Enhancement Of Fibrinolytic Activity Research Articles

In Vitro Study of the Fibrinolytic Activity via Single Chain Urokinase-Type Plasminogen Activator and Molecular Docking of FGFC1.

Fungi fibrinolytic compound 1 (FGFC1) is a rare marine-derived compound that can enhance fibrinolysis both in vitro and in vivo. The fibrinolytic activity characterization of FGFC1 mediated by plasminogen (Glu-/Lys-) and a single-chain urokinase-type plasminogen activator (pro-uPA) was further evaluated. The binding sites and mode of binding between FGFC1 and plasminogen were investigated by means of a combination of in vitro experiments and molecular docking. A 2.2-fold enhancement of fibrinolytic activity was achieved at 0.096 mM FGFC1, whereas the inhibition of fibrinolytic activity occurred when the FGFC1 concentration was above 0.24 mM. The inhibition of fibrinolytic activity of FGFC1 by 6-aminohexanoic acid (EACA) and tranexamic acid (TXA) together with the docking results revealed that the lysine-binding sites (LBSs) play a crucial role in the process of FGFC1 binding to plasminogen. The action mechanism of FGFC1 binding to plasminogen was inferred, and FGFC1 was able to induce plasminogen to exhibit an open conformation by binding through the LBSs. The molecular docking results showed that docking of ligands (EACA, FGFC1) with receptors (KR1–KR5) mainly occurred through hydrophilic and hydrophobic interactions. In addition, the binding affinity values of EACA to KR1–KR5 were −5.2, −4.3, −3.7, −4.5, and −4.3 kcal/moL, respectively, and those of FGFC1 to KR1–KR5 were −7.4, −9.0, −6.3, −8.3, and −6.7 kcal/moL, respectively. The findings demonstrate that both EACA and FGFC1 bound to KR1–KR5 with moderately high affinity. This study could provide a theoretical basis for the clinical pharmacology of FGFC1 and establish a foundation for practical applications of FGFC1.

Extracellular histones promote fibrinolysis by single-chain urokinase-type plasminogen activator in a factor seven activating protease-dependent way

IntroductionExtracellular histones inhibit tissue plasminogen activator (t-PA)-mediated fibrinolysis by modifying fibrin structure and rheological properties. However, other plasminogen activators involved in intravascular and extravascular fibrinolysis have not been considered yet. ObjectivesWe investigated the effect of histones on fibrinolysis driven by different plasminogen activators. MethodsClot lysis induced by t-PA, urokinase (u-PA) and its single chain precursor (scu-PA) was evaluated by turbidimetry. Conversion of scu-PA to u-PA and activation of factor seven activating protease (FSAP) were assessed by fluorogenic and chromogenic assays, respectively. ResultsHistones delayed t-PA- and u-PA-mediated fibrinolysis but strongly accelerated scu-PA-driven clot lysis through the enhancement of scu-PA to u-PA conversion. This effect required a plasma factor identified as FSAP by the following findings: 1) histones enhanced neither scu-PA activation nor scu-PA-mediated clot lysis under purified conditions; 2) in plasma, the enhancement of fibrinolytic activity by histones was abolished by a neutralizing anti-FSAP antibody; and 3) histones promoted the activation of plasma FSAP. The effect of the natural mixture of histones on scu-PA-driven fibrinolysis was differentially recapitulated by the individual recombinant histones, H4 displaying the strongest activity. When complexed to DNA, histones still accelerated scu-PA-mediated fibrinolysis but with a lesser efficiency due to a reduced FSAP activation. Finally, preincubation of histones with heparin or activated protein C, two known inhibitors of histones, further amplified histone-mediated boost of scu-PA-driven fibrinolysis. ConclusionsEnhancement of FSAP-mediated scu-PA activity by histones may play yet unforeseen roles in intravascular fibrinolysis and contribute to extravascular proteolysis and tissue damage.

