
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Thrombin Inhibition in discordant xenograft rejection 总被引:1,自引:0,他引:1
Beth-Ann Lesnikoski Daniel Candinas Ichiro Otsu Rainer Metternich Fritz H. Bach Simon C. Robson 《Xenotransplantation》1997,4(3):140-146
Abstract: Microvascular thrombosis and the associated platelet and endothelial cell activation are prominent observations in xenograft rejection. This pathological picture could be related to the excessive generation of thrombin in the context of either inflammation or putative inter-species molecular incompatibilities between activated coagulation factors and their natural anticoagulants. Relatively selective thrombin Inhibition with the serine protease inhibitor SDZ MTH 958 (MTH-958) are independent of heparinoids and anti-thrombin III. MTH-958 has been shown to significantly prolong porcine cardiac function during perfusion with human blood in an ex vivo model. The aim of this study was to validate the role of thrombin generation in a rodent model of discordant xenograft rejection in vivo. The effect of thrombin inhibition with MTH-958 was tested in both hyperacute rejection (HAR) and delayed xenograft rejection (DXR) after decomplementation with cobra venom factor (CVF) in normal Lewis (Lew) rats and Intrinsic C6 deficiency In PVG (C6-/PVG) recipient rats. Recipient rats received heterotopic guinea pig cardiac xenografts and were treated with titrated doses of MTH-958 until the time of graft rejection. Plasma samples at selected time points were examined to confirm effective thrombin inhibition, and rejected grafts were analyzed by immunohistology. MTH-958 significantly improved graft survival in HAR albeit the extent of prolongation was not marked, but the agent failed to prolong survival In CVF-treated Lew rats. In C6-/PVG rats receiving MTH-958, a significantly reduced graft survival time was observed when compared with C6-/PVG controls. The grafts from MTH-958-treated animals showed dense deposits of C3, IgM, and IgG with fibrin levels similar to controls. The thrombin antagonist tested could prolong xenograft survival during HAR but had no benefit in DXR. The relative non-specificity of the serine protease inhibitor MTH-958 with the potential activation of alternative pathway of complement via the inhibition of factor I could account for the failure to prolong xenograft survival in DXR. The pathogenetic significance of thrombin generation in this situation remains to be determined by the use of more selective and pharmacologically acceptable I anti-thrombin agents. 相似文献
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凝血酶(thromibin,Ⅱa)是一种生成于损伤处血管内皮细胞多功能蛋白酶,它是参与凝血过程各个环节反应中的关键酶。在发挥止血作用的同时,还可能诱导炎症、增生及修复等反应。最近发现的凝血酶受体(thrombin receptor,TR)分子可能为解释上述现象提供了一个理论框架。同时,TRN端被Ⅱa切下的41个氨基酸片段是否具有特殊功效,值得研究探讨。 相似文献
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Objective To determine the concentrations of cholesterol sulfate ( CS) in human sera and pla-cental villi during the course of pregnancy. And to analyze its inhibitory activity on thrombin and further characterize the functional significance of CS. Methods The concentrations of CS were determined by thin-layer chromatog-raphy (TLC) on 60 cases of normal pregnant women and 30 cases of normal placental villi. The effect of CS in human sera on the activity of thrombin was analyzed. Results The concentrations of CS in human sera gradually increased from the first to third trimester of gestation with a correlation coefficient of 0. 69, and a correlation between the concentration of CS and weeks of gestation (P <0. 01). CS was also contained in the placental villi, and its concentrations at the second and third trimester of gestations were 4. 7 and 6. 2-fold of that at the first trimester of gestation. CS inhibited the activity of thrombin. Conclusion Placental CS is one of the sources of CS in the serum , pr 相似文献