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.     

Effect of diluents on blood clot lysis

One of the current methods used for measuring natural fibrinolytic activity in blood involves dilution of the blood with phosphate buffer. An adaptation of this method using a solution of sodium acetate as diluent is described. Evidence is given to show that dilute blood clots lyse not only because of the dissociation of an antiplasmin-plasmin complex, but also by virtue of a reduction of the intrinsic chloride ion concentration.     

Fibrinolytic activity of tissue surfaces during surgery.

A non-invasive method has been developed for the study of tissue surface fibrinolytic activity during surgery in man. Surface exudate was collected using a filter-paper disc which was then applied directly to a fibrin plate or used to prepare a euglobulin fraction. The method detected the intraoperative increase in fibrinolytic activity. At 45 minutes from the start of surgery the increase in lytic activity for tissues was four to eight times greater than that of venous blood taken simultaneously. The technique may be used to compare regional differences in tissue fibrinolysis, to determine the effect of excess local fibrinolysis on postoperative bleeding, and to study the relation between low fibrinolytic activity and postoperative adhesion formation.     

On the significance of the fibrinolytic system of the blood

Summary Subject to a study in experiments on dogs (involving intravenous administration of nicotinic acid and thromboplastin, blood loss) and in observations of patients (operative interventions, atherosclerosis, thromboembolic disease, etc.) were interrelationships between the blood coagulation, fibrinogen concentration, fibrinolytic and fibrinogenolytic activity. The blood of healthy individuals and dogs showed fibrinolytic activity to be present regularly. Fibrinogenolytic activity, on the other hand, was absent. In stress conditions (surgical intervention, blood loss, nicotinic acid administration) the blood coagulation was accelerated with blood fibrinolytic activity increasing at the same time (no fíbrinogenolysis was present). Intensified fibrinolytic activity of the blood is a normal protective body reaction, occurring with acceleration of blood coagulation. This regularity is disturbed in pathological conditions. Association of accelerated blood coagulation with diminished fibrinolysis (atherosclerosis, thromboembolic disease) creates favorable conditions for thrombosis. There is no direct relationship between blood fibrinogen concentration and its fibrinolytic activity. This is explained by the fact that, as a rule, activation of fibrinolytic system in the body leads to the intensification of its fibrinolytic properties, but not of fibrinogenolytic ones.(Presented by Active Member of the AMN SSSR A. V. Lebedinskii) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 54, No. 9, pp. 35–39, September, 1962     

ANTIFIBRINOLYTIC ACTIVITY IN NORMAL PREGNANCY

It is found that serum antifibrinolytic activity is inhibitory both to the spontaneous fibrinolytic activity and to the activity of streptokinase-activated plasminogen.     

Reaction pattern to three stresses—electroplexy, surgery, and myocardial infarction—of fibrinolysis and plasma fibrinogen

The effects of three types of stress-electroplexy, surgery, and myocardial infarction-on blood fibrinolytic activity and plasma fibrinogen levels were studied in 10, eight, and six patients respectively. The fibrinolytic response to electroplexy consisted of an initial short increase followed in half the patients by reduced fibrinolytic activity lasting two to four days. After surgery and myocardial infarction normal fibrinolytic activity was followed by a period of reduced activity; the timing of the measurements on these patients may have precluded recognition of an initial increase in fibrinolysis similar to that following electroplexy. The fibrinolytic ;shutdown' which lasted for about 10 days in the coronary patients was evidently due to reduction of plasminogen activator, as judged by prolongation of the euglobulin lysis times as well as of the blood clot lysis times. Plasma fibrinogen levels rose in the surgical and coronary patients but not in the patients given electroplexy which indicates that fibrinolytic activity changes independently of plasma fibrinogen level. The results suggest that the fibrinolytic system exhibits a common reaction pattern to stress, irrespective of its nature and of tissue damage. They call for caution in assuming a specific causal association in acute diseases such as pancreatitis and haematemesis where similar fibrinolytic changes may be encountered.     

Timed fibrin digestion: A simplified technique for the measurement of the fibrinolytic activity of the blood

When the rate of fibrinolysis in vitro was measured by an isotopic technique, it was found that the amount of fibrin digested after incubation for three hours correlated closely with the overall rate of the digestion process. It is suggested that, using an isotopic technique, estimation of fibrin digestion after three hours' incubation provides a useful method for measuring the fibrinolytic activity of blood. A recommended technique is described and its advantages are discussed briefly.     

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.     

Advances in clinical fibrinolysis

The discovery of a fast-acting plasminogen activator inhibitor has resulted in the notion that the balance between tissue-type plasminogen activator and its inhibitor determines the net fibrinolytic activity of blood. The inhibitor shows a rapidly fluctuating acute-phase pattern, which may be important in relation to thrombosis in acute disease. Other newly discovered modulators of the fibrinolytic system include histidine-rich glycoprotein, tetranectin and thrombospondin. The role of fibrin as a cofactor in its own dissolution is further elucidated with emphasis on local aspects. Therapeutic inhibition of overactive fibrinolysis by various drugs needs careful monitoring. Prophylactic stimulation of fibrinolysis is possible, e.g. by stanozolol or other drugs that lower inhibitor levels, but its proven value is as yet limited. Results of clinical trials with activators of the fibrinolytic system as thrombolytic agents are discussed in relation to the physiology of the fibrinolytic system.     

