Importance of the antithrombin III-heparin complex in the protective reaction to thrombin formation in animal and human blood

The antithrombin III-heparin complex was isolated from albino rat and human blood. The non-enzymatic fibrinolytic activity of the complex during anticoagulation system activation was higher, as compared to analogous activity in normal conditions or in depressed function of anticoagulation system.     

Nonenzymatic fibrinolytic reaction to the intravenous administration of small doses of Salmonella endotoxin to rabbits

Intravenous injection of a low dose of Salmonella endotoxin (10 micrograms/kg bw) into rabbits results in an increase in the non-enzymatic fibrinolytic activity of blood at early stages of a pathological process followed by depression of this response at later stages of the pathology. At higher degrees of non-enzymatic fibrinolysis activation the morphological and ultrastructural changes in renal tissues caused by endotoxin injection are the least pronounced. Intravenous injection of heparin after injection of a lethal dose of Salmonella endotoxin (100-200 micrograms/kg) enhances non-enzymatic fibrinolysis activation and decreases the morphological and ultrastructural lesions in renal tissues.     

The action of timoptin peptides on the hemostatic system

Repeated introduction of thymus polypeptide drug thymoptine (8 times every 24 hours, 1 microgram/kg) led to the increase in total and non-enzymatic fibrinolytic activity of blood plasma and the decrease in fibrinogen concentration. Thymoptine could lyse non-stabilized fibrin and cause depolymerization of aggregates of fibrin monomer in virto. Highest depolymerization activity was observed at thymoptine concentration of 10 micrograms/ml.     

Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.     

Correction of impairments in functions of anticoagulation and insular systems of an organism by the regulatory peptide Leu-Pro-Gly-Pro

It has been established that fivefold intranasal administration of the peptide Leu-Pro-Gly-Pro (1 mg/kg) to rats with developing refractory hyperglycemia leads to restoration and normalization of the functions of anticoagulation and insular systems. In the blood of experimental animals, there was a decrease in the sugar level and platelet aggregation and an increase in anticoagulant and all kinds of fibrinolytic (total, enzymatic, non-enzymatic, Hageman-dependent) activity.     

Fibrinolytic properties of a heparin-ocrase complex]

The obtained heparin-ocrase complex includes properties of inhibiting coagulation and a high fibrinolytic activity in vitro and in vivo. The fibrinolytic activity of the heparin-ocrase complex is higher than the equivalent amount of ocrase. The fibrinolytic activity of the complex is not only evident on non-stabilized fibrin plates, but likewise on stabilized ones and at the presence of inhibitors of enzymatic fibrinolysis, such as Trasylol and epsilon-aminocaproic acid. The complex formation between heparin and protease was confirmed by means of cross-paper electrophoresis and spectrophotometry.     

Increases in factor VIII complex and fibrinolytic activity are dependent on exercise intensity

Components of the factor VIII complex increase and activation of the fibrinolytic system occur during exercise. The relation between the duration and intensity of exercise and the relative changes in the VIII complex and fibrinolytic system have not been previously examined. Five healthy male subjects were exercised with three protocols: a graded progressive exercise test to exhaustion on a cycle ergometer with 50-W increments every 4 min, steady-state exercise, 15 min at 5 and 125 W each, and an acute 30-s maximal exercise test on a cycle ergometer. Venous blood samples were drawn at base line, during the last 30 s of each power output in the graded exercise, at 5-min intervals for the steady-state exercise, and for up to 1 h after completion of exercise in all three protocols. At the maximum exercise intensities, increases in plasma lactate concentration ([La]), O2 uptake, and [H+] were observed. Components of the VIII complex [VIII procoagulant, VIII procoagulant antigen, VIII-related antigen (VIIIR:Ag), VIII ristocetin cofactor activity] abruptly rose at only the highest work intensities, whereas the whole blood clot lysis time began to gradually shorten much earlier at low work intensities. There were no qualitative changes in the factor VIIIR:Ag on crossed immunoelectrophoresis nor was there evidence of thrombin generation as determined by fibrinopeptide A generation. We conclude that during exercise the changes observed in the coagulation and fibrinolytic systems are related to the intensity of the exercise, which is reflected by increases in plasma [La] and [H+], and that the fibrinolytic system is activated before the changes in the VIII complex are observed.     

Mechanism of inhibition of non-enzymatic fibrinolysis by a spleen factor

Some data on the mechanism of inhibition of non-enzymatic fibrinolysis by a spleen factor are presented. It is demonstrated that the spleen protein factor which interacts with heparin at certain ratios forms a complex with the latter. As a result, the anticoagulating activity and properties of the spleen factor as a non-enzymatic fibrinolysis inhibitor are blocked.     

Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies

Remodeling of extracellular matrix (ECM) is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR) is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA) promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.     

