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.     

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。     

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.     

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。     

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.     

Glutathione Peroxidase Activity in Porcine Blood

Determination of the seleno-enzyme glutathione peroxidase (GSH-Px) in blood from Danish Landrace pigs was done using a quantitative, spectrophotometric method and a simple “spot test”. A close correlation between the net reaction rate measured spectrophotometrically (Δ A/min.) and time for defluores-cence (minutes) was obtained (r2 = 0.72—0.77, P < 0.0005). From these results the factors used for a conversion of defluorescence time to u/g hemoglobin were evaluated. The results further showed that the “spot test” can be used as a screening method for detection of subnormal GSH-Px levels in pigs. While red cell GSH-Px seems independent of the sex, an elevation of both plasma and red cell GSH-Px was found with increasing age of pigs. The normal range of red cell GSH-Px activity was wide, contrasting the small variations observed in the individual pig. Some evidence that porcine red cell GSH-Px is under genetical control was found and discussed in relation to the possible use of GSH-Px as an indicator of the pig's selenium status.     

Blood Muramidase Activity in Colorectal Cancer

The serum muramidase levels were measured in 128 patients with primary or metastatic colorectal cancer, 166 tumour-free patients after resection of a colorectal cancer, and 172 controls. Muramidase levels over 10 μg/ml were detected in 30%-39% of the tumour-bearing patients, in 8·2% of the tumour free, and in only 1·7% of the controls (normal level 6·68 ± 1·42 μg/ml). Long-term follow up indicated that raised levels may occur as a transient phenomenon in recurrent or metastatic disease. The likely relation of abnormal serum muramidase activity and stimulation of the reticuloendothelial system is discussed.     

Discrepant Fibrinolytic Response in Plasma and Whole Blood during Experimental Endotoxemia in Healthy Volunteers

Background

Sepsis induces early activation of coagulation and fibrinolysis followed by late fibrinolytic shutdown and progressive endothelial damage. The aim of the present study was to investigate and compare the functional hemostatic response in whole blood and plasma during experimental human endotoxemia by the platelet function analyzer, Multiplate and by standard and modified thrombelastography (TEG).

Methods

Prospective physiologic study of nine healthy male volunteers undergoing endotoxemia by means of a 4-hour infusion of E. coli lipopolysaccharide (LPS, 0.5 ng/kg/hour), with blood sampled at baseline and at 4 h and 6 h. Physiological and standard biochemical data and coagulation tests, TEG (whole blood: TEG, heparinase-TEG, Functional Fibrinogen; plasma: TEG±tissue-type plasminogen activator (tPA)) and Multiplate (TRAPtest, ADPtest, ASPItest, COLtest) were recorded. Mixed models with Tukey post hoc tests and correlations were applied.

Results

Endotoxemia induced acute SIRS with increased HR, temperature, WBC, CRP and procalcitonin and decreased blood pressure. It also induced a hemostatic response with platelet consumption and reduced APTT while INR increased (all p<0.05). Platelet aggregation decreased (all tests, p<0.05), whereas TEG whole blood clot firmness increased (G, p = 0.05). Furthermore, during endotoxemia (4 h), whole blood fibrinolysis increased (clot lysis time (CLT), p<0.001) and Functional Fibrinogen clot strength decreased (p = 0.049). After endotoxemia (6 h), whole blood fibrinolysis was reduced (CLT, p<0.05). In contrast to findings in whole blood, the plasma fibrin clot became progressively more resistant towards tPA-induced fibrinolysis at both 4 h and 6 h (p<0.001).

Conclusions

Endotoxemia induced a hemostatic response with reduced primary but enhanced secondary hemostasis, enhanced early fibrinolysis and fibrinogen consumption followed by downregulation of fibrinolysis, with a discrepant fibrinolytic response in plasma and whole blood. The finding that blood cells are critically involved in the vasculo-fibrinolytic response to acute inflammation is important given that disturbances in the vascular system contribute significantly to morbidity and mortality in critically ill patients.     

