融合蛋白SRH的构建及治疗动脉粥样硬化的作用机制研究
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摘要
动脉粥样硬化( atherosclerosis, AS)是众多心脑血管疾病共同的病理基础,也是心血管系统疾病中最常见的疾病,严重危害人类健康。AS的发病机制存在多种学说,但任何一种学说均不能全面的解释AS的发生发展。近年来大量细胞及分子水平的实验研究表明尽管AS的致病因素多种多样,但是AS所导致的是一条相似的发展途径即慢性炎症病理过程。血管内皮受损,脂质氧化及浸润,血小板、炎性细胞活化、粘附并浸润,平滑肌细胞迁移、增殖等伴随着AS发生、进展、恶化及斑块破裂并血栓形成的全过程。针对AS的发展特点,我们考虑是否可以通过构建多靶点的治疗药物,针对不同病理过程从多个方面抑制AS的进展。
研究表明炎症贯穿于动脉粥样硬化发生和发展的全过程,是动脉粥样硬化过程中许多病理生理改变的共同基础。凝血酶和血小板作为参与凝血过程的重要因子是多种抗凝药物作用的靶点,同时越来越多的研究表明在动脉粥样硬化发生发展过程中血管损伤处聚集的凝血酶,血小板对血管壁炎症反应具有重要促进作用。由于局部凝血功能亢进在损伤内皮表面常常有纤维蛋白形成的网状结构出现,在这种网状结构中聚集了大量血小板,白细胞,凝血酶等,这些物质的存在加剧了损伤血管处的炎症反应促进了动脉粥样硬化的发展。由此可见凝血酶和血小板同时参与了凝血及炎症两个反应过程。在AS发展的晚期,患者常常伴有冠状动脉综合征的症状,临床应用抗凝及溶栓治疗。因此我们设计构建了多功能融合蛋白SRH,针对抗凝、抗炎及溶栓三个方面实现抗AS的作用。
天然葡激酶(staphylokinase, SAK)可以选择性地作用于纤维蛋白原,具有纤维蛋白特异性高,对血小板富集型血栓作用强,副作用小,价格便宜等优点。我室研制的rSAK已经完成临床试验评价,研究结果表明,rSAK的再通率显著高于r-tPA,显示出良好的临床应用前景。因此我们设计了以SAK为骨架的多功能融合蛋白SRH,其分子结构主要包括三个部分:SAK分子,RGD序列,水蛭素12肽。SAK主要发挥其溶解纤维蛋白的作用,RGD序列具有抑制血小板聚集功能,水蛭素12肽具有抗凝血酶功能。本研究的主要内容包括以下四个部分
一利用重叠延伸PCR方法,在SAK突变体的基础上构建了SRH融合蛋白基因,将编码融合基因的DNA片段重组入表达载体pBV220,转化E.coli BL21感受态细胞进行诱导表达。结果显示,SRH在E.coli/pBV220受体菌中主要以可溶性形式表达。将E.coli BL21/pBV220-SRH菌株进行发酵,大量表达后,进行纯化。经过阴离子交换层析和凝胶过滤层析两步纯化,SRH纯度达95%以上。我们进一步对融合分子SRH的各个功能域进行了功能检测分析,使用纤维蛋白平板溶圈法,底物发色法等检测SRH溶栓活性,结果显示SRH蛋白溶栓活性为(1.3±0.15)×104 AU/mg低于SAK(4.1±0.12)×104 AU/mg;ELISA检测SRH蛋白结合凝血酶能力,结果显示SRH蛋白具有结合凝血酶的能力,进一步使用纤维蛋白凝块法检测SRH的抗凝血酶功能,结果显示与SAK相比SRH蛋白具有显著抗凝血酶功能;检测SRH的抗血小板聚集功能结果显示SRH蛋白保持了RGD序列的抗血小板聚集功能。以上研究结果表明我们构建的SRH蛋白保持了各功能域的活性,具有溶栓,抑制血小板聚集和抗凝血酶的功能。
二药效学评价,使用apoE-/-小鼠为动物模型,高脂饲料喂养,观察连续静脉注射SRH后其动脉粥样硬化进展情况。结果显示SRH给药组具有抗AS的作用。主动脉窦部HE染色结果显示,模型组主动脉窦部粥样硬化病变非常显著,AS斑块占管腔面积为91%,SRH治疗组:1mg/kg体重组斑块占管腔面积为:39%,0.125mg/kg体重组斑块占管腔面积为:43%。结果显示与模型组相比,治疗组AS斑块病变有明显减轻的趋势。对小鼠CRP检测显示SRH可以显著降低apoE-/-小鼠CRP水平,这一结果提示我们SRH治疗动脉粥样硬化的功能可能通过抗炎性机制实现。
三为进一步研究SRH蛋白的抗动脉粥样硬化机制,我们选择血管内皮细胞系HUVEC,检测了SRH蛋白对HUVEC细胞的作用。研究发现SRH蛋白可以抑制凝血酶诱导的动脉粥样硬化相关基因PAI-1,ICAM-1,VCAM-1,IL6,IL8,MCP-1,TF的mRNA表达水平,抑制IL6,MCP-1的分泌,抑制细胞膜粘附分子ICAM-1,VCAM-1的表达,抑制凝血酶诱导的单核细胞与血管内皮细胞的粘附。这些研究结果提示SRH蛋白可以通过抑制凝血酶对内皮细胞的作用发挥其抗炎功能,进一步抑制动脉粥样硬化。
四由于SRH蛋白在SAK基础上融合了其它分子,这种变化可能会造成在体内代谢的变化。为了初步检测SRH蛋白在体内的代谢情况并与SAK进行比较,我们制备了SRH的兔源及鼠源多克隆抗体,建立了双抗夹心ELISA方法检测SRH及SAK含量的方法,并使用该方法检测了动物体内SRH蛋白及SAK蛋白的代谢情况,结果显示这两种蛋白在动物体内代谢存在一定的差异,SRH半衰期有延长的趋势,为进一步研究SRH蛋白的药学功能奠定了基础。
总之,我们针对AS发生和发展的特点构建了多功能融合蛋白SRH,以实现多靶点治疗AS。在本研究中我们完成了SRH的构建表达和纯化,对各功能域的体外活性检测;在AS动物模型中评价了SRH的对AS治疗作用,并初步探讨了其作用机制;建立了SRH含量检测方法,并在动物体内初步检测了SRH的代谢情况。这些工作为SRH的成药性做了初步评价,也为新型抗AS药物的研制奠定基础。
Atherosclerosis (AS) is the common pathological basis of many cardio and cerebral vascular diseases, and also one of the most frequent disease with serious threat to human health. Although many researchers proposed different theories about AS pathogenesis however, anyone of which could not alone elucidate the genesis and development of AS completely. In the recent years, lots of experimental researches at cellular and molecular levels indicated that AS resulted in a similar development pathway, i.e. chronic inflammatory pathological process. The development of AS was accompanied by some similar pathophysiological process, including hemoendothelial damage, oxidation and infiltration of lipid, the activation, adhension and infiltration of platelet and inflammatory cells, the immigration and proliferation of smooth muscle cells etc.. On the basis of the AS development characteristics, we consider whether the multi-targeted drug could be constructed and applied, and then the effect against AS could be better.
