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It is known that in vitro plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may lead to artefactually low fibrinogen and alpha 2-antiplasmin values. To mimic this phenomenon, pooled normal plasma was supplemented with 2.5 micrograms/ml t-PA and incubated at various temperatures. The rates of fibrinogen degradation and alpha 2-antiplasmin consumption were most pronounced at 37 degrees C, were less pronounced at 25 degrees C, but surprisingly, did not further decrease at 10 degrees C, 0 degrees C or -8 degrees C. In contrast, when plasma was supplemented with 160 IU/ml urokinase or 30 IU/ml streptokinase, the rates of fibrinogen degradation and alpha 2-antiplasmin consumption gradually decreased with incubation temperature and were negligible at 10 degrees C and lower temperatures. The rate of plasminogen activation also decreased gradually with temperature in mixtures of purified fibrinogen, plasminogen, alpha 2-antiplasmin and t-PA. These results imply that, in a plasma milieu, additional factors with a stimulatory activity are involved in t-PA-induced plasminogen activation at around 0 degrees C. The abnormally high reaction rate at low temperatures explains in vitro plasminogen activation observed during the processing of t-PA-containing blood samples. In contrast to the activation of plasminogen by t-PA, the slow inhibition of t-PA (2.5 micrograms/ml) by proteinase inhibitors in plasma could be minimized to a negligible level by keeping the plasma samples at 0 degrees C. This makes it possible to reliably monitor t-PA activity during thrombolytic therapy.

Objective: To study the influence of α- and β-adrenoceptor stimulation in vivo on fibrinolysis in healthy male volunteers. Design: Infusions were given in a single blind fashion and in a randomized order if placebo infusions were included. Setting: University Department (Karolinska Hospital). Subjects: Thirty-four healthy, non-smoking male volunteers aged 21-38 years. Interventions: Study 1 - The volunteers participated in two or three separate experiments; 30 min infusions of saline placebo, phentolamine (500 μg/min) or propranolol (bolus dose of 140 μg/kg + 0.5 μg/kg/min) followed by the addition of a 30 min adrenaline infusion (0.4 nmol/kg/min), n=10 in each group. Study 2 - Infusion of a low and an intermediate-high dose of noradrenaline (0.15 and 0.75 nmol/kg/min; 20 min at each dose level), n=11. Study 3 - Infusion of a low and high dose of isoprenaline (5 and 20 ng/kg/min), each during 20 min, n=10. Main outcome measures: Venous plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigen and activity levels, and plasma levels of urokinase (u-PA) antigen. Results: t-PA activity and antigen levels were markedly increased during adrenaline infusion (by 309±35 and 81±16% respectively, both P

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in (factor XH-dependent) fibrinolysis in plasma is still unclear. The aim of our study was to investigate plasminogen activator activity (PAA) of both FXIIa in a purified system and endogenous FXIIa in plasma by measuring fibrinolysis in a clot lysis assay. During activation of purified FXII by trypsin, PAA was generated and remained stable even when FXIIa was completely cleaved to smaller fragments. Far more PAA was generated during autoactivation of purified FXII in the presence of dextran sulphate (DXS), a negatively charged component. However, PAA of autoactivated FXIIa decreased when it was cleaved to smaller fragments and reached a plateau comparable to PAA after activation by trypsin, while amidolytic activity of FXIIa remained constant. The same result was found when purified FXII was activated by kallikrein in the presence of DXS, while PAAwas reduced even more rapidly due to faster cleavage of FXII. This finding suggested that DXS may potentiate PAA of FXIIa in the clot lysis assay and not of the FXIIa fragments, missing the binding domain for DXS. Indeed, DXS was found to potentiate PAA of FXIIa more than tenfold according to a template model. Factor XII-dependent fibrinolysis is routinely measured in a dextran sulphate euglobulin fraction (DEF) of plasma and accounts for about 50% of total PAA. Since DXS was found to potentiate PAA of purified FXIIIa, FXI1 itself may be an important candidate for factor XIIdependent PAA in the DEF, a milieu in which DXS is present. Therefore, a DEF was prepared from normal human plasma and immunodepleted of FXIIa using an anti-FXII IgG Sepharose column. About 20% of factor XII-dependent PAA in the DEF could be ascribed to FXIIa. This study demonstrates that the fibrinolytic activity of FXIIa is substantially potentiated by DXS. Due to this potentiation, FXIIa significantly contributes to factor XII-dependent fibrinolysis in plasma as measured in a dextran sulphate euglobulin fraction.