Enhancement of fibrinolytic activity in vascular endothelial cells by heterologous expression of adenine nucleotide translocase-1

The fibrinolytic activity of blood is regulated by expressing tissue-type plasminogen activator (t-PA) and its specific inhibitor, type-1 plasminogen activator inhibitor (PAI-1), from vascular endothelial cells. Since t-PA is a major plasminogen activator in blood, it is considered that the binding protein for t-PA, which exists on endothelial cell membrane, immobilizes t-PA on the surface of endothelial cells and enhances their antithrombotic property. Recently, we have found a new t-PA binding protein in endothelial cells. Its amino acid sequence has matched that of human adenine nucleotide translocase-1 (ANT1). The aims of this study are to confirm the binding of t-PA to ANT1, and to clarify the effect of ANT1 on fibrinolytic activity around endothelial cells. ANT1 is prepared from recombinant glutathione S-transferase (GST)-ANT1 fusion protein, and reveals t-PA binding activity in a ligand blot assay. In addition, ANT1 is exclusively expressed on endothelial cell membrane by using pDisplay vector. Interaction of t-PA with ANT1, which is expressed on the surface of endothelial cells, is confirmed by IAsys binding analysis and chromogenic assay. The heterologous expression of ANT1 on endothelial cell membrane enhances the t-PA binding ability of endothelial cells and the effect of ANT1 expression on fibrinolytic activity is demonstrated by increasing t-PA-catalyzed plasminogen activation. These results suggest that a novel t-PA-binding protein, ANT1, may concentrate t-PA on the surface of cells and enhance fibrinolytic properties around endothelial cells; therefore, ANT1 can be a powerful tool for regulating the plasminogen activation system in the vessel.

On stimulation of fibrinolytic activity by pneumoencephalography.

The fibrinolytic activity of blood and cerebrospinal fluid (CSF) after pneumoencephalography (PE) has been investigated in ten patients. Samples of blood and CSF were collected and assayed before and 10, 20, 30, and 60 minutes after PE. It was noted that the rise in fibrinolytic activity in CSF preceded the increased fibrinolytic activity which occurred in blood and was of shorter duration. It is suggested that two different mechanisms are involved in the enhancement of fibrinolytic activity in blood and CSF. Injection of air may produce the release of fibrinolytic agents into the CSF from meninges and choroidal plexus which are very rich in plasminogen activator. The stimulation of systemic fibrinolytic activity seems instead mediated by the action of the neurovegetative system.

Historical Perspective on Garlic and Cardiovascular Disease

Cardiovascular disease is a complex and multifactorial disease characterized by such factors as high cholesterol, hypertension, reduced fibrinolysis, increase in blood-clotting time and increased platelet aggregation. Dietary therapy is the first step in the treatment of hyperlipidemia; garlic has been used medicinally for centuries and is still included in the traditional medicine of many cultures. Historically, there has been great interest in the role of garlic in reducing cardiovascular risk factors. Evidence from numerous studies points to the fact that garlic can bring about the normalization of plasma lipids, enhancement of fibrinolytic activity, inhibition of platelet aggregation and reduction of blood pressure and glucose. However, some contradictory results have also emerged as a result of methodological shortcomings, the use of different formulations/preparations of garlic and different time scales of the studies. Accordingly, further clinical studies are required in which standardized formulations of garlic with known compositions can be used. Such formulations (e.g., Aged Garlic Extract) are now available and are being investigated. Evidence obtained from these studies indicates that garlic has potential in the prevention and control of cardiovascular disorders and is beneficial when taken as a dietary supplement.

Haemostatic system and arterial disease: future perspectives

The thrombotic component in ischaemic heart disease (IHD) is now generally accepted. Two main questions to be answered are, first, how far it is necessary to further improve our understanding of the biological processes involved and, secondly, the implications of what we have already learned for prevention, both secondary and primary. As far as life-style modification is concerned, distinguishing between the lipid and haemostatic contribution is of limited practical significance since the recommendations to be made are the same from either viewpoint. Implications for pharmacological prevention are more complex. The strong and independent relation of fibrinogen not only with IHD but also with stroke and lower extremity arterial disease raises the question of whether lowering fibrinogen levels may be beneficial. In trials now in progress it may be possible to approtion any clinical benefits between the lipid-modifying and fibrinogen-lowering properties of fibrates. Selective fibrinogen-lowering agents and the enhancement of fibrinolytic activity should be developed but uncertainties about the long-term safety of all new medicines indicates the need to make the best use of those already available. The re-evaluation of oral anticoagulants in lower than conventional doses and in combination with platelet-active agents are two such possibilities. Finally, the potential value of agents that simultaneously influence lipids and the haemostatic system should be assessed. Besides fibrates, other lipid-modifying regimens including the statins, recently show to be clinically effective, have these combined properties.

