Circadian Variation of Fibrinolytic Activity in Blood

Approximately 35 years ago, it was discovered that spontaneous fibrinolytic activity in blood showed a sinusoidal variation with a period of 24 h; it increased severalfold during the day, reaching a peak at 6:OO p.m. and then dropped to trough levels at 3:00–4:00 a.m. The range of the fluctuation and the 24-h mean levels were highly reproducible within an individual; moreover, the timing of the oscillation was remarkably consistent among individuals, with a fixed phase relationship to external clock time. The biorhythm could not be accounted for simply by variations in physical activity, body posture, or sleepfwake schedule. Gender, ethnic origin, meals, or resting levels of blood fibrinolytic activity also did not influence the basic features of the rhythm. Older subjects, compared to younger ones, showed a blunted diurnal increase in fibrinolytic activity in blood. Recent studies have established that, of the known components of the fibrinolytic system, only tissue-type plasminogen activator (tPA) and its fast-acting inhibitor, plasminogen activator inhibitor- 1 (PAL l), show a marked circadian variation in plasma. In contrast, levels of plasminogen, α₂-antiplasmin, urinarytype plasminogen activator, and a reversible tPA inhibitor vary little or none during the 24 h. Quenching antibodies to tPA have shown that the circadian rhythm of fibrinolytic activity in blood is due exclusively to changes in tPA activity. However, the 24-h fluctuation of plasma tPA activity is phase shifted in relation to the rhythm of immunoreactive tPA, but shows a precise phase inversion with respect to the 24-h variation of PAL 1 activity and antigen. Therefore, plasma tPA activity, as currently measured in vitro, is tightly and inversely related to the levels of PAL 1 throughout the 24-h cycle. The factors controlling the rhythmicity of plasma PAI-1 are not fully elucidated but probably involve a humoral mechanism; changes in endothelial function, circulating platelet release. products, corticosteroids, catecholamines, insulin, activated protein C, or hepatic clearance do not appear to be responsible. Shift workers on weekly shift rotations show a disrupted 24-h rhythm of plasma tPA and PAL 1. In acute and chronic diseases, the circadian rhythmicity of fibrinolytic activity may show a variety of alterations, affecting the 24-h mean, the amplitude, or the timing of the fluctuation. It is advisable, therefore, to define the 24-h pattern of plasma tPA and PAI- 1 in patient groups, before levels based on a single blood sampling time are compared to those of a control population. In normal conditions, the 24-h variation of plasma tPA and PAI- 1 is not associated with parallel circadian changes in effective fibrinolysis, assessed as plasma D-dimer concentrations, presumably because fibrin generation in the circulation is low. In diseases in which fibrin formation is increased, however, the physiological drop of fibrinolytic activity in the morning hours may favour thrombus development at this time of day, in agreement with the reported higher morning frequency of acute thrombotic events.     

Matrix Metalloproteinases and Cellular Fibrinolytic Activity

Several molecular interactions between the matrix metalloproteinase (MMP) and the plasminogen/plasmin (fibrinolytic) system may affect cellular fibrinolysis. MMP-3 (stromelysin-1) specifically hydrolyzes urokinase (u-PA), yielding a 17 kD NH₂-terminal fragment containing the functionally intact receptor (u-PAR)-binding sequence and a 32 kD COOH-terminal fragment containing the intact serine proteinase domain. MMP-3 generates an angiostatin like fragment (containing kringles 1-4 with the cellular binding domains) from plasminogen. Treatment with MMP-3 of monocytoid THP-1 cells saturated with bound plasminogen, resulted in a dose-dependent reduction of the amount of u-PA-activatible plasminogen. Treatment with MMP-3 of cell-bound u-PA, in contrast, did not alter cell-associated u-PA activity. These data thus indicate that MMP-3 may downregulate cell-associated plasmin activity by decreasing the amount of activatible plasminogen, with out affecting cell-bound u-PA activity. MMP-3 also specifically interacts with the main inhibitors of the fibrinolytic system. Thus, MMP-3 specifically hydrolyzes human ₂-antiplasmin (₂-AP), the main physiological plasmin inhibitor. ₂-AP cleaved by MMP-3 no longer forms a stable complex with plasmin and no longer interacts with plasminogen. Cleavage and inactivation of ₂-AP by MMP-3 may constitute a mechanism favoring local plasmin-mediated proteolysis. Furthermore, MMP-3 specifically hydrolyzes and inactivates human plasminogen activator inhibitor-1 (PAI-1). Stable PAI-1 bound to vitronectin is cleaved and inactivated by MMP-3 in a comparable manner as free PAI-1; the cleaved protein, however, does not bind to vitronectin. Cleavage and inactivation of PAI-1 by MMP-3 may thus constitute a mechanism decreasing the antipro teolytic activity of PAI-1 and impairing the potential inhibitory effect of vitronectin-bound PAI-1 on cell adhesion and/or migration. These molecular interactions of MMP-3 with enzymes, substrates and inhibitors of the fibrinolytic system may thus play a role in the regulation of (cellular) fibrinolysis. Furthermore, the temporal and topographic expression pattern of MMP components, as well as studies in gene-deficient mice, suggest a functional role in neointima formation after vascular injury.     

