Measurement Of Fibrinolytic Activity Research Articles

Importance of Endogenous Fibrinolysis in Platelet Thrombus Formation.

The processes of thrombosis and coagulation are finely regulated by endogenous fibrinolysis maintaining healthy equilibrium. When the balance is altered in favour of platelet activation and/or coagulation, or if endogenous fibrinolysis becomes less efficient, pathological thrombosis can occur. Arterial thrombosis remains a major cause of morbidity and mortality in the world despite advances in medical therapies. The role endogenous fibrinolysis in the pathogenesis of arterial thrombosis has gained increasing attention in recent years as it presents novel ways to prevent and treat existing diseases. In this review article, we discuss the role of endogenous fibrinolysis in platelet thrombus formation, methods of measurement of fibrinolytic activity, its role in predicting cardiovascular diseases and clinical outcomes and future directions.

Alcohol-induced up-regulation of fibrinolytic activity and plasminogen activators in human monocytes.

Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0-1 hr), followed by incubations in the absence of alcohol (0-24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). Brief exposure (15 min, 4 degrees C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 +/- 5.6 fmol/1 x 10 cells versus 656.0 +/- 94.0 fmol/1 x 10 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 +/- 174.7 vs. 965.33.0 +/- 104.8 fmol/1 x 10 cells) and an approximately 3- to 4-fold (20.5 +/- 2.14 vs. 74 +/- 2.28 fmol/2 x 10 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37 degrees C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 +/- 0.02 vs. 0.42 +/- 0.02) and an approximately 2- to 3-fold (0.89 +/- 0.04 vs. 2.07 +/- 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.

Indicators of depressed fibrinolytic activity in pre-operative prediction of deep venous thrombosis.

Euglobulin lysis time (ELT), tissue plasminogen activator (tPA), and the fast-acting inhibitor of tPA, were measured pre-operatively in 128 patients who underwent elective major abdominal surgery. Deep venous thrombosis (DVT) was detected by 125I-labelled fibrinogen scan in 37 patients (29 per cent) after operation. Pre-operatively, there was diminished euglobulin lysis activity (332 +/- 197 versus 255 +/- 156 min, mean +/- s.d.; P less than 0.025), and tissue plasminogen activator activity (4.2 +/- 9.9 versus 7.7 +/- 14.3 milliunits/ml, mean +/- s.d.; P = 0.094) in patients who subsequently developed postoperative DVT compared with those who did not. There was no significant difference between the two groups in the level of inhibition of tissue plasminogen activator (160.6 +/- 75.4 per cent versus 152.5 +/- 77.5 per cent, mean +/- s.d.; n = 47). Stepwise logistic discriminant analysis of the data obtained preoperatively showed that tissue plasminogen activator, a more specific measure of fibrinolytic activity, was a weaker predictor of DVT than euglobulin lysis time. The results confirm other observations which indicate that lowered fibrinolytic activity is a risk factor for postoperative DVT. In addition, they suggest that this is not due entirely to low levels of activity of tissue plasminogen activator in plasma.

Metabolic Syndrome Accompanied by Hypercholesterolemia Is Strongly Associated With Proinflammatory State and Impairment of Fibrinolysis in Patients With Type 2 Diabetes

To determine whether plasma concentrations of thrombin-activatable fibrinolysis inhibitor (TAFI) in patients with type 2 diabetes were associated with components of metabolic syndrome (MS), including high-sensitivity C-reactive protein (hs-CRP), plasminogen activator inhibitor (PAI)-1, and LDL cholesterol. We studied 136 consecutive patients with type 2 diabetes. Diagnosis of MS was diagnosed by current criteria. Hypercholesterolemia (HC) was defined as serum LDL cholesterol >140 mg/dl (3.6 mmol/l) or treatment with a statin. For comparisons, diabetic patients were divided into four groups: those with no MS and no HC (n = 38), with MS but not HC (n = 39), with no MS but with HC (n = 26), and with both MS and HC (n = 33). Considering all patients with type 2 diabetes, plasma PAI-1 was strongly associated with MS components such as BMI, triglyceride, alanine aminotransferase, a homeostasis model assessment of insulin resistance, and hs-CRP. Plasma TAFI only correlated positively and independently with LDL cholesterol. Plasma concentrations of plasmin-alpha2-antiplasmin complex (PAP), a measure of fibrinolytic activity in blood, showed a significant negative correlation with plasma PAI-1 but not TAFI. Diabetic patients with both MS and HC had the highest serum hs-CRP concentrations and the lowest plasma PAP concentrations. LDL cholesterol is a main determinant of plasma TAFI in patients with type 2 diabetes. Coexistence of MS and HC synergistically accelerates inflammation and impairment of fibrinolysis via elevated concentrations of both TAFI and PAI-1, which inhibit fibrinolysis.