Therapeutic Effect of a Low Molecular Weight Dermatan Sulphate (Desmin 370) in Rat Venous Thrombosis - Evidence for an Anticoagulant-Independent Mechanism

We evaluated the capacity of a low molecular weight dermatan sulphate (D370) to prevent thrombus formation and to induce a reduction of a stabilized thrombus in a rat venous thrombosis model. Injection of D370, 10 min before induction of venous stasis (prevention model), prevented thrombus formation in a dose-dependent way (ED50: 2.3 mg/kg). When given to rats 6 h after induction of venous stasis (therapeutic model), D370 caused a time- and dose-dependent reduction in thrombus size (60% to 70% reduction 2 h after injection of 10 mg/kg). At comparable antithrombotic dosages (i.e. minimum dose giving complete inhibition of thrombus formation), heparin (0.5 mg/kg) only caused 40% reduction of a preformed thrombus while hirudin (1 mg/kg) was virtually ineffective (less than 10% reduction in weight). All three compounds inhibited 125I-fibrin(ogen) deposition on 6-h aged thrombi by more than 85%, suggesting that D370 and, to a lesser extent, heparin reduce thrombus size via mechanisms other than inhibition of thrombus accretion. The involvement of a fibrinolysis-mediated mechanism in the D370-induced effect is suggested by the following. EACA (1 g/kg), when given to thrombus-bearing control animals, did not influence thrombus weight. However, when administered before D370 treatment, it prevented the expected reduction in thrombus weight by more than 80%, without influencing the effect of D370 on 125I-fibrin(ogen) accumulation onto preexisting thrombi. D370 injection caused neither an enhancement of fibrinolytic activity nor a reduction of PAI in plasma. In vitro, D370 (200 microns/ml) was unable to potentiate the spontaneous or PA-induced lysis of 125I-fibrinogen labelled blood, plasma, or purified fibrin clots.(ABSTRACT TRUNCATED AT 250 WORDS)

TFC-612, a prostaglandin E 1 derivative, enhances fibrinolytic activity in rats

TFC-612 inhibited thrombus formation on wire coils inserted into the lumen of the inferior vena cava of rats after 5 oral doses of 1.0 and 3.2 mg/kg and subcutaneous doses of 1.0 and 3.2 pg/rat/hr. This compound showed slight inhibition of platelet aggregation induced by collagen at 1.0 and 3.2 mg/kg (po) and significant inhibition at 10 mg/kg. TFC-612 had no effect on the plasma coagulation system at 3.2 mg/kg. Conversely, oral doses of 0.32– 3.2 mg/kg dose- dependently enhanced fibrinolytic activity as measured by euglobulin clot lysis time and lysis area on fibrin plates by euglobulin fraction. TFC-612 did not enhance fibrinolytic activity in vitro. These results suggest that the enhancement of fibrinolytic activity by TFC-612, which may be due to an increase in tissue plasminogen activator release or reduction of plasminogen activator inhibitors release, contributes to its inhibition of thrombus formation.

Diabetic retinopathy in non-insulin-dependent diabetes mellitus patients: The role of gliclazide

Diabetic retinopathy is the most common cause of human blindness between the ages of 30 and 67 in the industrialized world. Retinopathy has a multifactorial etiology. Standard treatment has aimed at correcting only disturbed glucose metabolism, but this may lead only to the partial amelioration of certain hemobiologic factors. In addition to its metabolic action, gliclazide has been shown to have specific hemobiologic properties; studies with gliclazide in animals and humans have shown significant improvements of platelet abnormalities, stimulation of prostaglandin I 2 synthesis, and enhancement of fibrinolytic activity. In humans, open-label studies have shown that gliclazide treatment leads to stabilization of background retinopathy in non-insulin-dependent diabetics and, more recently, these beneficial effects have been confirmed in controlled studies lasting up to 37 months in which other sulfonylureas were used. The Japanese Diabetic Retinopathy Program studied the progression of retinopathy over a 5-year period, comparing gliclazide with other sulfonylureas and with placebo. This study showed that, with equivalent metabolic control, there was a trend toward a lower rate of deterioration and a significantly lower incidence of preproliferative retinopathy in the group receiving gliclazide compared with those receiving other sulfonylureas. Overall, the specific hemobiologic actions of gliclazide appear to offset or retard the progression of diabetic retinopathy and may have the advantage of lowering the incidence of preproliferative retinopathy.

