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Tissue plasminogen activator in cultured human trabecular meshwork cells. Predominance of enzyme over plasminogen activator inhibitor.

Maintenance of patency of the trabecular meshwork, the major outflow channel of the anterior chamber of the eye, is necessary to prevent an excessive rise in intraocular pressure. Obstruction of flow due to clot formation results in severe glaucoma and damage to the optic nerve. We have found that human trabecular meshwork cells which have been passaged in tissue culture synthesize large amounts of tissue plasminogen activator (t-PA), based on functional, immunologic and molecular weight analysis. Trabecular cells express substantially more t-PA activity than vascular endothelium which produces t-PA for clot dissolution in the systemic circulation. Vascular cells produce excess t-PA inhibitor while trabecular cells make comparatively little. Trabecular meshwork cells are the first normal cell type reported in which the balance between t-PA and inhibitor is weighted towards the activator, indicating that fibrinolysis may be more important than clotting in the anterior chamber of the eye.

Cells, Cultured↗

Fibrinolytic and haemostatic responses to desamino-D-arginine vasopressin (DDAVP) administered by intravenous and subcutaneous routes in healthy subjects.

DDAVP was administered at 0.4 microgram kg-1 intravenous (i.v.) and subcutaneous (s.c.) routes to 6 healthy subjects in a double blind crossover study. Both study treatments were well tolerated. Flushing occurred after both treatments but was more prominent after i.v. than after s.c. DDAVP. Mild transient local discomfort at the s.c. injection site occurred in 5 of 6 subjects. The mean peak factor VIII (FVIII) response was 369% and 247% of baseline after i.v. and s.c. DDAVP respectively and the maximum increase in FVIII occurred earlier with the i.v. route. Changes in FVIII antigen (FVIII:Ag) and von Willebrand factor antigen (vWF:Ag) were also monitored. Tissue-type plasminogen activator (t-PA) activity measured by a chromogenic assay employing soluble fibrin had a median peak value of 2.9 IU ml-1 at 20 min after i.v. and of 1.9 IU ml-1 at 60 min after s.c. DDAVP. t-PA antigen was also measured so that the specific activity of circulating t-PA could be determined. Preinjection median values of 14,650 and 13,700 IU mg-1 increased to peak median values of 236,200 IU mg-1 at 20 min after i.v. and 202,400 IU mg-1 at 60 min after s.c. DDAVP. Plasminogen activator inhibitor (PAI) activity fell following DDAVP and became undetectable in some subjects during the sampling period. The ratio of maximum fibrinolytic response was similar to the ratio of maximum haemostatic responses obtained by two routes of injection. Our results indicate that s.c. DDAVP might successfully replace i.v. DDAVP in several applications such as confirmation of haemostatic or fibrinolytic responsiveness in patient groups; for obtaining FVIII enriched plasma; as well as its obvious potential usefulness in home treatment of haemophilia A and von Willebrand's disease.

Adult↗

Plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2 in various disease states.

The association of increased PA-inhibitor (PAI) activity and of PAI-1 and PAI-2 antigen levels with different pathological conditions was studied in a collective of over 300 patients. PAI-1 and PAI-2 levels were measured by specific radioimmunoassays. A good correlation was observed of PAI activity with PAI-1 antigen (r = 0.718; p less than 0.0001) but not with PAI-2 (r = 0.070; n.s.). Both in the controls and in the patients, PAI activity and PAI-1 antigen showed an extremely large range of values. PAI activity ranged from 0.5 to 68 U/ml and PAI-1 antigen from 6 to 600 ng/ml. Increased PAI activity and PAI-1 antigen was observed in patients with malignant tumors, cardiovascular or thromboembolic disease, in the postoperative phase, with hepatic insufficiency, after trauma and after extracorporeal circulation. The large spectrum of disease states with increased PAI activity and PAI-1 antigen reinforces previous suggestions that PAI-1 is an acute phase reactant. After extracorporeal circulation, PAI activity and PAI-1 concentrations strongly increased within one hour, remained elevated for at least one week and returned to preoperation values within 7 days. PAI-2 values ranged from below detection limit (15 ng/ml), observed in half of the plasmas, to 485 ng/ml in a pregnant woman. High values of PAI-2 were only observed in pregnancy.

Glycoproteins↗

Fibrinolytic properties of a human endothelial hybrid cell line (Ea.hy 926).