Studies displayed that inflammation penetrated through all the process of AS genesis and development, which was a common basis of many pathophysiological changes. Thrombin and platelet as the targeting sites for many anti-coagulant drugs were the important factors involved in blood coagulation, while more and more researches indicated that thrombin and platelet accumulated at damaged site of vessel played an important promoting role for the inflammatory response of vessel wall. Because of the hyperfunction of local blood coagulation, a large amount of platelet, leukocyte and thrombin accumulated, the existence of which exacerbated the inflammatory reaction at the damaged blood vessel and then accelerate the development of atherosclerosis, therefore thrombin and platelet simultaneously participated in both blood coagulation and inflammation. In the late period of AS development, the patients were often accompanied by the symptoms of coronary syndrome, to which the anticoagulant and thrombolysis therapies were applied .Therefore, we designed and constructed a multifunctional fusion protein SRH to perform the anti -AS effect through its anticoagulation, anti-inflammation and thrombolysis activity.
Natural staphylokinase (SAK) as a selective fibrinogen activator possessed some advantages, such as higher fibrinogen specificity, stronger action on platelet-enriched thrombus, lower side-effect, and cheaper price. The clinical trial evaluation of our rSAK has been completed, and the results indicated that its recanalization rate was significantly higher than that of r-tPA, thus showing a good perspective of clinical application. We designed a multifunctional fusion protein SRH by using the modified SAK as the backbone. The molecular structure of SRH consisted of three functional domains: the modified SAK molecule, RGD sequence, and dodecapeptide of hirudin. SAK displayed mainly the dissolution of fibrin, RGD sequence possessed inhibition effect on platelet aggregation, and the dodecapeptide of hirudin had anti-thrombin function.
This study mainly includes the following four parts:
1. Overlapping extension PCR methods were used to construct the recombinant gene encoding SRH fusion protein, which was inserted in the expression vector pBV220, and the resulted recombinant plasmid was used to transform the competent cells of E.coli BL21 to obtain an engineering bacteria strain for inducing expression of SRH. The results showed that SRH could be expressed in the soluble form by the engineering strain E.coli BL21/pBV220-SRH, and SRH could be purified after expression in large scale. The purity of SRH reached to 95% after two steps of purification with anion-exchange chromatography and gel filtration. The functions of different domains of the fusion molecule SRH were analyzed by using the lysis circle assay on fibrin agarose gel plate and chromogenic substrate method. The results showed that the thrombolytic activity of SRH was (1.3±0.15)×104 AU/mg, lower than that of SAK((4.1±0.12)×104 AU/mg). Binding ability analysis indicated that SRH was able to bind with thrombin, and further assay with fibrin clotting method showed SRH had stronger anti-coagulation function than that of SAK. Assay of SRH anti-platelet aggregation function displayed that SRH maintained the function of RGD anti-platelet aggregation. The above results demonstrated the fusion protein constructed in our research maintained the functions of three domains, possessing thrombolysis, inhibition of platelet aggregation and anti-thrombin.
2. We utilized apoE-/-mouse as the animal model feeding with high fat diet to observe the progression of atherosclerosis after injection with SRH. H.E. staining of aorta sinus demonstrated the model group showed a serious lesion of AS, and AS plaque occupied 91% of the aorta lumen area. The group treated with SRH displayed different results: 39% of that for SRH 1mg/kg body weight, and 43% for SRH 0.125 mg/kg. These results suggested that the plaque lesion of AS in the SRH-treating group had tendency of attenuation.
3. In order to elucidate the anti-atherosclerosis mechanism of SRH protein, the effect of SRH protein on HUVEC cell was tested. The results showed that SRH protein could down-regulate expression of AS-related genes including PAI, ICAM-1, VCAM-1, IL6, IL8, MCP-1 and TF. SRH also inhibited the secretion of IL6 and MCP-1.These results suggested that SRH protein could carry out its anti-atherosclerosis through the inhibition of thrombin.
4. In order to examine the metabolism of SRH in vivo, polyclonal antibodies to SRH in rabbit and mice were prepared. And we established double antibody-sandwich ELISA to determine the contents of SRH and SAK. The results indicated that half-life of SRH tended to be extended. This laid a foundation for the further exploration of its pharmacy.