Fibrinolysis in Normal Subjects - Comparison Between Plasminogen Activator Inhibitor and Other Components of the Fibrinolytic System

We analyzed fibrinolytic parameters in 20 healthy men and 20 healthy women, aged from 25 to 59, before and after 10 and 20 min venous occlusion. The 10 min post-occlusion fibrinolytic activity measured directly in diluted unfractionated plasma by a highly sensitive 125I-fibrin plate assay correlated well with the activity of euglobulins determined by the classical fibrin plate assay (r = 0.729), but pre-stasis activities determined with these two methods did not correlate (r = 0.084). The enhancement of fibrinolytic activity after venous occlusion was mainly due to an increase of t-PA in the occluded vessels (4-fold increase t-PA antigen after 10 min and 8-fold after 20 min venous occlusion). Plasminogen activator inhibitor (PAI) activity and plasminogen activator inhibitor 1 (PAI-1)1 antigen levels at rest showed considerable dispersion ranging from 1.9 to 12.4 U/ml, respectively 6.9 to 77 ng/ml. A significant increase of PAI-1 antigen levels was observed after 10 and 20 min venous occlusion. At rest no correlation was found between PAI activity or PAI-1 antigen levels and the fibrinolytic activity measured by 125I-FPA. However, a high level of PAI-1 at rest was associated with a high prestasis antigen level of t-PA and a low fibrinolytic response after 10 min of venous stasis. Since the fibrinolytic response inversely correlated with PAI activity at rest, we conclude that its degree depends mainly on the presence of free PAI.

Effects of Antibiotics on Fibrinolytic Activity In Vivo

The effects of latamoxef, cefamandole, carbenicillin and cefotaxime on fibrinolytic system in rats were examined under feeding of an ordinary diet or a vitamin K-deficient diet for eight days. No obvious change was observed in UK-induced plasma ciot lysis time, euglobulin lysis time and antiplasmin level in plasma. These findings of the in vitro and ex vivo studies suggest that these antibiotics cause no enhancement of fibrinolytic activity.

Effects of antibiotics on fibrinolytic activity in vivo.

The effects of latamoxef, cefamandole, carbenicillin and cefotaxime on fibrinolytic system in rats were examined under feeding of an ordinary diet or a vitamin K-deficient diet for eight days. No obvious change was observed in UK-induced plasma clot lysis time, euglobulin lysis time and antiplasmin level in plasma. These findings of the in vitro and ex vivo studies suggest that these antibiotics cause no enhancement of fibrinolytic activity.

In vitro stimulation of plasminogen activator release from vein walls by adrenaline.

The effect of adrenaline on plasminogen activator release was studied in vitro in human vein biopsy specimens, in which the fibrinolytic activity was determined according to the fibrin slide technique. The tissue slides were covered with a thin fibrin film containing 10(-9) and 10(-7) M adrenaline and exposed for 30 to 60 minutes. In both concentrations highly significant (p less than 0.001) enhancement of fibrinolytic activity was shown, and the enhancement of fibrinolysis was most pronounced during the first 30 minutes of exposure. Stimulation of fibrinolysis was maximal after exposure to the physiological concentration of 10(-9) M, while no further increase was seen using the pharmacological concentration. These results show that adrenaline has a stimulant effect on tissue fibrinolysis in vitro, and this effect may account for the direct stimulation of fibrinolysis by adrenaline in vivo.

Effect of danazol on clotting factor levels, bleeding incidence, factor infusion requirements, and immune parameters in hemophilia

Several recent studies have reported conflicting results on the effectiveness of danazol, an attenuated androgen, in raising plasma levels of clotting factors VIII and IX in patients with hemophilia. We undertook a randomized, double-blind cross-over trial using 8 weeks' administration of danazol (D), 600 mg/d, and 8 weeks' administration of placebo (P) separated by 2 weeks of rest in 12 patients with hemophilia A and four patients with hemophilia B. Plasma factor VIII and IX levels, frequency and type of bleeding episodes, amount of factor concentrate infused, fibrinogen, fibrinolysis assays, antithrombin III, liver function, and immune parameters were followed. During the danazol phase a minimal increase was noted in the average clotting factor levels, an increase that, although statistically significant, was of hemostatically marginal magnitude. Significant increases in protein C and plasminogen levels, however, were observed during the danazol period, suggestive of danazol-mediated enhanced fibrinolysis. Clinically, bleeding frequency was significantly increased, and more clotting factor was consumed during the danazol period. Furthermore, eight episodes of hematuria and oral mucosal bleeding was reported during the danazol phase in contrast to only one episode of hematuria during the placebo phase, consistent with an enhancement of fibrinolytic activity with danazol. We conclude that danazol does not have a hemostatically significant effect on plasma levels of factor VIII and IX but may be associated with enhancement of fibrinolytic activity, resulting in increased bleeding frequency and requiring more clotting factor infusions. Therefore, danazol is not a viable alternative in the treatment of hemophilia.