Localization of plasminogen activator inhibitor-1 production in inflamed appendix by in situ mRNA hybridization

The peritoneum has been shown to possess fibrinolytic activity which is thought to play a role in the prevention of intra-abdominal adhesion formation. Recently inflamed peritoneal tissue has been shown to have reduced fibrinolytic activity secondary to increased levels of plasminogen activator inhibitor-1 (PAI-1). The aim of this study was to localize the production of PAI-1 in appendix tissue using in situ mRNA hybridization. Sections of normal and inflamed appendix were hybridized with a digoxigenin-labelled cDNA probe. PAI-1 production was localized to both mcsothelium and serosal blood vessel endothelium in all inflamed appendix samples. Cell identities were confirmed using immunohistochemistry directed against mesothelial and endothlial cell markers. Staining was not seen on sections of normal appendix or on negative control slides of inflamed appendix (hybridization with plasmid DNA, PAI-1 probe following ribonuclease digestion). The identification of the cells expressing the PAI-1 gene in peritoneum increases our understanding of the pathophysiological changes in fibrinolytic activity which occur in inflammation and may lead to adhesion formation.     

Tranexamic acid reduces early post-operative blood loss after total knee arthroplasty: a prospective randomised controlled trial of 29 patients

INTRODUCTION: Extensive blood loss related to knee arthroplasty is quite normal and many patients require blood transfusions. Surgery and the use of pneumatic tourniquets lead to an increase in the activity of the fibrinolytic system, which in turn may accentuate the blood loss. Drugs that inhibit the fibrinolytic system may thus be used to reduce blood loss. Tranexamic acid (TA) acts by binding to one of the enzymes at the start of the coagulation cascade, so inhibiting the fibrinolytic system. A concern is that this inhibition may have the side effect of increasing thromboembolic disease, a common complication of joint replacement surgery. We aimed to confirm the reductions in blood loss and to assess the impact of TA usage on clinical and sub-clinical DVT. METHOD: We performed a prospective, randomised, double blind, controlled trial, using patients due to undergo primary unilateral total knee arthroplasty. Patients were randomised to receive either 15 mg/kg of tranexamic acid or a similar volume of normal saline at the time of cementing of the prosthesis. Perioperative blood loss was recorded and patients were screened for DVT with duplex ultrasound assessment of both legs on the fifth post-operative day. RESULTS: A statistically significant (p=0.006) decrease in blood loss in the early post-operative period was noted in the group receiving tranexamic acid. This was not associated with a significant difference in total blood loss (p=0.55) or in transfusion requirements. There was no of evidence in DVT in either group on duplex ultrasound screening of the lower limbs. INTERPRETATION: One injection of 15 mg/kg of tranexamic given at the time of cementing the prosthesis in total knee arthroplasty, before deflation of the tourniquet, significantly decreases the amount of blood loss in the early post-operative period. The treatment was not associated with an increase in thromboembolic complications.     

Effect of local exercise and vessel occlusion on fibrinolytic activity.

Arterial occlusion of the upper limb did not affect the fibrinolytic activity in the venous blood of that limb; venous occlusion associated with venous distension resulted in a marked rise in activity. Local fibrinolytic activity was increased substantially by active exercise; passive exercise induced a smaller increase. Active exercise undertaken during arterial occlusion did not result in a rise in activator level. It is concluded from these results that blood flow into the venous system rather than accumulation of metabolites is responsible for the increased release of activator into the blood during local exercise and vessel occlusion.     

The fibrinolysis system in recurrent myocardial infarction]

Studies of blood fibrinolytic activity in 112 patients with repeated acute myocardial infarction or injury to the myocardium have revealed reduced fibrinolytic activity on non-heated fibrin plates, decreased plasmin activity and euglobulin+ fraction lysis, lowered levels of plasminogen activator, total nonenzymic fibrinolysis, antithrombin III, ++FFDP, and elevated soluble complexes of fibrin-monomer level. Complications of myocardial infarction presenting as thromboembolism; ciliary arrhythmia, chronic aneurysm with thrombosis are associated with still more marked disorders of the fibrinolysis system.     

Effects of changes in temperature (local and central) on plasma fibrinolytic activity

The effect of local and whole-body heating and cooling on plasma fibrinolytic activity has been investigated in 59 subjects. An increase in whole-body temperature and both local cooling and heating increased the fibrinolytic activity. The results demonstrate that the application of local heat or cold will produce a local increase in fibrinolytic activity which is independent of general factors such as emotion and stress.     

Fibrinolytic response of diabetics and non-diabetics to adrenaline

The fibrinolytic response of 30 diabetic patients and 35 non-diabetics to 0.5 mg. subcutaneous adrenaline was measured, using the dilute clot lysis test. With the exception of two patients out of six who were taking phenformin, who had a depressed response, the diabetics showed a similar increase in fibrinolytic activity to the non-diabetics. The diabetics in this series did not tend to have longer spontaneous lysis times than the non-diabetics, although this type of comparison may not be valid.     