Effect of Ethylene on Peroxidase Activity

Ethylene increased the peroxidase activity of nine out of ten varieties of sweet potato (Ipomoea batatas (L.) Lam.) root disks tested. The increase which was observed four hours after ethylene treatment was partially overcome by carbon dioxide. The increase was inhibited by actinomycin D and cycloheximide, indicating de novo protein synthesis. Electrophoretic separation on polyacrylamide gels indicated the appearance of two new peroxidase bands. Peroxidase activity in bean petiole explants was localized around the separation layer. Ethylene caused a small increase in peroxidase activity in the petiolar portion of the explant. Phenolic substances had no effect on abscission consistent with their proposed roles as cofactors for auxinoxidase, indicating that auxin-oxidase does not play a role in abscission of Coleus blumei Benth. abscission zone explants.     

Effect of Cyclanilide on Auxin Activity

Cyclanilide is a plant growth regulator that is registered for use in cotton at different stages of growth, to either suppress vegetative growth (in combination with mepiquat chloride) or accelerate senescence (enhance defoliation and boll opening, used in combination with ethephon). This research was conducted to study the mechanism of action of cyclanilide: its potential interaction with auxin (IAA) transport and signaling in plants. The activity of cyclanilide was compared with the activity of the auxin transport inhibitors NPA and TIBA. Movement of [³H]IAA was inhibited in etiolated corn coleoptiles by 10 μM cyclanilide, NPA, and TIBA, which demonstrated that cyclanilide affected polar auxin transport. Although NPA inhibited [³H]IAA efflux from cells in etiolated zucchini hypocotyls, cyclanilide had no effect. NPA did not inhibit the influx of IAA into cells in etiolated zucchini hypocotyls, whereas cyclanilide inhibited uptake 25 and 31% at 10 and 100 μM, respectively. Also, NPA inhibited the gravitropic response in tomato roots (85% at 1 μM) more than cyclanilide (30% at 1 μM). Although NPA inhibited tomato root growth (30% at 1 μM), cyclanilide stimulated root growth (165% of control at 5 μM). To further characterize cyclanilide action, plasma membrane fractions from etiolated zucchini hypocotyls were obtained and the binding of NPA, IAA, and cyclanilide studied. Cyclanilide inhibited the binding of [³H]NPA and [³H]IAA with an IC₅₀ of 50 μM for both. NPA did not affect the binding of IAA, nor did IAA affect the binding of NPA. Kinetic analysis indicated that cyclanilide is a noncompetitive inhibitor of both NPA and IAA binding, with inhibition constants (K _i) of 40 and 2.3 μM, respectively. These data demonstrated that cyclanilide interacts with auxin-regulated processes via a mechanism that is distinct from other auxin transport inhibitors. This research identifies a possible mechanism of action for cyclanilide when used as a plant growth regulator.     

Effect of Immobilization of the Micromycete Aspergillus ochraceus VKM-F4104D in Polymeric Carriers on the Production of the Fibrinolytic Protease Activator of Blood Plasma Protein C

Applied Biochemistry and Microbiology - The best known polymers (alginate, carrageenan, and acrylamide) were examined for their potential use as carriers of mycelium cells of Aspergillus ochraceus...     

The Effect of Magnetite Nanoparticles and Bacteria on the Activity of NADPH-Oxidase and Myeloperoxidase in Neutrophils of Human Blood

Differences in the oxygen-dependent reactions of neutrophil granulocytes (NGs) depending on the nature of the agent affecting the cells were revealed. In vitro magnetite nanoparticles (MNPs) cause suppression of the NADPH–oxidase activity of NGs, which manifests itself in falling rates of reactions (NBT test) both with the effect of MNPs on NGs alone and a combined effect (MNPs and zymosan), as well as in the reduction of the index of activation (IA) and functional reserve of neutrophils (FRN). However, the introduction of MNPs dose-dependently stimulates the activity of myeloperoxidase (MPO). Gram-positive (S. aureus 2879 M) and gram-negative (E. coli 321) bacteria caused a respiratory burst of neutrophils, which manifested itself in a significant increase in the number of NBT-positive cells in single and combined influences (bacteria and zymosan). The lack of differences in the reaction of cells on opsonized and nonopsonized bacteria and the decrease in IA and FRN suggest that NGs are at the maximum level of functionality. Both strains of bacteria caused activation of the MPO.