Effect of physical exercise on fibrinolytic properties of erythrocytes

The fibrinolytic properties of blood and erythrocytes were studied before and after physical exercise in male volunteers. Their fibrinolytic responses were of two distinct types. In type 1 response, fibrinolytic activities of blood and erythrocytes increased; the plasminogen activator and active plasmin contents in erythrocytes also increased, whereas the profibrinolysin content correspondingly decreased. In addition, physical exercise increased the erythrocyte adsorption properties for plasma activators of fibrinolysis. Type 2 response was characterized by a decrease in the fibrinolytic activity of blood; neither fibrinolytic activity nor adsorption properties of erythrocytes increased. The type of blood and erythrocyte response to muscular activity was determined by the pre-exercise level of red blood cell fibrinolytic activity. It was low in type 1 response due to a lesser content of plasmin activators and greater content of antiplasmin. In type 2 response, the initially high lytic capacity is connected with a greater reserve of activators and lesser reserve of inhibitors of the fibrinolytic system. A conclusion was made that individual differences in fibrinolytic responses to physical exercise were largely accounted for by the properties of erythrocytes.     

Position and developmental trends in fibrinolytic therapy

Increasing insight into the mechanism of fibrinolysis and particularly into the formation and release of plasminogen activator has led to more effective thrombolytic therapy. The understanding of the mechanism of thrombolysis has provided the possibility to improve the therapeutic effects of the fibrinolytic agents streptokinase and urokinase. Further advances in thrombolytic therapy are expected by the use of the plasminogen activator from tissue endothelium and pro-urokinase. Acylation of fibrinolytic enzymes will lead to beneficial effects (depot effect, protection from intrinsic inhibitors). Due to the extensive research into substances with fibrinolytic and thrombolytic effects a new generation of activators of fibrinolysis is expected that interfere with the biosynthesis and release of plasminogen activator of the vessel wall and that are suited for treatment of hypofibrinolytic states.     

Blocking of the fibrinolytic antiactivator by substituted benzoates

The effects of substituted benzoates on the inhibition of the urokinase-activation of profibrinolysin by the fibrinolytic antiactivator in plasma were investigated. It was found that substituted benzoates block the inhibition of urokinase by plasma fibrinolytic antiactivators. The urokinase which is bound by the antiactivator appears to be released and hence is available to activate profibrinolysin to fibrinolysin. The nitrobenzoates were the most effective of all compounds tested.     

Secreted Bacillus anthracis proteases target the host fibrinolytic system

The fibrinolytic system is often the target for pathogenic bacteria, resulting in increased fibrinolysis, bacterial dissemination, and inflammation. The purpose of this study was to explore whether proteases NprB and InhA secreted by Bacillus anthracis could activate the host's fibrinolytic system. NprB efficiently activated human pro-urokinase plasminogen activator (pro-uPA), a key protein in the fibrinolytic cascade. Conversely, InhA had little effect on pro-uPA. Plasminogen activator inhibitors (PAI)-1, 2 and the uPA receptor were also targets for NprB in vitro. InhA efficiently degraded the thrombin-activatable fibrinolysis inhibitor (TAFI) in vitro. Mice infected with B. anthracis showed a significant decrease in blood TAFI levels. In another mouse experiment, animals infected with isogenic inhA deletion mutants restored TAFI levels, while the levels in the parent strain decreased. We propose that NprB and InhA may contribute to the activation of the fibrinolytic system in anthrax infection.     

Growth factor-dependent proliferation and invasion of muscle satellite cells require the cell-associated fibrinolytic system

The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFbeta) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cell-associated fibrinolytic system. We have observed that the urokinase plasminogen activator (u-PA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly up-regulated by TGFbeta, mainly in dystrophic myoblasts. bFGF up-regulated u-PA in both normal and dystrophic myoblasts grown in primary culture, while a striking down-regulation was observed with TGFbeta. TGFbeta was the only growth factor able to exceptionally up-regulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factor-dependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cell-associated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of u-PA and of its receptor. Noteworthy, TGFbeta induced a dose-dependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFbeta-challenged dystrophic myoblasts undergo an exceptional up-regulation of the receptor and of PAI-1, we propose the possibility that the TGFbeta-induced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.     

Effect of exercise and oral contraceptive agents on fibrinolytic potential in trained females

It has been shown that physical exercise increases blood fibrinolytic potential, primarily by inducing a release of extrinsic plasminogen activator from the vessel wall. Synthetic estrogens have also been reported to influence fibrinolytic activity. The effect of exercise and the possible additional effect of oral contraceptive agents (OCA) on the fibronolytic system were studied in 20 competitive female rowers. Ten females used OCA (users), and 10 others did not (nonusers). All participants were subjected to standardized exhaustive exercise. Preexercise data revealed higher factor XII, total plasminogen, and free plasminogen levels together with a significantly lower C1-inactivator level in the group of users. No differences were observed in prekallikrein, high-molecular-weight kininogen, alpha 2-antiplasmin, alpha 2-macroglobulin, antithrombin III, and histidine-rich glycoprotein plasma levels. The factor XII-dependent fibrinolytic activator activity and the extrinsic (tissue-type) plasminogen activator were significantly higher; however, the urokinase-like fibrinolytic activator activity was significantly lower. These observations suggest a greater susceptibility to activation of the fibrinolytic pathways during OCA medication. Exercise resulted in a decrease of all factors under study but an increase in all fibrinolytic activities. No differences were observed between the two groups in the percentages of change that occurred with exercise.     