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a two-chain form (tcu-PA/T), which is virtually inactive in plasminogen activator assays. Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was examined in the plasma of 27 healthy individuals and of 17 sepsis patients, and in the synovial fluid of 16 rheumatoid arthritis patients. In addition, the concentration of urokinase antigen and scu-PA were measured in all three groups. In the plasma of the healthy individuals no measurable amounts of tcu-PA/T could be found (< detection limit of 0.2 ng/ml). In the plasma of almost all sepsis patients tcu-PA/T could be detected (median value 0.4 ng/ml). The amount of tcu-PA/T was 12% of the amount of scu-PA and accounted for about 9% of urokinase antigen. In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml) at a concentration which was twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% of the urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of large amounts of thrombin. This way thrombin may regulate fibrinolysis and extracellular proteolysis. The BIA for tcu-PA/T can be of use for further research on the physiological role of tcu-PA/T. Chemicals/CAS: Thrombin, EC 3.4.21.5; Urinary Plasminogen Activator, EC 3.4.21.73

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T). Little is known about the physiological importance of tcu-PA/T. To examine the occurrence of tcu-PA/T in vivo, we developed a sensitive and specific bioimmunoassay (BIA) for the assessment of tcu-PA/T in human body fluids. In this BIA, urokinase antigen was immuno-immobilized in microtiter plates and treated with cathepsin C, a specific activator of tcu-PA/T, after which plasminogen activator activity was measured. The occurrence of tcu-PA/T was assessed in the plasma of healthy individuals and sepsis patients, and in the synovial fluid of rheumatoid arthritis patients. In addition, the concentrations of urokinase antigen and scu-PA were measured. In the plasma of the healthy individuals the concentration of tcu-PA/T was below the detection limit of 0.2 ng/ml. In the plasma of almost all sepsis patients tcu-PA/T was found (median value 0.4 ng/ml). In the synovial fluid of all rheumatoid arthritis patients tcu-PA/T could be measured (median value 5.4 ng/ml), and its concentration was about twofold higher than the concentration found for scu-PA. In this group tcu-PA/T contributed to about 47% (median value) of urokinase antigen. From these data we conclude that inactivation of scu-PA by thrombin can take place in vivo under pathological conditions which involve the production of thrombin. In this way thrombin may regulate fibrinolysis and extracellular proteolysis.

Neoplastic growth and metastatic spread of adenocarcinomas is characterized by a marked increase of urokinase-type plasminogen activator (u-PA) and a decrease of tissue-type plasminogen activator (t-PA). In this study, the authors determined the activity and antigen levels of u-PA and t-PA, and their inhibitors, plasminogen-activator inhibitors types 1 and 2 (PAI-1 and PAI-2), in normal mucosa, adenomatous polyps, and adenocarcinomas of the human colon. The decrease in t-PA activity in the neoplastic tissues, determined enzymatically and zymographically, was significantly correlated with an increase in PAI-1 and PAI-2, in particular in carcinomas. In spite of significantly higher inhibitor levels in the neoplastic tissues, u-PA was found to be increased as well, both in antigen level and in activity. The authors conclude that PAI-1 and PAI-2 are significantly increased in neoplastic tissue of the human colon and contribute considerably to the decrease of t-PA activity in carcinomas. However, the malignancy-associated increase in u-PA seems not to be affected by the plasminogen activator inhibitors. Thus, it appears that there is an imbalance between plasminogen activators and their inhibitors in colonic neoplasia in favor of u-PA, which may contribute to plasmin-mediated growth, invasiveness, and metastasis. This feature was also noticed in adenomatous polyps, supporting the malignant potency of adenomas.