研究表明炎症贯穿于动脉粥样硬化发生和发展的全过程,是动脉粥样硬化过程中许多病理生理改变的共同基础。凝血酶和血小板作为参与凝血过程的重要因子是多种抗凝药物作用的靶点,同时越来越多的研究表明在动脉粥样硬化发生发展过程中血管损伤处聚集的凝血酶,血小板对血管壁炎症反应具有重要促进作用。由于局部凝血功能亢进在损伤内皮表面常常有纤维蛋白形成的网状结构出现,在这种网状结构中聚集了大量血小板,白细胞,凝血酶等,这些物质的存在加剧了损伤血管处的炎症反应促进了动脉粥样硬化的发展。由此可见凝血酶和血小板同时参与了凝血及炎症两个反应过程。在AS发展的晚期,患者常常伴有冠状动脉综合征的症状,临床应用抗凝及溶栓治疗。因此我们设计构建了多功能融合蛋白SRH,针对抗凝、抗炎及溶栓三个方面实现抗AS的作用。
天然葡激酶(staphylokinase, SAK)可以选择性地作用于纤维蛋白原,具有纤维蛋白特异性高,对血小板富集型血栓作用强,副作用小,价格便宜等优点。我室研制的rSAK已经完成临床试验评价,研究结果表明,rSAK的再通率显著高于r-tPA,显示出良好的临床应用前景。因此我们设计了以SAK为骨架的多功能融合蛋白SRH,其分子结构主要包括三个部分:SAK分子,RGD序列,水蛭素12肽。SAK主要发挥其溶解纤维蛋白的作用,RGD序列具有抑制血小板聚集功能,水蛭素12肽具有抗凝血酶功能。本研究的主要内容包括以下四个部分
一利用重叠延伸PCR方法,在SAK突变体的基础上构建了SRH融合蛋白基因,将编码融合基因的DNA片段重组入表达载体pBV220,转化E.coli BL21感受态细胞进行诱导表达。结果显示,SRH在E.coli/pBV220受体菌中主要以可溶性形式表达。将E.coli BL21/pBV220-SRH菌株进行发酵,大量表达后,进行纯化。经过阴离子交换层析和凝胶过滤层析两步纯化,SRH纯度达95%以上。我们进一步对融合分子SRH的各个功能域进行了功能检测分析,使用纤维蛋白平板溶圈法,底物发色法等检测SRH溶栓活性,结果显示SRH蛋白溶栓活性为(1.3±0.15)×104 AU/mg低于SAK(4.1±0.12)×104 AU/mg;ELISA检测SRH蛋白结合凝血酶能力,结果显示SRH蛋白具有结合凝血酶的能力,进一步使用纤维蛋白凝块法检测SRH的抗凝血酶功能,结果显示与SAK相比SRH蛋白具有显著抗凝血酶功能;检测SRH的抗血小板聚集功能结果显示SRH蛋白保持了RGD序列的抗血小板聚集功能。以上研究结果表明我们构建的SRH蛋白保持了各功能域的活性,具有溶栓,抑制血小板聚集和抗凝血酶的功能。
二药效学评价,使用apoE-/-小鼠为动物模型,高脂饲料喂养,观察连续静脉注射SRH后其动脉粥样硬化进展情况。结果显示SRH给药组具有抗AS的作用。主动脉窦部HE染色结果显示,模型组主动脉窦部粥样硬化病变非常显著,AS斑块占管腔面积为91%,SRH治疗组:1mg/kg体重组斑块占管腔面积为:39%,0.125mg/kg体重组斑块占管腔面积为:43%。结果显示与模型组相比,治疗组AS斑块病变有明显减轻的趋势。对小鼠CRP检测显示SRH可以显著降低apoE-/-小鼠CRP水平,这一结果提示我们SRH治疗动脉粥样硬化的功能可能通过抗炎性机制实现。
三为进一步研究SRH蛋白的抗动脉粥样硬化机制,我们选择血管内皮细胞系HUVEC,检测了SRH蛋白对HUVEC细胞的作用。研究发现SRH蛋白可以抑制凝血酶诱导的动脉粥样硬化相关基因PAI-1,ICAM-1,VCAM-1,IL6,IL8,MCP-1,TF的mRNA表达水平,抑制IL6,MCP-1的分泌,抑制细胞膜粘附分子ICAM-1,VCAM-1的表达,抑制凝血酶诱导的单核细胞与血管内皮细胞的粘附。这些研究结果提示SRH蛋白可以通过抑制凝血酶对内皮细胞的作用发挥其抗炎功能,进一步抑制动脉粥样硬化。
四由于SRH蛋白在SAK基础上融合了其它分子,这种变化可能会造成在体内代谢的变化。为了初步检测SRH蛋白在体内的代谢情况并与SAK进行比较,我们制备了SRH的兔源及鼠源多克隆抗体,建立了双抗夹心ELISA方法检测SRH及SAK含量的方法,并使用该方法检测了动物体内SRH蛋白及SAK蛋白的代谢情况,结果显示这两种蛋白在动物体内代谢存在一定的差异,SRH半衰期有延长的趋势,为进一步研究SRH蛋白的药学功能奠定了基础。
总之,我们针对AS发生和发展的特点构建了多功能融合蛋白SRH,以实现多靶点治疗AS。在本研究中我们完成了SRH的构建表达和纯化,对各功能域的体外活性检测;在AS动物模型中评价了SRH的对AS治疗作用,并初步探讨了其作用机制;建立了SRH含量检测方法,并在动物体内初步检测了SRH的代谢情况。这些工作为SRH的成药性做了初步评价,也为新型抗AS药物的研制奠定基础。
Atherosclerosis (AS) is the common pathological basis of many cardio and cerebral vascular diseases, and also one of the most frequent disease with serious threat to human health. Although many researchers proposed different theories about AS pathogenesis however, anyone of which could not alone elucidate the genesis and development of AS completely. In the recent years, lots of experimental researches at cellular and molecular levels indicated that AS resulted in a similar development pathway, i.e. chronic inflammatory pathological process. The development of AS was accompanied by some similar pathophysiological process, including hemoendothelial damage, oxidation and infiltration of lipid, the activation, adhension and infiltration of platelet and inflammatory cells, the immigration and proliferation of smooth muscle cells etc.. On the basis of the AS development characteristics, we consider whether the multi-targeted drug could be constructed and applied, and then the effect against AS could be better.