Fibrinolytic system of cultured endothelial cells: regulation by plasminogen activator inhibitor

Cultured bovine aortic endothelial cells have a relatively complex fibrinolytic system that is responsive to both the physiological state of the cell itself and to a variety of agents added to the culture medium. The fibrinolytic activity of these cells results from the production of both urokinase-type and tissue-type plasminogen activators and is regulated by an inhibitor capable of neutralizing their activities. The properties of these fibrinolytic components will be reviewed, and their respective roles in initiating and regulating the fibrinolytic activity of the cells will be summarized. A cDNA coding for the inhibitor has been isolated, and its sequence will be compared to that of other serine proteinase inhibitors.     

Comparative study of hemostatic properties of the complex and mixture of high molecular weight heparin and adenosine triphosphate

Anticoagulant and nonenzymatic fibrinolytic activities increased in blood plasma of 6–7-month-old rats after repeated intramuscular administration of the heparin-adenosine triphosphate complex (G-ATP). The mixture of heparin and ATP had no fibrin depolymerizing activity in vitro. Repeated intramuscular administration of the mixture had anticoagulant effect although it was 1.5–1.6 times less pronounced compared to the complex. A higher anticoagulant and fibrinolytic efficiency of the G-ATP complex compared to the mixture is concluded.     

Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells

We have examined the effects of bacterial lipopolysaccharide (endotoxin) on the fibrinolytic activity of bovine pulmonary artery endothelial cells. Endotoxin suppressed the net fibrinolytic activity of cell extracts and conditioned media in a dose-dependent manner (threshold dose, 0.1 ng/ml; maximal dose, 10-100 ng/ml). The effects of endotoxin required at least 6 h for expression. Cell extracts and conditioned media contained a 44-kDa urokinase-like plasminogen activator. Media also contained multiple plasminogen activators with molecular masses of 65-75 and 80-100 kDa. Plasminogen activators in extracts and media were unchanged by treatment of cells with endotoxin. Diisopropyl fluorophosphate (DFP) abolished fibrinolytic activity of extracts and conditioned media. DFP-treated samples from endotoxin-treated but not untreated cells inhibited urokinase and tissue plasminogen activator, but not plasmin. Inhibitory activity was lost by incubation at pH 3 or heating to 56 degrees C for 10 min. These treatments did not affect inhibitory activity of fetal bovine serum. Incubation of 125I-urokinase with DFP-treated medium from endotoxin-treated cells produced an inactive complex with an apparent molecular mass of 80-85 kDa. The complex could be detected by chromatography on Sephadex G-100, but not by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These findings suggest that low doses of endotoxin suppress fibrinolytic activity in endothelial cells by stimulating the production or expression of a fast-acting, relatively labile inhibitor of plasminogen activator.     

Fibrinolytic properties of protease complex isolated from the culture fluid of Nocardia minima

The protease complex isolated form the Nocardia minima culture liquid was studied in vitro. The preparation had two different activities (fibrinolytic and activating), i.e. it was able to convert plasminogen into plasmin. At a concentration of 250 micrograms/ml and above the preparation lysed experimental thrombi. The fibrinolytic activity of the preparation was completely inhibited with normal human plasma.     

高产纤溶酶菌株CNY16发酵条件优化及其酶学特性初步研究

【目的】通过响应面试验对产纤溶酶菌株CNY16发酵条件进行优化,并对其酶学特性进行初步研究。【方法】采用Plackett-Burman设计得出酵母膏、氯化钠、转速3个最重要影响因素,通过最陡爬坡实验逼近酶活的最高区域,然后根据Box-Behnken中心组合设计实验对显著因素进行优化分析,最后对该酶学性质进行初步分析。【结果】最终得到3个因素的最优组合:酵母膏3.28%,氯化钠1.14%,转速166 r/min,在此培养条件下,纤溶酶活达到875.932 U/mL,比优化前提高了46%;该菌株产纤溶酶最适温度为30°C,最适pH为6.5。【结论】确定了高产纤溶酶菌株CNY16的最优发酵条件及其部分酶学性质,为该酶的进一步深入研究及中试实验奠定基础。     

黏细菌抗凝溶栓双功能蛋白MF-1的纯化及其酶学性质

对黏细菌Angiococcus sp.的抗凝溶栓双活性蛋白MF-1进行纯化、鉴定并对其酶学性质进行初步研究。采用丙酮分级沉淀法、DEAE-Sepharose离子交换层析和Sephadex G-50分子筛层析对发酵液进行纯化,用SDS-PAGE和等电点聚焦电泳对其进行鉴定,并用纤维蛋白平板法和水解酪蛋白法对其酶学性质进行检测。结果表明：经过一系列的纯化步骤分离得到该蛋白相对分子质量为3.2×10^4,等电点为8.5,酶的比活力为30761.57U/mg,活性回收率为13.9%;溶栓活性的最适反应温度为35℃,最适反应pH为8.0;抗凝时间大于10min,且酶活性十分稳定,在35℃下保温72h后仍有89%活性。首次从黏细菌中分离得到具有较高抗凝和溶栓双活性的MF-1蛋白,且稳定不易失活,具有开发成为创新溶栓药物的潜力。     

Deep venous thrombosis of the arm: a study of coagulation and fibrinolysis.