Alcohol-Induced Up-Regulation of Fibrinolytic Activity and Plasminogen Activators in Human Monocytes

Background Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis. Methods Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0–1 hr), followed by incubations in the absence of alcohol (0–24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction). Results Brief exposure (15 min, 4°C) of U937 cells to low alcohol resulted in an approximately 2- to 3-fold increase (269.0 ± 5.6 fmol/1 × 106 cells versus 656.0 ± 94.0 fmol/1 × 106 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4- to 5-fold increase (414.0 ± 174.7 vs. 965.33.0 ± 104.8 fmol/1 × 106 cells) and an approximately 3- to 4-fold (20.5 ± 2.14 vs. 74 ± 2.28 fmol/2 × 106 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5- to 6-fold (0.06 ± 0.02 vs. 0.42 ± 0.02) and an approximately 2- to 3-fold (0.89 ± 0.04 vs. 2.07 ± 0.29) increase in t-PA and u-PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde-3-phosphate dehydrogenase ratio), respectively. Conclusions These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t-PA/u-PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.

Alcohol‐Induced Up‐Regulation of Fibrinolytic Activity and Plasminogen Activators in Human Monocytes

Background Moderate alcohol consumption is associated with reduced risk for coronary heart disease. This may due, in part to increased fibrinolysis. Monocytes synthesize fibrinolytic proteins, tissue plasminogen activator (t‐PA), urokinase plasminogen activator (u‐PA), and their receptors. These studies were carried out to determine the effect of low alcohol on monocyte fibrinolytic activity and PA messenger RNA (mRNA) synthesis.Methods Peripheral blood monocytes and U937 cells were incubated in absence/presence of low alcohol (0.1%, v/v) for various times (0–1 hr), followed by incubations in the absence of alcohol (0–24 hr) before measurement of fibrinolytic activity and PA mRNA levels (reverse transcriptase polymerase chain reaction).Results Brief exposure (15 min, 4°C) of U937 cells to low alcohol resulted in an approximately 2‐ to 3‐fold increase (269.0 ± 5.6 fmol/1 × 106 cells versus 656.0 ± 94.0 fmol/1 × 106 cells) in fibrinolytic activity. Preincubation of U937 cells and peripheral blood monocytes in low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (24 hr) resulted in a sustained approximately 4‐ to 5‐fold increase (414.0 ± 174.7 vs. 965.33.0 ± 104.8 fmol/1 × 106 cells) and an approximately 3‐ to 4‐fold (20.5 ± 2.14 vs. 74 ± 2.28 fmol/2 × 106 cells, respectively) increase in fibrinolytic activity. Preincubation of monocytes with low alcohol (1 hr, 37°C) followed by incubation in the absence of alcohol (6 hr) resulted in an approximately 5‐ to 6‐fold (0.06 ± 0.02 vs. 0.42 ± 0.02) and an approximately 2‐ to 3‐fold (0.89 ± 0.04 vs. 2.07 ± 0.29) increase in t‐PA and u‐PA mRNA (reverse transcriptase polymerase chain reaction; PA/glyceraldehyde‐3‐phosphate dehydrogenase ratio), respectively.Conclusions These data suggest that low alcohol exerts a rapid, direct, and sustained effect on monocyte fibrinolytic activity, which may be, due in part, to increased monocyte t‐PA/u‐PA expression. These data provide a feasible molecular mechanism by which alcohol effects on monocyte fibrinolysis may contribute to the cardioprotective benefit associated with moderate alcohol consumption.