Studies displayed that inflammation penetrated through all the process of AS genesis and development, which was a common basis of many pathophysiological changes. Thrombin and platelet as the targeting sites for many anti-coagulant drugs were the important factors involved in blood coagulation, while more and more researches indicated that thrombin and platelet accumulated at damaged site of vessel played an important promoting role for the inflammatory response of vessel wall. Because of the hyperfunction of local blood coagulation, a large amount of platelet, leukocyte and thrombin accumulated, the existence of which exacerbated the inflammatory reaction at the damaged blood vessel and then accelerate the development of atherosclerosis, therefore thrombin and platelet simultaneously participated in both blood coagulation and inflammation. In the late period of AS development, the patients were often accompanied by the symptoms of coronary syndrome, to which the anticoagulant and thrombolysis therapies were applied .Therefore, we designed and constructed a multifunctional fusion protein SRH to perform the anti -AS effect through its anticoagulation, anti-inflammation and thrombolysis activity.
Natural staphylokinase (SAK) as a selective fibrinogen activator possessed some advantages, such as higher fibrinogen specificity, stronger action on platelet-enriched thrombus, lower side-effect, and cheaper price. The clinical trial evaluation of our rSAK has been completed, and the results indicated that its recanalization rate was significantly higher than that of r-tPA, thus showing a good perspective of clinical application. We designed a multifunctional fusion protein SRH by using the modified SAK as the backbone. The molecular structure of SRH consisted of three functional domains: the modified SAK molecule, RGD sequence, and dodecapeptide of hirudin. SAK displayed mainly the dissolution of fibrin, RGD sequence possessed inhibition effect on platelet aggregation, and the dodecapeptide of hirudin had anti-thrombin function.
This study mainly includes the following four parts:
1. Overlapping extension PCR methods were used to construct the recombinant gene encoding SRH fusion protein, which was inserted in the expression vector pBV220, and the resulted recombinant plasmid was used to transform the competent cells of E.coli BL21 to obtain an engineering bacteria strain for inducing expression of SRH. The results showed that SRH could be expressed in the soluble form by the engineering strain E.coli BL21/pBV220-SRH, and SRH could be purified after expression in large scale. The purity of SRH reached to 95% after two steps of purification with anion-exchange chromatography and gel filtration. The functions of different domains of the fusion molecule SRH were analyzed by using the lysis circle assay on fibrin agarose gel plate and chromogenic substrate method. The results showed that the thrombolytic activity of SRH was (1.3±0.15)×104 AU/mg, lower than that of SAK((4.1±0.12)×104 AU/mg). Binding ability analysis indicated that SRH was able to bind with thrombin, and further assay with fibrin clotting method showed SRH had stronger anti-coagulation function than that of SAK. Assay of SRH anti-platelet aggregation function displayed that SRH maintained the function of RGD anti-platelet aggregation. The above results demonstrated the fusion protein constructed in our research maintained the functions of three domains, possessing thrombolysis, inhibition of platelet aggregation and anti-thrombin.
2. We utilized apoE-/-mouse as the animal model feeding with high fat diet to observe the progression of atherosclerosis after injection with SRH. H.E. staining of aorta sinus demonstrated the model group showed a serious lesion of AS, and AS plaque occupied 91% of the aorta lumen area. The group treated with SRH displayed different results: 39% of that for SRH 1mg/kg body weight, and 43% for SRH 0.125 mg/kg. These results suggested that the plaque lesion of AS in the SRH-treating group had tendency of attenuation.
3. In order to elucidate the anti-atherosclerosis mechanism of SRH protein, the effect of SRH protein on HUVEC cell was tested. The results showed that SRH protein could down-regulate expression of AS-related genes including PAI, ICAM-1, VCAM-1, IL6, IL8, MCP-1 and TF. SRH also inhibited the secretion of IL6 and MCP-1.These results suggested that SRH protein could carry out its anti-atherosclerosis through the inhibition of thrombin.
4. In order to examine the metabolism of SRH in vivo, polyclonal antibodies to SRH in rabbit and mice were prepared. And we established double antibody-sandwich ELISA to determine the contents of SRH and SAK. The results indicated that half-life of SRH tended to be extended. This laid a foundation for the further exploration of its pharmacy.
引文
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7) Rudel, L.L. J.S. Parks, et al. Low density lipoproteins in atherosclerosis. J. Lipid Res. 1986; 27: 465–474.
8) Hackamd G. Emerging risk factors for atherosclerotic vascular disease. J AMA 2003 ; 290(7) :932-940.
9) Yehuda S,Yaniv S, Dror H. Atheroselerosis as an infections, inflammatory and antoimmune disease. Trends in Immunol 2001; 22: 293-295.
10) Ross R. Atherosclerosis an inflammatory disease. N Engl J Med, 1999; 340 (2): 115-126.
11) Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med, 2005; 352 (16): 1685-1695
12) Kriszbacher I, Koppan M, Bodis J. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med, 2005; 353(4): 429-430.
13) Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor.Circulation, 2004; 109[SuppleⅡ]:Ⅱ-2-Ⅱ-10.
14) Libby P. Inflammation and cardiovascular disease mechanisms. Am J Clin Nutr 2006; 83: 456S–460S.
15) Tabas I. Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. J Clin Invest 2002; 110: 905–911.
16) Hackett D, Davies G, Maseri A. Pre-existing coronary stenoses in patients with first myocardial infarction are not necessarily severe.Eur Heart J 1988; 9: 1317–23.
17) Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001; 104: 365–72.
18) Amento EP, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb 1991; 11: 1223–30.
19) Sukhova GK, Schonbeck U, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatousplaques. Circulation 1999; 99: 2503–9.
20) Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994; 94: 2493–503.
21) Gough PJ, Gomez IG, Wille PT. Raines EW. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest 2006; 116: 59–69.
22) Gold HK. Conjunctive antithrombotic and thrombolytic therapy for coronaryartery occlusion. N Engl J Med 1990; 323: 1483-1485
23)王敏,王晓军,崔连群凝血酶与血管内皮细胞.细胞生物学杂志2005;27:s57-60
24)陈荣琳,钱传云,凝血酶及其受体与动脉粥样硬化医学综述,2005;11(10)870-871
25) Steinberg SF. The cardiovascular actions of protease-activated receptors. Mol Pharmacol 2005; 67:2–11.
26) Coughlin SR. Thrombin signalling and protease-activated receptors.Nature. 2000; 407: 258–264.
27) Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functionalthrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057–1068.
28) Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci U S A. 1994; 91:9208–9212.
29) Ishihara H, Connolly AJ, Zeng D, et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997; 386: 502–506.
30) Kahn ML, Zheng YW, et al. A dual thrombin receptor system for platelet activation. Nature. 1998; 394: 690–694.
31) Coughlin SR. Thrombin receptor structure and function Thromb Haemost, 1993; 70(1): 184-187.
32) Hirano K.The roles of PARS in the vascular physiology and pathophysiology. Arteriscler Thromb Vasc Biol 2007; 27: 27-36
33) Kahn Ml, Zheng YW, et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-694
34) Andrews RK. The glycoproteinⅠb-Ⅸ-Ⅴcomplex in platelet adhesion and signaling. Thromb Haemost 1999; 82: 367-364
35) Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18s-25s
36) Rabiet MJ et al. Arterioscler Thromb Vasc Biol, 1996; 16: 488
37) McLaughlin JN, Mazzoni MR, Cleator JH, et al Thrombinmodulates the expression of a set of genes including thrombospondin-1 in human microvascular endothelial cells. J Biol Chem. 2005; 280: 22172–22180.
38) Jorgensen L. The role of platelets in the initial stages of atherosclerosis.J Thromb Haemost 2006; 4: 1443–9.
39) Nieswandt B, Aktas B, Moers A, Sachs UJ. Platelets in atherothrombosis: lessons from mouse models. J Thromb Haemost 2005; 3: 1725–36.
40) David Varga-Szabo, Irina Pleines, Bernhard Nieswandt. Cell Adhesion Mechanisms in Platelets Arterioscler. Arterioscler Thromb Vasc Biol 2008; 28: 403-412;
41) Philipp von Hundelshausen, Christian Weber. Platelets as Immune Cells Bridging Inflammation and Cardiovascular Disease Circ. Res. 2007; 100: 27-40
42) M. Gawaz. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium, Cardiovasc. Res. 2004; 61: 498–511.
43) Jorgensen L. ADP-induced platelet aggregation in the microcirculation of pig myocardium and rabbit kidneys. J Thromb Haemost 2005; 3: 1119–1124.
44) Theilmeier G, Michiels C, Spaepen E, et al. Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia. Blood 2002; 99: 4486–4493.
45) Bombeli T, Schwartz BR, Harlan JM. Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha. J Exp Med 1998; 187: 329–339.29.
46) Gawaz M, Neumann FJ, Dickfeld T, et al. Vitronectin receptor (alpha(v)beta3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation 1997; 96: 1809–1818.
47) Frenette PS, Johnson RC, Hynes RO, et al. Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA 1995; 92: 7450–7454.
48) Massberg S, Enders G, Leiderer R, et al. Plateletendothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood 1998; 92: 507–515.
49) Frenette PS, Denis CV, Weiss L, et al. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med 2000; 191: 1413–1422.
50) Johnson RC, Mayadas TN, Frenette PS, et al. Blood cell dynamics in P-selectin deficient mice. Blood 1995; 86: 1106–1114.
51) Subramaniam M, Frenette PS, Saffaripour S, et al. Defects in hemostasis in P-selectin-deficient mice. Blood 1996; 87: 1238–1242.
52) Frenette PS, Moyna C, Hartwell DW, et al. Plateletendothelial interactions in inflamed mesenteric venules. Blood 1998; 87: 1238-1324.
53) Gawaz MP, Loftus JC, Bajt ML, et al. Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIbIIIa) dependent homotypic and heterotypic cell-cellinteractions. J Clin Invest 1991; 88: 1128–1134.
54) Massberg S, Enders G, Matos FC, et al. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood 1999; 94: 3829–3838.
55) Massberg S, Schurzinger K, Lorenz M, et al. Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb. Circulation 2005; 112: 1180–1188.
56) Kleinschnitz C, Pozgajova M, Pham M, et al. Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarctsize, functional outcome, and intracranial bleeding. Circulation 2007; 115: 2323–2330.
57) Gershlick AH. Antiplatelet therapy. Hosp Med. 2000 Jan; 61(1): 15-23
58) Rydel, T. J., Ravichandran, K. G., Tulinsky, A., et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science, 1990; 249: 277-280.
59) Vu, T.-K. H., Hung, D. T., Wheaton, V. I., Coughlin, S. R. . Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell, 1991; 64: 1057-1068.
60) Vu, T.-K. H., Wheaton, V. I., Hung, D. T. and Coughlin, S. R.. Domains specifying thrombin-receptor interaction. Nature,1991; 353: 674-677.
61) Bagdy D, Barabas E, Gráf L, Petersen TE, Magnusson S, Hirudin. Methods Enzymol, 1976; 45: 669-678
62) Maraganore JM, Chao B, Joseph ML, et al, Anticoagulant activity of synthetic hirudin peptides. J Biol Chem 1978; 264: 8692-8698
63) Lefkovits J, Topol E J. Platelet glycoproteinⅡb /Ⅲa receptor and tagonists in coronary artery disease Eur Heart J, 1996; 17(1): 9-18.
64) Lijnen HR, Van HB, Vandenbossche L,et al. Biochemical properties of natural and recombinant staphylokinase. Fibrinolysis, 1992, 6: 214-225.
65) Collen D,Franks DC,Jean S, et al. Comparative immunogenicity and throbolytic properties toward arterial and venous thrombi of streptokinase and recombinant staphylokinase in baboons.Circulation, 1993; 87: 996-1006.
66) Vanderschueren S, Dens J, Kerdsinchia P, et al.Randeomized coronary potency trail of double-bolusrecombinant staphylokinase verus front-loaded alteplase in acute myocardial infarction.Am Heart J, 1997; 134: 213-219.
67) Vanderschueren S, Vlaendern I,Collen D,A randomized trail of recombinant staphylokinase versus alteplase for coronary artery patency inacute myocardial infarction.Circulation,1995; 92: 2044-2049.