Effect of Danazol on Clotting Factor Levels, Bleeding Incidence, Factor Infusion Requirements, and Immune Parameters in Hemophilia

Several recent studies have reported conflicting results on the effectiveness of danazol, an attenuated androgen, in raising plasma levels of clotting factors VIII and IX in patients with hemophilia. We undertook a randomized, double-blind cross-over trial using 8 weeks' administration of danazol (D), 600 mg/d, and 8 weeks' administration of placebo (P) separated by 2 weeks of rest in 12 patients with hemophilia A and four patients with hemophilia B. Plasma factor VIII and IX levels, frequency and type of bleeding episodes, amount of factor concentrate infused, fibrinogen, fibrinolysis assays, antithrombin III, liver function, and immune parameters were followed. During the danazol phase a minimal increase was noted in the average clotting factor levels, an increase that, although statistically significant, was of hemostatically marginal magnitude. Significant increases in protein C and plasminogen levels, however, were observed during the danazol period, suggestive of danazol-mediated enhanced fibrinolysis. Clinically, bleeding frequency was significantly increased, and more clotting factor was consumed during the danazol period. Furthermore, eight episodes of hematuria and oral mucosal bleeding was reported during the danazol phase in contrast to only one episode of hematuria during the placebo phase, consistent with an enhancement of fibrinolytic activity with danazol. We conclude that danazol does not have a hemostatically significant effect on plasma levels of factor VIII and IX but may be associated with enhancement of fibrinolytic activity, resulting in increased bleeding frequency and requiring more clotting factor infusions. Therefore, danazol is not a viable alternative in the treatment of hemophilia.

A FIBRINOLYTIC DEFECT IN CHRONIC BACK PAIN SYNDROMES

Blood fibrinolytic activity was measured in 18 subjects with severe chronic back pain and 18 age and sex matched controls. The patients showed evidence of defective fibrinolysis—namely, significant prolongation of the euglobulin clot-lysis time, reduction in fibrin-plate lysis-area and plasminogen levels, and increase in levels of the fibrinolytic inhibitors α2 antiplasmin and α2 macroglobulin. This defect could be associated with fibrin deposition and scar formation and be responsible for the development and/or perpetuation of chronic inflammation and scarring at sites of damage in the spine. Enhancement of fibrinolytic activity may offer a new approach to the management of these back problems, and a double-blind controlled trial is in progress.

Effects of dilazep on fibrinolytic system in animals. I. Enhancement of fibrinolytic activity by administered dilazep in ex vivo experiment.

The effects of dilazep on blood fibrinolytic system were studied in ex vivo experiment. Fibrinolytic activity in plasma was determined by the fibrin plate method at designed times after the administration of dilazep in guinea pigs, rabbits and mongrel dogs. In guinea pigs, fibrinolytic activity of euglobulin (plasminogen activator level, 1.10+/-0.08 CU/ml in control animals) rose to 1.53+/-0.24, 1.92+/-0.13 and 2.35+/-0.15 CU/ml 2 h after the p.o. administration of 30, 100 and 300 mg/kg of dilazep, respectively. Plasminogen levels in euglobulin were unchanged, however, plasmin inhibitory activities were lowered following the increase of plasminogen activator levels. About 2-fold increase of plasminogen activator level was also observed in rabbits 60 min after the p.o. administration of 300 mg/kg dilazep and returned to the initial level (0.054 CU/ml) within 3 h after the administration. A rapid increase of plasminogen activator level was observed in a case of the i.v. administration of dilazep into rabbits and mongrel dogs. In in vitro experiment, dilazep added to whole blood or plasma of guinea pigs ranging 0.1 to 1000 microgram/ml did not reveal any effects on plasminogen activator level or plasmin inhibitory activity in plasma.

ENHANCEMENT OF FIBRINOLYTIC ACTIVITY

ENHANCEMENT OF FIBRINOLYTIC ACTIVITY