Several investigators have reported that tumor necrosis factor (TNF) can alter the production of plasminogen activator type-1 (PAI-1) and plasminogen activators (PAs) by endothelial cells in vitro. We have examined the in vivo effects of recombinant human TNF administration on fibrinolysis as assessed by parameters in plasma during a 24-hour period of continuous TNF infusion to 17 cancer patients with active disease. The plasma levels of PAI activity increased sevenfold after 3 and 24 hours of TNF infusion. This was the result of an increase of PAI-1 antigen; PAI-2 antigen was not detectable. Plasma concentrations of tissue-type PA (t-PA) antigen increased twofold to fivefold after 3 and 24 hours of TNF infusion, whereas urokinase-type PA antigen levels in plasma remained unaltered. After 3 hours of TNF infusion the plasma levels of α2-antiplasmin were slightly decreased, 5% on average, suggesting that fibrinolysis continued. After 24 hours of TNF infusion a highly significant increase in fibrin- plus fibrinogen-degradation products, and separately of fibrin degradation products and fibrinogen degradation products, was found. This indicates that fibrinolysis persisted, at least partly, in the presence of high levels of PAI activity. Whereas PAI-1 production increased, t-PA production by human endothelial cells in vitro remains unaltered or even decreases on TNF addition. It has been shown previously that TNF infusion in our patients results in thrombin and fibrin generation. Therefore, it is possible that thrombin, not TNF, is the actual stimulus for t-PA production in our patients. We speculate that fibrin is formed during TNF infusions and that plasmin is generated by t-PA action immediately on the initial formation of (soluble) fibrin molecules. Such a process may explain the generation of degradation products of both fibrin and fibrinogen during infusion of TNF in patients. Chemicals/CAS: plasminogen activator inhibitor 1, 140208-23-7; tissue plasminogen activator, 105913-11-9; alpha-Macroglobulins; Fibrin Fibrinogen Degradation Products; Plasmin, EC 3.4.21.7; plasmin-alpha(2)-macroglobulin complex; Plasminogen Inactivators; Recombinant Proteins; Tissue Plasminogen Activator, EC 3.4.21.68; Tumor Necrosis Factor; Urinary Plasminogen Activator, EC 3.4.21.73

Endothelial cells play an important role in the regulation of fibrinolysis by the production of several key regulatory proteins. The cytokines tumor necrosis factor (TNF), lymphotoxin, and interleukin-1 (IL-1), but not interleukin-6, increase the production of plasminogen activator inhibitor-1 (PAI-1) by endothelial cells, whereas they have no stimulatory effect on the production of tissue-type plasminogen activator (t-PA). Primary cultures of human endothelial cells release very little urokinase-type plasminogen activator (u-PA). We report here that TNF and lymphotoxin induce, in a concentration-dependent way, the production of both cellular and secreted u-PA antigen in primary and subcultured human endothelial cells. The TNF-induced increase was accompanied by a more than 10-fold increase in u-PA mRNA. Upon stimulation of early passage umbilical vein endothelial cells by TNF, u-PA was predominantly secreted at the basolateral side, whereas PAI activity and t-PA were found in more equal amounts at the apical and basolateral sides of the cell monolayers. TNF-stimulated u-PA secretion by subcultured human aorta endothelial cells showed only a marginal polarity. The u-PA antigen was present in a plasmin-activatable form (single chain u-PA) and in a nonactivatable form (probably u-PA: PAI-1 complex). During the induction of u-PA by TNF, the ratio between plasmin-activatable u-PA and total u-PA decreased markedly. This may indicate that TNF also increases the degree of u-PA activation. The parallel induction of the synthesis and secretion of both u-PA and PAI-1 by endothelial cells adds a new aspect to the alterations of the fibrinolytic system caused by inflammatory mediators. This aspect may be significant for the regulation of cell-associated and interstitial plasminogen activator activity.

SummaryPlasminogen activators were determined in intestinal tissue, obtained after surgery from patients with Crohn’s disease and ulcerative colitis, and compared with normal intestinal tissue from colorectal cancer patients.The activity and quantity of tissue-plasminogen activator (t-PA) was found to decrease with the severity of inflammation in the patients with inflammatory bowel disease. Urokinase (u-PA) activity, however, was not changed compared with controls or in relation with severity of inflammation. In contrast, the level of u-PA antigen was found to be increased significantly in the inflammatory bowel disease tissues and was also related with severity of inflammation. The difference between u-PA activity and antigen in inflammatory bowel disease tissue could be attributed to an increase in inactive pro-u-PA and u-PA-inhibitor complexes.This increase in u-PA and the concomitant decrease in t-PA, are similar to those found in premalignant colonic adenomas, and might be related to the known increased cancer risk in inflammatory bowel disease.