68)王旻,王圆圆,邹民吉,徐涛,刘深,吴琛,王嘉玺,徐东刚新型低抗原性葡激酶的构建表达及功能分析军事医学科学院院刊2007; 31: 118-121
69) Saksela O. Radial caseinolysis in agarose: a simple method for detection of plasminogen activator in the presence of inhibitory substances and serum.Anal Biochem, 1981; 111: 276-282.
70) DiMaio J, Gibbs B, Munn D, et al. Bifunctional thrombin inhibtors based on the sequence of hirudin. J Biol Chem 1990; 265: 21698-21703
71) Collen D,Van Hoef B,Schlott B. Of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase. Eur J Biochem, 1993; 15; 216(1) :307
72) Collen D, Schlott B, Engelborghs Y J. On the mechanism of the activation of human plasminogen by recombinant staphylokinase. J Biol Chem,1993; 268 (11): 8284
73) Jespers L, Vanwetswinkel S, Lijnen HR, Structural and functional basis of plasmingen activation by staphylokinase. Thromb Hoaemost, 2002; 87: 666-673
74) Ken-ichi A, Hiroyuki A,et al.Heparin cofactorⅡas a novel vascular protective factor against atherosclerosis.J Atheroscler Thromb,2009; 16: 523-531
75) Paigen B,Morrow A,et al. Quantitative assessment of atherosclerotic lesions in mice.Atherosclerosis, 1987; 68: 231-240
76) Gamble JR, Vadas MA. A new assay for the measurement of the attachment of neutrophils and other cell types to endothelial cells. J Immunol Methods, 1988, 109: 175-184
77) Rene′R. S. Packard and Peter Libby Inflammation in Atherosclerosis: FromVascular Biology to Biomarker Discovery and Risk Prediction. Clinical Chemistry 2008; 54:124–38
78)化学药物非临床药代动力学研究技术指导原则(编号GTP5-1),2005年3月
79) Sophie Vanwetswinkel, Stephane Plaisance,et al.Pharmacokinetic and thrombolytic properties of cysteine-linked polyethylene glycol derivatives of staphylokinase. Blood 2000; 95: 936-942
80) DésiréCollen, Peter Sinnaeve, et al. Polyethylene Glycol–Derivatized Cysteine-Substitution Variants of Recombinant Staphylokinase for Single-Bolus Treatment of Acute Myocardial Infarction Circulation 2000; 102; 1766-1772
1) Vu T-HK, Hung DT, et al. Molecular of functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.Cell 1991; 64: 1057-1068
2) Ishihare H,Connllly AJ,et al. PAR3 is a second thrombin receptor in humans.Nature 1997; 386: 502-506
3) O Brien PJ. Thrombin responses in human endothelial cells.Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1.J Biol Chem 2000; 275: 13502-13509
4) Hirano K. The roles of PARS in the vascular physiology and pathophysiology. Arteriscler Thromb Vasc Biol 2007; 27: 27-36
5) Coughlin SR.Thrombin signaling and protease-activated receptors.Nature 2000;407:258-264
6) Coughlin SR. Protease-activated receptors in haemostasis,thrombosis and vascular biology. J Thromb Haemost 2005; 3: 1800-1814
7) Macfarlane SR, Seatter MJ, et al. Protease-activated receptors. Pharmacolev 2001; 53: 245-282
8) Treio J,Altschuler Y,et al. Protease-activaated receptor-1 down regulation:a mutant Hela cell line suggests novelrequirements for PAR1 phophorylation and recruitment to clathrin-coated pits. J Biol Chem 2000; 275: 1255-31265
9) Hein L,Ishii K, et al. Intracellular targeting and trafficking of thrombin receptors.J Biol Chem 1994; 269: 27719-27726
10) Yufu T,Hirano K,et al.Rac1regulation of surface expression of Protease-activated receptor-1 and responsiveness to thrombin in vascular smooth muscle cells.Arterioscler Thromb VascBiol 2005; 25: 1506-1511
11) Kahn Ml,Zheng YW,et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-694
12) Shapiro MJ, Weiss EJ, et al. Protease-activated receptor-1 and Protease-activated receptor-4 are shut off with distinct kinetics after activation by thrombin. J Biol Chem 2000; 275: 25216-25221
13) Andrews RK, The glycoproteinⅠb-Ⅸ-Ⅴcomplex in platelet adhesion and signaling. Thromb Haemost 1999; 82: 367-364
14) Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18s-25s
15) Weiss EJ, et al. Protection against thrombosis in mice lacking PAR3. Blood 202; 100: 3240-3244
16) Lin Z et al. Kruppel-like factor 2 inhibits protease activated receptor-1 expression and thrombin-mediated endothelial activation. Arterioscler Thromb Vasc Biol 2006;26:1185-1189
17) Mizuno O et al. Mechanism of endothelium-dependent relaxation induced by thrombin in the pig coronary artery. Eur J Pharmacol 1998; 351: 67-77
18) Nobe K,et al. Thrombin-induced force development in vascular endothelial cells.J Pharmacol Sci 2005; 99: 252-263
19) Ku DD,et al. Expression of thrombin receptors in human atherosclerotic coronary arteries leads to an exaggerated vasoconstrictory response in vivo. J Cardiovasc Pharmacol 1997; 30: 649-657
20) Bretschneider E, et al. Evidence for PAR4 in human vascular smooth muscle cells.Br J Pharmacol 2001; 132: 1441-1446
2) Fry, D.L. Acute vascular endothelial changes associated with increased blood velocity gradients. Circ. Res 1968; 22: 165–197.
3) Fry, D.L. Certain histological and chemical responses of the vascular interface to acutely induced mechanical stress in the aorta of the dog. Circ. Res. 1969; 24: 93–108.
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6) Musliner, T.A. C. Giotas ,R.M. Krauss. Presence of multiple subpopulations of lipoproteins of intermediate density in normal subjects. Arteriosclerosis 1986; 6: 79–87.
7) Rudel, L.L. J.S. Parks, et al. Low density lipoproteins in atherosclerosis. J. Lipid Res. 1986; 27: 465–474.
8) Hackamd G. Emerging risk factors for atherosclerotic vascular disease. J AMA 2003 ; 290(7) :932-940.
