Staphylococcus aureus,master manipulator of the human hemostatic system

Summary

The coagulation system does not only offer protection against bleeding, but also aids in our defense against invading microorganisms. The hemostatic system and innate immunity are strongly entangled, which explains why so many infections are complicated by either bleeding or thrombosis. Staphylococcus aureus (S. aureus), currently the most deadly infectious agent in the developed world, causes devastating intravascular infections such as sepsis and infective endocarditis. During these infections S. aureus comes in close contact with the host hemostatic system and proves to be a master in manipulating coagulation. The coagulases of S. aureus directly induce coagulation by activating prothrombin. S. aureus also manipulates fibrinolysis by triggering plasminogen activation via staphylokinase. Furthermore, S. aureus binds and activates platelets and interacts with key coagulation proteins such as fibrin(ogen), fibronectin and von Willebrand factor. By manipulating the coagulation system S. aureus gains a significant advantage over the host defense mechanisms. Studying the interplay between S. aureus and the hemostatic system can therefore lead to new innovative therapies for battling S. aureus infections.

     

葡激酶研究进展

葡激酶是由金黄色葡萄球菌分泌的新一代天然的纤溶酶原激活剂,具有高效的溶栓活性和纤维蛋白特异性.进入机体后引起的免疫反应是限制其临床使用的主要原因,近年来生物工程技术的发展为解决这些问题提供了新的重要途径.现将从结构特点、溶栓机制、免疫原性及分子结构改造等方面重点介绍葡激酶的研究进展.     

单次静滴重组葡激酶在健康人体的药代动力学

目的研究重组葡激酶（r-SAK）（抗急性心肌梗死药）单次静脉给药后在健康人体内的药代动力学。方法24例健康受试者随机分为3组（5，10，15mg剂量组），建立双抗体酶联免疫吸附法（ELISA），测定r-SAK的血药浓度，用3P97药代动力学软件进行数据处理。结果r-SAK符合二房室一级消除模型，％为6．68-23．19L，AUC0-t为1．39-3．59μg·h·mL^-1,为1．35～4．37h。结论建议临床给药方法为r-SAK10mg、30min内静脉输注。     

微球的制备和表征

目的制备葡激酶突变体(K35R，DGR)的聚乳酸-羟基乙酸(PLGA)微球，使其在包封和释放过程中都能保持活性。方法使用复乳溶剂挥发法制备DGR的PLGA微球，研究了搅拌速度、PLGA浓度、内水相和外水相中的添加剂对蛋白包封率以及微球性质的影响，并进行了DGR微球的体外和体内释放试验。结果2%聚乙烯醇可以有效抑制超声乳化时DGR在水/二氯甲烷界面上的变性，将DGR的活性回收率从16%提高到几乎100%。在外水相中加入NaCl可以显著提高蛋白包封率，同时对微球的粒径分布和表面形态也产生了重要影响。DGR微球的体外释放呈现两个时相，15 d释放大约DGR总活性的50%。DGR微球在体内持续释放5 d。结论制备的PLGA微球，DGR包封率高，稳定性较好，是DGR的良好载药系统。     

Preformulation development of recombinant pegylated staphylokinase SY161 using statistical design

The goal of this study was to perform preformulation development of SY161 by using statistical design methods to understand the effects of buffer strength, NaCl concentration, and pH on conformation and stability of the protein. It was also important to elucidate interactions between these factors. A central composite design using a 2-level full-factorial study was performed. Secondary structure was evaluated using circular dichroism. Stability toward unfolding was investigated using high-sensitivity differential scanning calorimetry. Depegylation, aggregation, and protein loss were evaluated using SEC-HPLC with on-line light scattering, at time zero and after a 2-week stability study. Response surface plots clearly show optimal pH, NaCl, and buffer conditions. Interactions between pH and NaCl as well as pH and buffer concentration are observed. T_m is seen to be predictive of SY161 stability. Secondary structure changes were minimal and did not influence stability. Statistical design was very effective in providing an understanding of the effects of the formulation components on SY161 stability.     