Influence of beta-sitosterol on the fibrinolytic potential in rabbits

Long-term treatment of rabbits with beta-sitosterol (40 mg/kg over 3 months) caused an increased fibrinolytic activity in blood, an increased fibrinolytic capacity and an enhanced plasminogen activator activity in tissue of lungs and kidneys. The 3-months lasting beta-sitosterol administration did not influence the content of plasminogen activator inhibitor, plasminogen, alpha 2-antiplasmin, antithrombin III and fibrinogen.     

Inhibition of fibrinolytic enzymes by thrombin inhibitors

Thrombin inhibitors have recently advanced to the stage of preclinical testing as anticoagulants. However, little is known about the effects of these inhibitors on the enzymes of the fibrinolytic system. In the present study we evaluated the effect of two protein and two synthetic inhibitors of thrombin on tissue plasminogen activator (tPA), urokinase, and plasmin. We found that hirudin inhibited the amidolytic activity of plasmin but had no effect on tPA or urokinase. Antithrombin III inhibited plasmin and urokinase but had no effect on tPA. D-Phe-Pro-Arg-CH2Cl inhibited plasmin and tPA but had no effect on urokinase. Thromstop inhibited all three fibrinolytic enzymes: plasmin, urokinase, and tPA. Thus each thrombin inhibitor tested had different inhibitory effects on the fibrinolytic enzymes. These effects should be carefully considered when thrombin inhibitors are used as antithrombotic drugs.     

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.     

White coat hypertension and haemostatic/fibrinolytic balance disorders

White coat hypertension (WCH) or isolated clinic hypertension is generally accepted to be a benign condition, although some reports have suggested that it may be associated with an increased cardiovascular event rate or other cardiovascular alterations. It has been previously shown that essential hypertension (EH) is associated with abnormalities in haemostatic/fibrinolytic balance and endothelial function. The aim of our study was to assess the impact of WCH on fibrinolytic balance and endothelial function by measuring plasma levels of plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator antigen (tPA), fibrinogen, and thrombomodulin. These markers were determined in 71 patients with EH, 26 with WCH and 87 normotensive healthy control subjects. The three groups were not different with respect to age, gender, smoking habits, BMI and blood lipids. Subjects with WCH were found to have increased plasma levels of PAI-1, tPA, fibrinogen and thrombomodulin compared to controls, but less compared to hypertensive ones. Our results suggest that WCH may be associated with decreased fibrinolytic potential and endothelial dysfunction, indicating that WCH may not be a completely harmless trait.     

Decreased fibrinolytic activity in porcine-to-primate cardiac xenotransplantation.

BACKGROUND: One major barrier to successful xenotransplantation is acute vascular rejection, a process pathologically characterized by microvascular thrombosis and diffuse fibrin deposition in transplant blood vessels. This pathologic picture may result from a disturbance in the coagulant or fibrinolytic pathways that regulate normal vascular patency. This study evaluated the regulation of fibrinolytic activity defined by tissue plasminogen activator and plasminogen activator inhibitor-1 as it may exist in the setting of acute vascular rejection. MATERIALS AND METHODS, RESULTS: Serial biopsies from cardiac xenotransplants evaluated by immunofluorescence microscopy demonstrated progressive decreases in tissue plasminogen activator and increases in plasminogen activator inhibitor-1. In vitro studies measuring fibrinolytic activity of cell culture medium from porcine aortic endothelial cells stimulated with human serum or autologous porcine serum revealed that human serum triggered as much as 93% increase in antifibrinolytic activity. CONCLUSIONS: These findings demonstrate that porcine vascular endothelial cells change toward an antifibrinolytic state following stimulation with human xenoreactive antibodies and complement. The shift is at least partly explained by an increased ratio of plasminogen activator inhibitor-1 to tissue plasminogen activator, and is at least in part mediated by the activation of complement. This increased antifibrinolytic activity may contribute to the thrombotic diathesis seen in acute vascular rejection in pig-to-primate xenografts.     

Highly potent fibrinolytic serine protease from Streptomyces

We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis.     

Effect of chromatographic fractions of blood ultrafiltrates of patients with chronic uremia on the fibrinolytic activity]

Ultrafiltrates obtained from the patients with chronic uremia were chromatographically separated in the column filled with Sephadex A25. "Middle" molecular weight substances were localized mainly in a single peak. Dialysis decreased their contents. Determination of the fibrinolytic activity of each chromatographic fractions with caseinolytic and fibrinplate techniques has shown activating effect of the fractions 10-12. Dialysis decreased fibrinolytic activity statistically significantly.     