Effect of Cultural Environment on the Blood Group Activity of Microorganisms

A method for the formation of the trimethylsilyl (TMS) derivatives of the whole-cell hydrolysate of bacteria was developed. The TMS derivatives were examined by gas-liquid chromatography. TMS profiles of various bacteria at the genus and species level were compared. Differences in TMS profiles of Listeria, Neisseria, and Clostridium were significant as were differences between the TMS profiles of C. perfringens and C. sporogenes. Two types of C. perfringens, two serotypes of L. monocytogenes, and one culture of C. sporogenes and N. meningitidis were studied. The possible application of TMS profiles as an aid in differentiating closely related organisms which are troublesome to separate by conventional means is discussed.     

蛇毒纤溶酶的神经生长因子活性

目的证明蛇毒生长因子具有神经生长因子活性,并初步探讨其原因.方法分实验组和对照组,定量和定性测定两组的神经生长因子活性,对比蛇毒纤溶酶和神经生长因子的氨基酸序列.结果蛇毒纤溶酶在20U/ml时具有明显的神经生长因子活性,N-末端氨基酸序列与神经生长因子同源性达80～90%.结论蛇毒纤溶酶具有明显的神经生长因子活性,其结构与神经生长因子相似.     

脱氧核糖核酸(DNA)对过氧化氢酶(CAT)活性的影响研究

机体在新陈代谢过程中,不断产生自由基,自由基对细胞结构损伤较大,机体内存在清除自由基的酶类,正常条件下,自由基的产生和清除是动态平衡的。过氧化氢酶是重要抗自由基酶类之一。试验企图探讨DNA对对氧化氢酶活性影响,从而探索核酸对抗自由基和抗衰老的作用。     

不同化疗方案对非小细胞肺癌患者凝血纤溶功能及血小板参数的影响及其临床意义

目的:探讨不同化疗方案对非小细胞肺癌患者凝血纤溶功能及血小板参数的影响及临床意义。方法:回顾性分析2013年2月至2016年2月于我院进行治疗的96非小细胞肺癌患者的临床资料,按照化疗方案的不同分为TP组(50例)及GP组(46例),TP组患者给予多西他赛联合卡铂方案(TP方案)进行化疗,GP组患者给予吉西他滨联合卡铂方案(GP方案)进行化疗,另选择45例健康体检者作为对照组。比较三组治疗前者血浆凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、血浆凝血酶时间(TT)、血浆纤维蛋白原含量(FIB)以及D-二聚体(D-dimer)、血小板数(PLT)、血小板比积(PCT)、血小板平均体积(MPV)、血小板体积分布宽度(PDW)以及大体积血小板比率(P-LCR)及TP组和GP组治疗后以上指标的差异。结果:TP组和GP组患者化疗前PT、APTT、TT、FIB、D-dimer、PLT、PCT、MPV、PDW以及P-LCR水平均明显高于对照组(P0.05),而TP组与GP组以上指标比较无显著差异(P0.05)。治疗后,TP组和GP组患者PT、APTT、TT、FIB、D-dimer、PLT、PCT、MPV、PDW以及P-LCR水平均较治疗前有所下降,且GP组TT明显长于TP组(P0.05),PLT、PCT明显低于TP组(P0.05)。结论:凝血纤溶功能及血小板参数对于评价不同化疗方案治疗非小细胞肺癌患者预后具有积极的临床价值,不同化疗方案选择对于患者凝血系统均具有一定的影响。     