Relationship between soluble thrombomodulin in plasma and coagulation or fibrinolysis in type 2 diabetes

Serum concentration of soluble thrombomodulin (TM) is thought to be a marker for endothelial damage. Although several studies have reported that serum TM concentrations are increased in patients with diabetes mellitus, there is little information on the physiological function of soluble TM in human plasma. To evaluate the relationship of soluble TM in plasma between coagulation and/or fibrinolysis system in patients with diabetes, we measured plasma soluble TM, protein C activity (a natural anticoagulant induced by thrombin–TM complex), prothrombin F1+2 (a direct marker of thrombin generation), and plasmin–α2-antiplasmin complex (PAP) and D dimer (measures of fibrinolytic activity) in 55 patients with type 2 diabetes mellitus. The plasma concentrations of soluble TM ( P<0.01), protein C activity ( P<0.01), prothrombin F1+2 ( P<0.05), PAP ( P<0.001) and D dimer ( P<0.001) were significantly higher in the diabetic patients than the 48 age-matched control subjects. The plasma concentrations of TM and PAP were obviously increased in patients with diabetic nephropathy. In the diabetic patients, the plasma concentrations of soluble TM were inversely correlated with the protein C activity ( r=−0.43, P<0.005), and were positively correlated with the plasma concentrations of prothrombin F1+2 ( r=0.63, P<0.0001) and the plasma PAP concentrations ( r=0.30, P<0.05). The present study demonstrated that both coagulation and fibrinolysis are enhanced concomitantly in patients with type 2 diabetes mellitus, and that an increase in plasma concentration of soluble TM is associated not only with hypercoagulability but also with enhanced fibrinolysis in diabetic patients.

Insulin resistance syndrome and fibrinolytic activity: the northern Sweden MONICA study.

Many studies have, in small and highly selected study populations, described how cardiovascular risk factors tend to cluster in subjects with insulin resistance. Recently, interest has focused on possible relationships between this insulin resistance syndrome and fibrinolysis, and the role of triglycerides in this association. The present study addresses these issues in a general population. A subsample of participants in the population-based Northern Sweden MONICA (MONItoring of trends and determinants in CArdiovascular diseases) Study, consisting of 353 men and 403 women in the 25-64 year age range, was investigated. Insulin resistance was estimated indirectly from the fasting levels of insulin and glucose. Fibrinolytic activity was measured both as plasminogen activator inhibitor type 1 (PAI-1) activity and tissue plasminogen activator ((t)PA) activity. Insulin resistance was highly correlated with those cardiovascular risk factors that have been associated with the insulin resistance syndrome, and to the measures of fibrinolytic activity. Subjects in the upper tertile of insulin resistance had a PAI-1 activity that was three times higher than that of the lower third men and twice as high in women. There was a strong interaction between insulin resistance and serum triglycerides. Low versus high levels of both variables together were associated with a fivefold difference in PAI-1 activity in men and a threefold difference in women. The (t)PA activity was inversely correlated to both insulin resistance and serum triglycerides. In a general population, the 'insulin resistance syndrome' is closely associated with low fibrinolytic activity. Serum triglyceride levels interact with insulin resistance to predict fibrinolytic activity.

Structural and functional characterization of the inhibition of urokinase by .alpha.2-macroglobulin

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity. According to gel electrophoretic analyses under reducing conditions, urokinase cleaved alpha 2M subunits and formed apparently covalent complexes with alpha 2M. Urokinase cleaved only about 60% of the alpha 2M subunits maximally at a mole ratio of 2:1 (urokinase: alpha 2M). Binding of urokinase to alpha 2M protected the urokinase active site from inhibition by antithrombin III-heparin and inhibited, to a significant extent, plasminogen activation by urokinase. Reaction of urokinase with alpha 2M caused an increase in intrinsic protein fluorescence and, thus, induced the conformational change in alpha 2M that is characteristic of its interactions with active proteinases. Our results indicate that both in plasma and in a purified system the alpha 2M-urokinase reaction is functionally significant.

Method for measuring fibrinolytic activity in a single layer of cells.

A method has been developed for measuring fibrinolytic activity in a single layer of cells--for example, venous endothelium or peritoneal mesothelium. A single layer of cells was collected on a gelatin disc, incubated on a fibrin plate for 24 h, and the resulting area of lysis measured. This was converted to a measure of fibrinolytic activity expressed in Ploug units of urokinase by reference to areas of lysis created by standard amounts of urokinase placed on similar fibrin plates. The method has been used for measuring fibrinolytic activity in venous endothelium and peritoneal mesothelium and has demonstrated that the activity in a single layer of endothelial cells is only about one-quarter of that in an equivalent area of whole vein wall. It has also shown that peritoneal fibrinolytic activity is reduced after peritoneal trauma. This method maybe useful in the investigation of the fibrinolytic system in a variety of pathological conditions--for example, thrombosis and intraperitoneal adhesions.