Activators and inhibitors of the plasminogen system in Alzheimer's disease

Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.     

水蛭素12肽与低分子量单链尿激酶融合基因的构建、表达及产物特性分析

用化学合成的方法合成了水蛭素12肽基因的编码序列,通过DNA重组技术将水蛭素12肽基因片段与低分子量单链尿激酶cDNA片段连接构建了融合基因。融合基因在大肠杆菌中获得表达。体外实验结果表明,表达的融合蛋白具有溶纤活性和抗凝活性。     

On the nature of fibrinolytic activity of arteries.

The plasminogen activator of the arterial wall was studied with the histochemical method of TODD. The plasminogen activator was removed from the sections after extraction with M-potassium thiocyanate. By this procedure we suggest that the activator demonstrated by the histochemical method is the same substance as that prepared by the extraction method with thiocyanate of ASTRUP and STAGE. However, a new fibrinolytic activity was restored after treatment of these extracted sections with streptokinase or urokinase. There were no differences in the different types of arteries examined and normal or atherosclerotic arteries. Similar findings were found when kidney or myocardial tissues were examined. It is suggested that the arterial and other tissues contain proactivator-plasminogen which is not extracted from the tissue by potassium thiocyanate and can adsorb streptokinase or urokinase.     

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      

Relationship of Plasminogen Activator to Fibrin

THERE are two principal theories of the mechanism of thrombus dissolution by the fibrinolytic system. Alkjaersig et al.¹ suggested that as fibrin polymerizes, plasminogen is adsorbed preferentially to the fibrin and is available in large quantities within a thrombus which is comparatively free of antiplasmin. When an activator enters the circulation it diffuses into the clot converting the plasminogen to plasmin in situ and so promotes lysis. Ambrus and Markus², however, proposed that when plasmin forms in the circulation naturally or during infusion of an activator it is normally bound to the excess antiplasmin present in blood. They suggested that this plasmin/antiplasmin complex is reversible and dissociates in the presence of fibrin, its preferred substrate, so allowing the plasmin to bring about fibrin dissolution by “external lysis”. Neither of these theories, however, is supported by an observed phenomena.     

CHARACTERIZATION AND PURIFICATION OF GUT FIBRINOLYTIC PROTEASE FROM TABANUS AMAENUS

Abstract After ammonium sulphate precipitation, Sephadex G-75 gel filtration, Lys-Sepharose 4B affinity chromatography and elution from electrophoresis, the fibrinolytic protease (TAFP) was isolated and purified from the extract of T. amaenus Walker gut. It appeared a single band corresponding to molecular weight of approximately 67kD on SDS-PAGE and an probably pI of 7.2 on IEF. On fibrin plate and plasminogen-free fibrin plate (heated at 85°C for 30 minutes to eliminate plasminogen), TAFP showed same fibrinolytic activity. The result might indicate that TAFP is a fibrinolytic enzyme degrading fibrin, as well as a plasminogen activator degrading fibrin via activating plasminogen. The result of chromogenic substrates indicated that TAFP possesses trypsin-like activity specifically degrading argininyl amide bond or peptide bond, but has no chymotrypsin activity. TAFP was almost inhibited powerfully by antipain, PMSF, soybean trypsin inhibitor and soybean Bowman-Birk inhibitor. However, leupeptin, antitrypsin and TLCK was more powerful effective inhibitors of TAFP. Optimal reaction pH of TAFP was 7.5, and it was stable in 5.5–7.0 of pH range.     

Identification of the human plasma protein which inhibits fibrinolysis associated with malignant cells

Mixed cultures of mouse fibroblasts and mouse fibroblasts transformed with Kirsten murine sarcoma virus were grown in petri dishes and overlayed with casein. The appearance of focal lysis zones required the presence of transformed cells in the culture and plasminogen in the overlay, indicating that caseinolysis was due to plasminogen activator released by the malignant cells. Caseinolysis was inhibited by addition of human plasma or bovine pancreatic trypsin inhibitor to the overlay, 1 ml of plasma being equivalent to 67 ± 18 (mean ± S.E.) kallikrein inhibitor (KI) units of trypsin inhibitor.The culture fluid of a human melanoma line induced lysis of a fibrin clot, 1 ml of culture fluid being equivalent to 250 CTA units of urokinase (EC 3.4.99.26). Fibrinolysis was inhibited by addition of human plasma or trypsin inhibitor, 1 ml of plasma being equivalent to 94 ± 34 KI units of trypsin inhibitor.Specific removal of antiplasmin, the fast-reacting plasmin inhibitor (Collen, D. (1976) Eur. J. Biochem. 69, 209), from plasma by immunoabsorption completely abolished its inhibitory activity, both in the caseinolytic and fibrinolytic assays. It is therefore concluded that antiplasmin is the only protein in human plasma capable of inhibiting the fibrinolytic activity associated with oncogenic transformation or neoplasia. Whether this effect is exclusively due to inhibition of formed plasmin or also to interference with plasminogen activvtion remains unsettled.