Sixty consecutive patients with phlebographically verified deep venous thrombosis of the upper arm were studied for disorders of coagulation and fibrinolysis. No appreciable increase in abnormalities of the factor VIII complex, antithrombin III, or inhibitors of activators of fibrinolysis were found. A decreased fibrinolytic defence mechanism, evident either as a deficient release capacity of fibrinolytic activators from the vein during stasis or as decreased fibrinolytic activity in the vein wall as determined histochemically, was found in 26 out of 53 patients studied (49%). It is concluded that deep venous thrombosis of the upper arm is a multifactorial disease. An impaired fibrinolytic defence mechanism is one of the factors that may be of pathogenetic importance.     

Effect of the enzyme preparation longolytin on fibrinolysis in animals

It has been established that fibrinolytically active enzyme longolytine isolated from the culture fluid of the saprophyte fungus Arthrobotrys longa at intravenous injection favours the prolonged increase of the plasma fibrinolytic properties as well as activation of endogenic plasminogen. Maximum values of fibrinolytic activity have been marked in 5 and 30 min after enzyme intravenous injection. The plasminogen activity is high in 120 min. The fibrinolysis indexes--fibrinolytic activity of the euglobulin fraction and amount of plasminogen activator--in 3,5 and 5 times higher than in vitro at intravenous injection. The activation of coagulation system does not occur.     

华广虻(Tabanusamaenus Walker)溶纤活性蛋白的纯化及生物活性分析

经过 ７５％ 饱和度硫酸铵沉淀、 Ｓｅｐｈａｄｅｘ Ｇ ７５ 凝胶过滤层析、 Ｌｙｓ Ｓｅｐｈａｒｏｓｅ ４ Ｂ 亲和层析和电泳制备洗脱,从华广虻（ Ｔａｂａｎｕｓ ａｍ ａｅｎｕｓ Ｗ ａｌｋｅｒ）腹部组织匀浆液中分离纯化出分子量约为 ６７ｋ Ｄ 的溶纤活性蛋白 Ｔ Ａ Ｆ Ｐ经纤维蛋白平板测定表明, Ｔ Ａ Ｆ Ｐ 只具有纤溶酶作用,不具有激活纤溶酶原的作用;但 Ｔ Ａ Ｆ Ｐ 能分解纤溶酶原激活剂的生色底物—— Ｃｈｒｏｍ ｏｚｙｍ Ｕ Ｋ 及 Ｓ ２２８８还能水解胰蛋白酶专一底物 Ｂｚ Ｐｈｅ Ｖａｌ Ａｒｇ Ｎ Ａ 及 Ｃ Ｂ Ｚ Ｇｌｙ Ｐｒｏ Ａｒｇ Ｎ Ａ,表明 Ｔ Ａ Ｆ Ｐ具有类胰蛋白酶活性,专一水解精氨酸形成的酰胺键（或肽键） Ｔ Ａ Ｆ Ｐ无胰凝乳蛋白酶活性      

A rapid and strong increase of plasminogen activator induced by experimental anaphylaxis in rabbits.

Anaphylactic shock was induced in rabbits by injecting bovine serum albumin (BSA) as an antigen. Measurements of the enzyme activities in the fibrinolytic system confirmed that a rapid and strong increase of plasminogen activator (PA) was induced during anaphylaxis. The euglobulin fibrinolytic activity (EFA) as estimated by the plasminogen-rich fibrin plate method rose significantly, peaking at 15 min after the BSA injection (when the arterial pressure was minimum). However, EFA was not detected by the plasminogen-poor fibrin plate method. The tissue-type PA (t-PA) activity using the natural substrate plasminogen increased significantly with a peak at 15 min. The amidolytic activity also simultaneously increased significantly using the t-PA substrate, H-D-Ile-Pro-Arg-pNA. The plasminogen activator inhibitor (PAI) activity remained at baseline levels until 30 min, but rose fourfold at 90 min. The main plasma fibrinolytic enzyme which increased in anaphylaxis was proved by zymography to be t-PA with a molecular weight (MW) of 69,000.     

Fibrinolytic activity of heart tissues and aorta of rats undergoing electroshock

In the first minute of shock caused by electric current passing through the rats parietal lobe the increase of fibrinolytic activity of euglobulin fraction of blood occurs. During the investigation of the right heart ventriculus tissue and aorta by histochemical method of Todd in our modification the decrease of fibrinolytic activity in wall of aorta (on 80%) and in tissue of heart ventriculus (on 30%) has been revealed in tentative animals as compared with control ones. It is suggested that the entering of plasminogen activator from vessel wall into the blood flow plays an important role in activation of fibrinolytic system in generalized reaction of organism to strong stress action.