通过定点突变提高纳豆激酶纤溶活性和热稳定性

纳豆激酶（Nattokinase, NK）是一种纤溶酶，可溶解血栓，常用于治疗心血管疾病（CVDs）。然而，野生型NK往往表现出很少的纤溶能力和较低的热稳定性，针对如何提高NK的热稳定性和活性开展研究。使用重叠延伸PCR法将NK中位于194位的Ser替换为Pro，为验证突变后的热稳定性，将野生型NK和突变体S194P均在不同温度下孵育相同时间，使用纤维蛋白板法测定二者酶活，验证热稳定性。成功构建了pET-26b-NK_S194P，测量酶活结果显示，野生型NK和突变体S194P酶活分别达到101.30 IU/mL和123.23 IU/mL，结果表明突变体S194P的酶活比野生型NK高（18.10±2）%，将野生型NK和突变体S194P在60~65 ℃温度下孵育30 min后测量酶活，野生型NK在63 ℃时丧失酶活，突变体S194P在65 ℃时丧失酶活，耐热温度提高2 ℃。结果表明，突变体S194p的蛋白结构在突变后发生了改变，使NK提高了耐热温度。     

几类天然产物对纤溶系统的作用

在初筛天然来源的小分子ＰＡＩ－１抑制剂过程中发现一些天然产物显示出活性苗头,进一步试验和选择性试验表明这些化合物都不同程度别对纤溶系统中存在的不同的酶具有抑制作用。化合物１（ｅｘｐｏｘｙｒｏｌｉｎ Ｂ）对纤溶酶有一定的抑制,化合物２（ｍｕｒｉｃａｔｉｎｃ）对ＰＡＩ－１具有一定的抑制作用且有较好的选择性。化合物４（ｃｈｅｒｉｍｏｌｉｎ－２）对纤溶酶较强的抑制。化合物５（ｍｏｒｉｎｄｏｎｅ）对凝血酶、     

单环刺螠纤溶酶UFE-Ⅰ的性质和溶栓活性

单环刺螠纤溶酶UFE-Ⅰ的最适反应温度为45 ℃;最适反应pH为7.0;Mg2+、Mn2+和Fe2+是该纤溶酶的强激活剂;Fe3+、Cu2+、Ag+、Hg+和Pb2+对该纤溶酶具有一定的抑制作用;SBTI和PMSF,完全抑制UFE-Ⅰ,说明该酶为丝氨酸蛋白酶;糜蛋白酶抑制剂部分抑制UFE-Ⅰ,亮抑酶肽、抑蛋白酶肽、苯甲脒较弱的抑制UFE-Ⅰ.与蚓激酶类似,UFE-Ⅰ不仅具有直接的纤溶活力更具有纤溶酶原激活活力(84.0%),另外分别将蚓激酶原料与该酶加入到家兔血栓块中,37 ℃孵育3 h,结果表明该酶具有比蚓激酶更为强大的溶栓能力.     

灵芝多糖对人脐血LAK细胞活性的影响

本文研究了灵芝多糖（ＧＬＰ）对人脐血ＬＡＫ（ＣＢ—ＬＡＫ）细胞活性的影响,结果发现,单独ＧＬＰ能刺激人脐血单个核细胞（ＣＢＭＣ）增殖,但不能诱导ＬＡＫ活性,当与５０ｕ／ｍｌｒＩＬ—２伍用时,可增殖ＣＢ—ＬＡＫ细胞诱导活性,不同剂量ＧＬＰ（０．５—１００μｇ／ｍｌ）影响作用不同,以１０μｇ／ｍｌ浓度最好．在不同浓度ｒＩＬ—２（１０—１００ｕ／ｍｌ）诱导ＣＢ—ＬＡＫ细胞过程中加入ＧＬＰ（１０μｇ／ｍｌ）,可明显提高细胞增殖能力,减少ｒＩＬ—２用量。ＧＬＰ亦能促进效应阶段ＣＢ—ＬＡＫ细胞对Ｒａｊｉ肿瘤靶细胞的杀伤作用（Ｐ＜０．００１）。由此看出,ＧＬＰ具有增强ＣＢ—ＬＡＫ细胞活性的作用,是一很好的生物反应调节剂（ＢＲＭ）,有必要进行深入的研究。     

内脂素对转基因小鼠血糖和运动性的影响

目的建立内脂素转基因小鼠动物模型,研究内脂素在转基因表达的情况下对小鼠的影响。方法把内脂素基因插入CMV启动子下游,构建转基因表达载体,通过显微注射法建立内脂素转基因小鼠。PCR鉴定内脂素转基因小鼠的基因型,Western Blot检测基因表达,通过血糖测定、血生化检测、转轮实验以及旷场观察,检测转基因小鼠在血糖和行为等方面的改变。结果建立了2个不同表达水平的内脂素转基因小鼠品系,转入的内脂素基因在骨骼肌和内脏脂肪组织中的表达高于内源性内脂素。血糖、血生化、代谢、疲劳度、协调性和旷场检查证实：内脂素转基因小鼠机体血糖降低,谷丙转氨酶降低,尿素氮升高,高密度脂蛋白胆固醇降低,低密度脂蛋白胆固醇升高,抗疲劳性和和协调性增高。结论成功建立了内脂素转基因小鼠,并证实内脂素对小鼠血糖和运动行为具有明显的影响,为研究脂肪细胞因子的作用机制提供了有价值的动物模型。     