冻干重组葡激酶对正常人凝血、纤溶系统影响的研究

目的　探讨冻干重组葡激酶 (r Sak)对正常人出、凝血及纤溶系统的影响 ,为进一步临床应用提供翔实的依据。方法　健康志愿者 2 0例静脉注射不同剂量r Sak(1、2 5、5、10、15mg) ,观察临床出血情况以及动态监测用药前后BT、BPC、APTT、PT、TT、Fg、D D、PL∶A、α2 PI∶A。结果　 2 0例中 4例有轻微出血 ,以皮肤粘膜为主 ,可自行止血 ,无 1例伴内脏出血。其中 3例牙龈渗血 ,2例穿刺部位出血。 4例有D D轻微异常 ,其余以上指标皆无变化。实验显示本药为高度选择性溶栓药 ,对正常人出凝血相无改变。结论　在本试验剂量范围内 ,该药是相对安全的 ,可很好耐受 ,但用于临床的最佳剂量有待进一步临床验证     

基因重组葡激酶和重组链激酶在猪急性脑栓塞模型中溶栓作用的比较研究

目的：比较局部动脉内灌注重组葡激酶和重组链激酶对脑血栓的溶化效果及对出凝血系统的影响。方法：制成１８只幼猪急性血栓性脑栓塞模型,４小时后分３组,２个实验组自颈内动脉灌注重组葡激酶（０．２ｍｇ／ｋｇ）或重组链激酶（３×１０４Ｕ／ｋｇ）,对照组自颈内动脉灌注５％葡萄糖;脑血管造影观察血栓是否溶化;并测定凝血系统指标。结果：２个实验组在用药后１．５小时血管再通均高于对照组,葡激酶组在０．５和１．０小时的再通率高于链激酶组（Ｐ均＜０．０５）。葡激酶组对纤维蛋白原、纤溶酶原影响小,凝血酶原时间、凝血酶时间及部分凝血活酶时间延长的幅度小于链激酶组。结论：重组葡激酶及链激酶均能溶化脑血栓,但前者溶栓效果较好,且对凝血功能影响较小     

葡激酶基因的分离及其在大肠杆菌中的高效表达

分离葡激酶基因并使其在Ｅ．ｃｏｌｉ中高效表达。方法：利用ＰＣＲ技术自溶源性金黄色葡萄球菌ＦＲ６１０株的染色体ＤＮＡ中分离葡激酶编码序，ＤＮＡ序列分析后，组入高效表害载体ｐＢＶ２２０中，转化至大肠杆菌。     

重组葡激酶对重症急性胰腺炎大鼠心肌损伤的治疗作用

[目的]研究重组葡激酶(r-Sak)对大鼠重症急性胰腺炎(SAP)的发展及心肌损伤的干预作用,探讨其作用机制.[方法]63只SD大鼠随机分为假手术(A)组(n=9),模型对照(B)组(n=27),r-Sak治疗(C)组(n=27).SAP模型采用5%牛磺胆酸钠胰胆管逆行注射方法建立.ELISA法测定6、12、18 h各时点血清肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、肌酸激酶同工酶(CK-MB)及心肌肌钙蛋白(CTn1)水平,免疫组化法检测术后6、12、18 h各时点心肌核转录因子-κB(NF-κB)的活化情况,光镜及电镜下观察胰腺及心肌组织的病理变化.[结果]大鼠从术后6 h开始出现CTn1、CK-MB升高,治疗组各时点较对照组明显下降(P＜0.05).IL-6、TNF-α水平亦从术后6 h开始升高,其变化在时点上与CTn1、CK-MB的变化具有一致性,治疗组各时点与对照组相比明显下降(P＜0.05).治疗组胰腺及心肌组织的出血坏死减轻,微血管血栓的形成减少.[结论]r-Sak能减轻SAP的病变程度和心肌损伤,可能具有治疗SAP及预防心肌损伤等并发症的作用,特异性溶栓剂的应用可能成为治疗SAP的新途径.     

Staphylokinase reduces plasmin formation by endogenous plasminogen activators

Hyperfibrinolysis is a consequence of imbalance between fibrinolytic activators and their inhibitors. Increased levels of circulating plasminogen (Plg) activators such as tissue- or urokinase-type plasminogen activators (tPA or uPA respectively) are the most common causes of hyperfibrinolysis, occasionally causing major hemorrhages. We found that staphylokinase (SAK), a well-known Plg activator of bacterial origin, inhibits Plg activation mediated by endogenous tPA and uPA. Furthermore, mixture of SAK with tPA led to a significantly reduced Plg-dependent fibrinolysis. This inhibitory effect was exerted through direct action of SAK on Plg rather than indirectly on tPA or uPA. Inhibition of Plg activation by SAK is readily abrogated by interaction of SAK with human neutrophil peptides (HNPs). Finally, we show that NH2-terminal residues of SAK are important for the inhibitory effect of SAK on tPA- and uPA-mediated Plg activation. In conclusion, SAK reduces tPA/uPA-mediated Plg activation by means of SAK.Plg complex formation, consequently downregulating tPA/uPA-induced fibrinolysis.