9) Yehuda S,Yaniv S, Dror H. Atheroselerosis as an infections, inflammatory and antoimmune disease. Trends in Immunol 2001; 22: 293-295.
10) Ross R. Atherosclerosis an inflammatory disease. N Engl J Med, 1999; 340 (2): 115-126.
11) Hansson GK. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med, 2005; 352 (16): 1685-1695
12) Kriszbacher I, Koppan M, Bodis J. Inflammation, atherosclerosis, and coronary artery disease. N Engl J Med, 2005; 353(4): 429-430.
13) Willerson JT, Ridker PM. Inflammation as a cardiovascular risk factor.Circulation, 2004; 109[SuppleⅡ]:Ⅱ-2-Ⅱ-10.
14) Libby P. Inflammation and cardiovascular disease mechanisms. Am J Clin Nutr 2006; 83: 456S–460S.
15) Tabas I. Consequences of cellular cholesterol accumulation: basic concepts and physiological implications. J Clin Invest 2002; 110: 905–911.
16) Hackett D, Davies G, Maseri A. Pre-existing coronary stenoses in patients with first myocardial infarction are not necessarily severe.Eur Heart J 1988; 9: 1317–23.
17) Libby P. Current concepts of the pathogenesis of the acute coronary syndromes. Circulation 2001; 104: 365–72.
18) Amento EP, Ehsani N, Palmer H, Libby P. Cytokines and growth factors positively and negatively regulate interstitial collagen gene expression in human vascular smooth muscle cells. Arterioscler Thromb 1991; 11: 1223–30.
19) Sukhova GK, Schonbeck U, et al. Evidence for increased collagenolysis by interstitial collagenases-1 and -3 in vulnerable human atheromatousplaques. Circulation 1999; 99: 2503–9.
20) Galis ZS, Sukhova GK, Lark MW, Libby P. Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. J Clin Invest 1994; 94: 2493–503.
21) Gough PJ, Gomez IG, Wille PT. Raines EW. Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice. J Clin Invest 2006; 116: 59–69.
22) Gold HK. Conjunctive antithrombotic and thrombolytic therapy for coronaryartery occlusion. N Engl J Med 1990; 323: 1483-1485
23)王敏,王晓军,崔连群凝血酶与血管内皮细胞.细胞生物学杂志2005;27:s57-60
24)陈荣琳,钱传云,凝血酶及其受体与动脉粥样硬化医学综述,2005;11(10)870-871
25) Steinberg SF. The cardiovascular actions of protease-activated receptors. Mol Pharmacol 2005; 67:2–11.
26) Coughlin SR. Thrombin signalling and protease-activated receptors.Nature. 2000; 407: 258–264.
27) Vu TK, Hung DT, Wheaton VI, Coughlin SR. Molecular cloning of a functionalthrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991; 64: 1057–1068.
28) Nystedt S, Emilsson K, Wahlestedt C, Sundelin J. Molecular cloning of a potential proteinase activated receptor. Proc Natl Acad Sci U S A. 1994; 91:9208–9212.
29) Ishihara H, Connolly AJ, Zeng D, et al. Protease-activated receptor 3 is a second thrombin receptor in humans. Nature. 1997; 386: 502–506.
30) Kahn ML, Zheng YW, et al. A dual thrombin receptor system for platelet activation. Nature. 1998; 394: 690–694.
31) Coughlin SR. Thrombin receptor structure and function Thromb Haemost, 1993; 70(1): 184-187.
32) Hirano K.The roles of PARS in the vascular physiology and pathophysiology. Arteriscler Thromb Vasc Biol 2007; 27: 27-36
33) Kahn Ml, Zheng YW, et al. A dual thrombin receptor system for platelet activation. Nature 1998; 394: 690-694
34) Andrews RK. The glycoproteinⅠb-Ⅸ-Ⅴcomplex in platelet adhesion and signaling. Thromb Haemost 1999; 82: 367-364
35) Brass LF. Thrombin and platelet activation. Chest 2003; 124: 18s-25s
36) Rabiet MJ et al. Arterioscler Thromb Vasc Biol, 1996; 16: 488
37) McLaughlin JN, Mazzoni MR, Cleator JH, et al Thrombinmodulates the expression of a set of genes including thrombospondin-1 in human microvascular endothelial cells. J Biol Chem. 2005; 280: 22172–22180.
38) Jorgensen L. The role of platelets in the initial stages of atherosclerosis.J Thromb Haemost 2006; 4: 1443–9.
39) Nieswandt B, Aktas B, Moers A, Sachs UJ. Platelets in atherothrombosis: lessons from mouse models. J Thromb Haemost 2005; 3: 1725–36.
40) David Varga-Szabo, Irina Pleines, Bernhard Nieswandt. Cell Adhesion Mechanisms in Platelets Arterioscler. Arterioscler Thromb Vasc Biol 2008; 28: 403-412;
41) Philipp von Hundelshausen, Christian Weber. Platelets as Immune Cells Bridging Inflammation and Cardiovascular Disease Circ. Res. 2007; 100: 27-40
42) M. Gawaz. Role of platelets in coronary thrombosis and reperfusion of ischemic myocardium, Cardiovasc. Res. 2004; 61: 498–511.
43) Jorgensen L. ADP-induced platelet aggregation in the microcirculation of pig myocardium and rabbit kidneys. J Thromb Haemost 2005; 3: 1119–1124.
44) Theilmeier G, Michiels C, Spaepen E, et al. Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia. Blood 2002; 99: 4486–4493.
45) Bombeli T, Schwartz BR, Harlan JM. Adhesion of activated platelets to endothelial cells: evidence for a GPIIbIIIa-dependent bridging mechanism and novel roles for endothelial intercellular adhesion molecule 1 (ICAM-1), alphavbeta3 integrin, and GPIbalpha. J Exp Med 1998; 187: 329–339.29.
46) Gawaz M, Neumann FJ, Dickfeld T, et al. Vitronectin receptor (alpha(v)beta3) mediates platelet adhesion to the luminal aspect of endothelial cells: implications for reperfusion in acute myocardial infarction. Circulation 1997; 96: 1809–1818.