一株纤维蛋白溶解酶产生菌的鉴定及其产酶条件初步研究

从亚洲传统发酵食品--虾酱中筛选到一株产纤维蛋白溶解酶能力较强的菌株,通过形态和常规生理生化性质鉴定,发现该菌株与芽孢杆菌属细菌的特征很相近,结合16S rDNA序列分析,构建系统发育树,确定其分类地位,由中国典型培养物保藏中心定名为Bacillus sp.nov.SK006(CCTCC No.M 205071),并优化了发酵培养基组成及培养条件,本研究为该菌株的深入研究和广泛应用提供了理论依据.     

The Effect of Tranexamic Acid on the Fibrinolytic System During Anaphylaxis in Rabbits; The Importance of the Fibrinolytic System During Anaphylaxis

Abstract

We previously reported (Shimaya et al. (1992) Enzyme, 46, 204) that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis, and that the main plasma fibrinolytic enzyme which increased in the anaphylaxis group was shown to be tissue-type plasminogen activator (t-PA). Anaphylaxis was induced in rabbits by giving BSA after t-AMCHA injection. 44% of those rabbits died within 3 h after BSA injection. In the dead group, the euglobulin fibrinolytic activity (EFA) could not be detected by the plasminogen-rich fibrin plate method and the t-PA activity, using the natural substrate plasminogen, did not rise significantly reaching a peak at 10–15 min. However, the EFA and t-PA activity increased significantly in the surviving group. A significant prolongation of the activated partial thromboplastin time (AFTT) and the prothrombin time (PT) was observed during anaphylaxis in both groups. These findings suggest that increased PA activity during anaphylaxis is an important defense mechanism against the rapid increase in the blood coagulation system.     

Clinical Importance of Angiogenic Cytokines,Fibrinolytic Activity and Effusion Size in Parapneumonic Effusions

Objective

To investigate the relationship among angiogenic cytokines, fibrinolytic activity and effusion size in parapneumonic effusion (PPE) and their clinical importance.

Methods

From January 2008 through December 2010, 26 uncomplicated (UPPE) and 38 complicated (CPPE) PPE were studied. Based on chest ultrasonography, there were non-loculated in 30, uni-loculated in 12, and multi-loculated effusions in 22 patients. The effusion size radiological scores, and effusion vascular endothelial growth factor (VEGF), interleukin (IL)-8, plasminogen activator inhibitor type-1 (PAI-1) and tissue type plasminogen activator (tPA) were measured on admission. Treatment outcome and pleural fibrosis, defined as radiological residual pleural thickening (RPT), were assessed at 6-month follow-up.

Results

The effusion size and effusion VEGF, IL-8 and PAI-1/tPA ratio were significantly higher in CPPE than in UPPE, and significantly higher in multi-loculated PPE than in non-locualted and uni-loculated PPE, respectively. VEGF (cutoff value 1975 pg/ml) and IL-8 (cutoff value 1937 pg/ml) seemed best to discriminate between UPPE and CPPE. VEGF, IL-8 and effusion size correlated positively with PAI-1/tPA ratio in both UPPE and CPPE. Moreover, the level of VEGF, but not IL-8, correlated positively with effusion size in all patients (r = 0.79, p<0.001) and in UPPE (r = 0.64, p<0.001) and CPPE (r = 0.71, p<0.001) groups. The patients with higher VEGF or greater effusion were prone to have medical treatment failure (n = 10; VEGF, odds ratio 1.01, p = 0.02; effusion size, odds ratio 1.26, p = 0.01). Additionally, ten patients with RPT had larger effusion size and higher levels of VEGF and PAI-1/tPA ratio than did those without.

Conclusions

In PPE, VEGF and IL-8 levels are valuable to identify CPPE, and higher VEGF level or larger effusion is associated with decreased fibrinolytic activity, development of pleural loculation and fibrosis, and higher risk of medical treatment failure.     

食品纤溶酶研究概况

溶栓疗法是血栓性疾病安全有效的治疗手段,开发安全高效、廉价的新型纤溶酶对于预防与治疗血栓性疾病具有重要意义。近年来,在许多亚洲传统发酵食品中如日本纳豆、韩国大豆酱、中国豆豉、发酵虾酱等中均发现有丰富的纤溶酶资源。本文重点介绍传统发酵食品中纤溶酶的研究概况及其开发前景。