The Effect of Plasma Fibrinogen Levels on Measures of Fibrinolytic Activity

It has been claimed that lysis time methods may be inappropriate as measures of fibrinolytic activity on the grounds that they are largely determined by plasma fibrinogen levels. The dilute blood clot lysis time (DBCLT) has therefore been measured in 103 subjects; fibrinolytic activity (expressed as 100/DBCLT) has been compared with the fibrin plate area (FPA) lysed by the euglobulin fraction, the latter method being, of course, virtually independent of plasma fibrinogen. Plasma fibrinogen levels have also been measured; they ranged from 167 to 416 mg%. Results have been analysed by the technique of multiple regression. The incorporation of plasma fibrinogen levels in the regression equation relating 100/DBCLT to FPA does not result in a significant increase in the proportion of the variance in 100/DBCLT that can be explained. It is concluded that there is no evidence that DBCLT is influenced by plasma fibrinogen levels within the physiological range.

Studies on the Fibrin-Agarose and Agar Plate Methods for Measurement of Fibrinolytic Activities

The methods for assessment of fibrinolytic activity has not been well established, although the fibrinogen plate method developed by Astrup et al. has been utilized as an assay method of fibrinolytic activity so far. There are, however, some difficulties associated with the preparation, use and interpretation of the Astrup fibrin plate. Our standard and plasminogen free fibrin agarose and agar plate method is made with a little modification of Bisohp's fibrin plate method.Standardized method is as follows. The solution is composed of 0.12% plasminogen free fibrinogen (Daiichi Seiyaku Co. Tokyo) and 1.25% agarose (1% agar) in 0.005M phosphate buffer (pH 7.5).Sodium azide is added at the rate of 0.1% to agarose solution as an antiseptic. Fibrin plate using 2.5×7.0×0.15cm plastic plate is made with 2.52ml of a above solution and added 0.5ml of thrombin solution which is adjusted to 50 unit per one ml of saline. Then washed in distilled water in a moment exactly 15 seconds after addition of thrombin. Plate is covered with a strip of filter paper and seald airtight in plastic bag. Those plates are kept at 4°C before use. Wells of 2mm of diameter are mechanically punched in the fibrin plate just before experiment.Using this method, several interesting findings are obtained in our study on the several preparatory codition, effect of pH, fibrinogen consentration, agarose consentration…etc, of the fibrin agarose plate.

A Comparative Ex Vivo Study of Plasminogen Activators and Proteases for Fibrinolytic Activity and Side Effects in Rabbits

Urokinase, streptokinase, Brinase, trypsin, and SN 687, a bacterial exoprotease, have been evaluated in an ex vivo assay system. These enzymes were injected into rabbits and the fibrinolytic activity as well as other coagulation parameters were measured by in vitro techniques. Dose-response correlations have been made using the euglobulin lysis time as a measure of fibrinolytic activity and the 50% effective dose has been determined for each enzyme. Loading doses, equal to four times the 50% effective dose were administered to monitor potential toxicity revealing that Brinase, trypsin and SN 687 were very toxic at this concentration. Having established the 50% effective dose for each enzyme, further testing was conducted where relevant fibrinolytic and coagulation parameters were measured for up to two days following a 50% effective dose bolus injection of each enzyme. Our results have demonstrated that urokinase and streptokinase are plasminogen activators specifically activating the rabbit fibrinolytic system while Brinase, trypsin and SN 687 increase the general proteolytic activity in vivo. The advantages of this ex vivo assay system for evaluating relative fibrinolytic potencies and side effects for plasminogen activators and fibrinolytic proteases have been discussed.

The relationship of the dilute whole blood lysis time to the fibrinolytic activity of blood: effect of change in plasma fibrinogen.

The rate of digestion of fibrin was measured in vitro by an isotopic technique in 140 blood samples of differing fibrinogen concentration; the assessment of fibrinolytic activity thus obtained was compared with a standard method of measurement, the dilute whole blood lysis time. The lysis time was related exponentially to the fibrinolytic activity as measured by the isotopic technique, and further was influenced markedly by alteration in the plasma fibrinogen concentration. The relevance of these observations to the use of lysis time methods for the measurement of fibrinolytic activity is discussed.