47) Frenette PS, Johnson RC, Hynes RO, et al. Platelets roll on stimulated endothelium in vivo: an interaction mediated by endothelial P-selectin. Proc Natl Acad Sci USA 1995; 92: 7450–7454.
48) Massberg S, Enders G, Leiderer R, et al. Plateletendothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood 1998; 92: 507–515.
49) Frenette PS, Denis CV, Weiss L, et al. P-Selectin glycoprotein ligand 1 (PSGL-1) is expressed on platelets and can mediate platelet-endothelial interactions in vivo. J Exp Med 2000; 191: 1413–1422.
50) Johnson RC, Mayadas TN, Frenette PS, et al. Blood cell dynamics in P-selectin deficient mice. Blood 1995; 86: 1106–1114.
51) Subramaniam M, Frenette PS, Saffaripour S, et al. Defects in hemostasis in P-selectin-deficient mice. Blood 1996; 87: 1238–1242.
52) Frenette PS, Moyna C, Hartwell DW, et al. Plateletendothelial interactions in inflamed mesenteric venules. Blood 1998; 87: 1238-1324.
53) Gawaz MP, Loftus JC, Bajt ML, et al. Ligand bridging mediates integrin alpha IIb beta 3 (platelet GPIIbIIIa) dependent homotypic and heterotypic cell-cellinteractions. J Clin Invest 1991; 88: 1128–1134.
54) Massberg S, Enders G, Matos FC, et al. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood 1999; 94: 3829–3838.
55) Massberg S, Schurzinger K, Lorenz M, et al. Platelet adhesion via glycoprotein IIb integrin is critical for atheroprogression and focal cerebral ischemia: an in vivo study in mice lacking glycoprotein IIb. Circulation 2005; 112: 1180–1188.
56) Kleinschnitz C, Pozgajova M, Pham M, et al. Targeting platelets in acute experimental stroke: impact of glycoprotein Ib, VI, and IIb/IIIa blockade on infarctsize, functional outcome, and intracranial bleeding. Circulation 2007; 115: 2323–2330.
57) Gershlick AH. Antiplatelet therapy. Hosp Med. 2000 Jan; 61(1): 15-23
58) Rydel, T. J., Ravichandran, K. G., Tulinsky, A., et al. The structure of a complex of recombinant hirudin and human alpha-thrombin. Science, 1990; 249: 277-280.
59) Vu, T.-K. H., Hung, D. T., Wheaton, V. I., Coughlin, S. R. . Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell, 1991; 64: 1057-1068.
60) Vu, T.-K. H., Wheaton, V. I., Hung, D. T. and Coughlin, S. R.. Domains specifying thrombin-receptor interaction. Nature,1991; 353: 674-677.
61) Bagdy D, Barabas E, Gráf L, Petersen TE, Magnusson S, Hirudin. Methods Enzymol, 1976; 45: 669-678
62) Maraganore JM, Chao B, Joseph ML, et al, Anticoagulant activity of synthetic hirudin peptides. J Biol Chem 1978; 264: 8692-8698
63) Lefkovits J, Topol E J. Platelet glycoproteinⅡb /Ⅲa receptor and tagonists in coronary artery disease Eur Heart J, 1996; 17(1): 9-18.
64) Lijnen HR, Van HB, Vandenbossche L,et al. Biochemical properties of natural and recombinant staphylokinase. Fibrinolysis, 1992, 6: 214-225.
65) Collen D,Franks DC,Jean S, et al. Comparative immunogenicity and throbolytic properties toward arterial and venous thrombi of streptokinase and recombinant staphylokinase in baboons.Circulation, 1993; 87: 996-1006.
66) Vanderschueren S, Dens J, Kerdsinchia P, et al.Randeomized coronary potency trail of double-bolusrecombinant staphylokinase verus front-loaded alteplase in acute myocardial infarction.Am Heart J, 1997; 134: 213-219.
67) Vanderschueren S, Vlaendern I,Collen D,A randomized trail of recombinant staphylokinase versus alteplase for coronary artery patency inacute myocardial infarction.Circulation,1995; 92: 2044-2049.
68)王旻,王圆圆,邹民吉,徐涛,刘深,吴琛,王嘉玺,徐东刚新型低抗原性葡激酶的构建表达及功能分析军事医学科学院院刊2007; 31: 118-121
69) Saksela O. Radial caseinolysis in agarose: a simple method for detection of plasminogen activator in the presence of inhibitory substances and serum.Anal Biochem, 1981; 111: 276-282.
70) DiMaio J, Gibbs B, Munn D, et al. Bifunctional thrombin inhibtors based on the sequence of hirudin. J Biol Chem 1990; 265: 21698-21703
71) Collen D,Van Hoef B,Schlott B. Of mammalian plasma fibrinolytic systems with streptokinase and with recombinant staphylokinase. Eur J Biochem, 1993; 15; 216(1) :307
72) Collen D, Schlott B, Engelborghs Y J. On the mechanism of the activation of human plasminogen by recombinant staphylokinase. J Biol Chem,1993; 268 (11): 8284
73) Jespers L, Vanwetswinkel S, Lijnen HR, Structural and functional basis of plasmingen activation by staphylokinase. Thromb Hoaemost, 2002; 87: 666-673
74) Ken-ichi A, Hiroyuki A,et al.Heparin cofactorⅡas a novel vascular protective factor against atherosclerosis.J Atheroscler Thromb,2009; 16: 523-531
75) Paigen B,Morrow A,et al. Quantitative assessment of atherosclerotic lesions in mice.Atherosclerosis, 1987; 68: 231-240
76) Gamble JR, Vadas MA. A new assay for the measurement of the attachment of neutrophils and other cell types to endothelial cells. J Immunol Methods, 1988, 109: 175-184
77) Rene′R. S. Packard and Peter Libby Inflammation in Atherosclerosis: FromVascular Biology to Biomarker Discovery and Risk Prediction. Clinical Chemistry 2008; 54:124–38
78)化学药物非临床药代动力学研究技术指导原则(编号GTP5-1),2005年3月
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