Complex compounds of heparin with blood proteins and their natural inhibitors in splenectomy in animals

In depression of the function of the anticoagulating system after splenectomy in rats the blood serum heparin level diminishes, the fibrinogen concentration increases, the total and nonenzymatic fibrinolytic activity decreases, and the activity of some heparin complexes (adrenaline-heparin and serotonin-heparin) increases. A low-molecular protein inhibiting nonenzymatic fibrinolytic activity was isolated from blood plasma of splenectomized animals, some of its properties were studied. No such protein inhibitor was found in blood plasma of false-operated and intact rats.     

Plasminogen Activator of the Blood Vessels in Tumours and in Carrageenin-induced Granulomas

Fibrinolytic activity in tumours was studied by the fibrin slide technique. The tumour cells were inactive and fibrinolysis was seen only in areas with young blood vessels. In carrageenin-induced granulomas at 6 days the fibrinolytic activity was small and confined to mature veins, but from 7-14 days activity was high in zones containing young vessels supplying the terminal capillary buds; these latter showed no activity. In old fibrosed granulomas there was no fibrinolytic activity. The vascular permeability changes of inflammation (detected by the colloidal carbon technique) showed no correlation with fibrinolytic activity, and systemic injection of inflammatory agents had no effect on the fibrinolytic activity of the vessels. These findings are discussed in relationship to tumour vascularization.     

Antithrombin III, plasminogen, plasmin, and alpha-2-antiplasmin in donor blood and plasma components

The functional levels of antithrombin III, plasminogen, plasmin, and alpha-2-antiplasmin were evaluated in sequentially derived fresh frozen plasma, cryoprecipitate, and cryo-poor plasma aliquots from 20 registered blood donors. Antithrombin III is the major plasma inhibitor of the serine proteases of the procoagulant system. Plasminogen, the proenzymatic form of plasmin, is the primary endogenous profibrinolytic moiety; alpha-2-antiplasmin is the principal intermediate acting inhibitor of plasmin. As congenital or acquired deficiencies of antithrombin III and/or plasminogen predispose to thrombosis, and as these agents may be consumed in acute thrombosis, the goal of this investigation was to discern those plasma components which potentially might maximize both antithrombotic and fibrinolytic activities if used therapeutically. Using enzyme specific synthetic substrate methods, it was determined that no spontaneous plasmin activity was evident through phlebotomy or component processing and storage. Analysis of variance showed antithrombin III to be significantly decreased in cryoprecipitate as compared to the other components (p less than 0.0001). Furthermore, the level of antithrombin III or plasminogen in cryo-poor plasma and fresh frozen plasma was not statistically different. Also, the alpha-2-antiplasmin level was not statistically different among the specimen groups. Since fresh frozen plasma and cryo-poor plasma contain comparable total quantities of antithrombin III and plasminogen and as most of the activity of factors I, V, VIII, and XIII is diverted into cryoprecipitate, it is suggested that cryo-poor plasma may be preferable to fresh frozen plasma for the treatment of thrombosis associated with or complicated by antithrombin III and/or plasminogen deficiency.     

The distribution of plasminogen activator in the male genital tract

The distribution of fibrinolytic activity in the tissues of the male genital tract was studied by a histological technique. Preparations made from testis, epididymis, vas deferens, seminal vesicle, prostate, bulbo-urethral gland, and urethra showed that most activity was related to the blood vessels. However, inconsistent fibrinolytic activity related to epithelium was found in all parts of the genital tract. This epithelial activity was least in the testis, greater in the seminal vesicle and prostate gland, and was greatest in the bulbo-urethral gland and terminal urethra. No fibrinolytic activity could be demonstrated in relation to spermatozoa.     

Studies on C1 inactivator resistant fibrinolytic activity

The fibrin lysis activity in the presence of enough quantity of C1 inactivator (C1.INA) is called C1.INA resistant fibrinolytic activity (C1.IRFA). C1.INA inhibits the activation of intrinsic fibrinolysis, therefore C1.IRFA is considered to reflect the extrinsic fibrinolytic activity mainly. We studied the effect of C1.INA using fibrin plate assay on fibrin lysis activity (FLA) and C1.IRFA in normal individuals and pregnant women, and studied the fluctuation of FLA and C1.IRFA during venous occlusion test (VO) or 1-deamino-8-D-arginine vasopressin (DDAVP) administration. After VO or DDAVP administration, the euglobulin lysis time (ELT) was reduced, FLA increased and C1.IRFA also increased. The increase in C1.IRFA accounted for most of the increase in FLA. Therefore, the increased fibrinolytic activity during VO or DDAVP administration is due to extrinsic fibrinolytic activity. In the pregnant women, the fibrinolytic activity was decreased during pregnancy and the low fibrinolytic activity is suggested to be involved in the low extrinsic fibrinolytic activity. The measurement of C1.IRFA is considered to be useful.