Increased fibrinolytic activity and fibrin degradation products after experimental intracerebral haemorrhage.

The fibrinolytic activity of the peripheral blood and cerebrospinal fluid was studied in 10 dogs in which intracerebral haematomas had been produced by injection of blood into the brain. Blood and cerebrospinal fluid samples were obtained at regular intervals before and after operation. The fibrinolytic activity of these two fluids were estimated by the fibrin plate method and by assays for fibrin degradation products. After the production of the haematoma, signs of increased fibrinolytic activity were found in blood and cerebrospinal fluid one day after the intracerebral injection in 6 animals. A correlation was found between the size of the haematoma and the level of the fibrinolytic activity of blood and cerebrospinal fluid. Determination of fibrin degradation products in blood and cerebrospinal fluid and the measurement of fibrinolytic activity by the plate method complement each other and are therefore recommended in this type of investigation.

Evaluation of fibrinolysis in hepatic cirrhosis. Relation of serial thrombin time and euglobulin lysis time.

For nearly half a century various observers have reported increased fibrinolytic activity in the blood of patients with hepatic cirr h o s i s . 3 ' 6 0 1 0 ' 1 6 1 6 The availability of «-aminocaproic acid (EACA), a potent inhibitor of plasminogen activators, has led to renewed interest in the mechanisms involved in the fibrinolysis of hepatic cirrhosis. 10 Recently, Fletcher and associates suggested that a major factor in this abnormal fibrinolysis was a failure of hepatic clearance mechanisms for plasminogen activator. In 13 instances of cirrhosis of the liver, Grossi and co-workers reported successful control of fibrinolysis and bleeding with intravenous EACA during and after portacaval shunt surgery. All of these cases demonstrated fibrinogenopenia and increased fibrinolysis; however, Lewis and Doyle indicated that although EACA therapy in cirrhosis of the liver decreased fibrinolytic activity, it did not improve the hemostatic mechanisms. In the following study, the fibrinolytic mechanism in hepatic cirrhosis was assayed by (1) the serial thrombin time (STT), (2) euglobulin lysis time, and (3) fibrinogen levels. These parameters were compared in normal persons and in patients with cirrhosis before and after the administration of nicotinic acid used to stimulate fibrinolysis. The euglobulin lysis measures plasminogen activators. The STT method was originally developed by Wilhelm and associates. Ingram and Matchett used this technic as a measure of fibrinolytic activity. They demonstrated a linear relation between proportionate loss of clot

Lysis of Arterial Clots

ADVANCES in vascular surgery techniques are largely responsible for the preservation of previously unsalvageable limbs. However, such techniques sometimes fail because of extensive clotting in the distal arterial tree which is not amenable to the usual operative approaches. A method has been developed for the dissolution of clots which may prove to be a useful adjunct to arterial surgery in the extremities and which may also be extended to other similar problems. This method utilizes large doses of fibrinolysin in an isolation perfusion system similar to that reported by Reid in 1962.3However, in this experiment, clot lysis is studied, in addition to the measurement of fibrinolytic activity, changes in peripheral pressure, and the long term results. Isolation perfusion techniques have been developed over the past few years mainly for the administration of cancer chemotherapeutic agents. Recently this method has been adapted for other uses. Isolation perfusion techniques have

The Measurement of Fibrinolytic Activity with I131-labelled Clots. II. Application

Summary1. The application has been described of a method, using I131-labelled clots as a substrate for the measurement of plasmin, urokinase and streptokinase.2. With this method the inhibition of these fibrinolytic agents by plasma has been studied.3. A striking difference in the fibrinolytic and fibrinogenolytic effect of urokinase and streptokinase was found. Urokinase appeared to be an effective fibrinolytic agent in the presence of plasma and gave rise to little fibrinogenolysis, while streptokinase appeared much less effective in this respect in the presence of plasma, but gave marked fibrinogenolysis.

The Measurement of Fibrinolytic Activity with I131-labelled Clots. I. The Methods

SummaryThe technical details of a method for the measurement of fibrinolytic activity with I131-labelled clots are described. The advantages of this modification of the method of Alkjaersig are discussed.

Selective inactivation of the plasminogen contaminant in thrombin.

THE contamination of preparations of thrombin and fibrinogen with plasminogen, the precursor of the fibrinolytic enzyme, has always presented great difficulties in the measurement of fibrinolytic activity.