The fibrinolytic characteristics of the endothelial hybrid cell line EA.hy 926, established by fusing a human umbilical vein endothelial cell with a human carcinoma cell line, were studied. The hybrid cell line produced large amounts of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1, and a small amount of urokinase. All plasminogen activator present in conditioned medium was complexed with inhibitor because the cells secreted plasminogen activator inhibitor in excess over plasminogen activator and no activator activity was detectable in conditioned media by direct activity assays. t-PA activator activity was, however, demonstrable in conditioned media after treatment with sodium dodecyl sulfate, in agreement with t-PA antigen determinations. Increased plasminogen activator inhibitor activity could be induced by incubating the cells in the presence of endotoxin or microtubule inhibitors, whereas increased t-PA activity could be induced by microtubule inhibitors. Interleukin-1 had no effect. The fibrinolytic characteristics of the hybrid cell line were stable for at least 30 passages. The perpetual human hybrid cell line EA.hy 926 therefore may be a useful tool for the study of fibrinolysis in cultured endothelial cells.

Cell Line↗

Daytime fluctuations in blood of tissue-type plasminogen activator (t-PA) and its fast-acting inhibitor (PAI-1).

Circadian fluctuation in blood fibrinolytic activity was studied in 10 volunteers in the day-time period at 09.00, 12.00 and 15.00 h. Activity of tissue-type plasminogen activator (t-PA) was found to increase from 09.00 to 15.00 h in accordance with known results with global assays of blood activity. Also, activity and antigen of plasminogen activator inhibitor 1 (PAI-1) and antigen of t-PA showed fluctuations but with an opposite pattern to that of t-PA activity, with highest levels in the morning and lowest ones in the afternoon. Activity of a reversible t-PA inhibitor was constant. The discordance between fluctuations in antigen and in activity of t-PA is attributed to the inhibitory effect of PAI-1 on t-PA activity. It is concluded that the appearance into the blood of t-PA and PAI-1 shows a daytime fluctuation with a similar pattern, suggesting a co-ordinated circadian fluctuation in production of both proteins by endothelial cells.

Adult↗

Increased plasma levels of urokinase-type plasminogen activator with endometrial and cervical cancer.

Plasma concentrations of urokinase-type plasminogen activator (competitive radioimmunoassay), tissue-type plasminogen activator (sandwich enzyme-linked immunosorbent assay), and plasminogen activator inhibitor (functional assay) were measured in 17 women with endometrial cancer and 52 women with cervical carcinoma. Significantly increased plasma urokinase-type plasminogen activator antigen levels were found (11.3 +/- 4.7 ng/mL) in cervical cancer patients when compared with an age-matched control group (7.4 +/- 0.6 ng/mL). Women with endometrial cancer (9.9 +/- 2.0 ng/mL) showed a similar pattern of plasma urokinase-type plasminogen activator antigen levels. Patients with advanced cervical cancer (International Federation of Gynecology and Obstetrics stages III and IV) revealed higher plasma urokinase-type plasminogen activator antigen levels than those with stages I and II disease. Compared with an age-matched control group, neither carcinoma group showed elevated plasma concentrations of tissue-type plasminogen activator and plasminogen activator inhibitor.

Adenocarcinoma↗

[Structure, function and use of fibrinolysis-promoting and inhibiting factors].

Our knowledge about the components of the fibrinolytic system has greatly increased during the last few years thanks to the cloning of the cDNA of the two plasminogen activators t-PA and u-PA, of the two plasminogen activator inhibitors PAI-1 and 2, and of other profibrinolytic and of inhibitory factors of this system. The two principal plasminogen activators (PA) and the two PA-inhibitors are present in infinitesimal concentrations in blood (pM to microM range) and in tissues. The expression of the t-PA and of the u-PA cDNA in mammalian cells and in E. coli bacteria has rendered it possible to produce sufficient amounts of these thrombolytic disorders for clinical studies. Over 2000 patients afflicted with acute myocardial infarction have been treated so far with recombinant t-PA. In general, the thrombolytic effect of t-PA appears to be superior to that obtained with streptokinase and the fibrin-specific-PA produces lesser fibrinogenolysis than the first generation thrombolytic agents streptokinase and two-chain urokinase.

Chemical Phenomena↗

Renal and hepatic handling of endogenous tissue-type plasminogen activator (t-PA) and its inhibitor in man.

In eight male patients with normal liver and kidney function fibrinolytic components were measured in arterial blood and in renal and hepatic vein blood, obtained during catheterization for analysis of hypertension. Blood samples were collected simultaneously from veins und corresponding arteries before and 5 minutes after the completion of intravenous injection of desmopressin (DDAVP), 0.4 micrograms/kg body weight over a 10 minute period. DDAVP induced a rise in t-PA antigen and activity, and in von Willebrand factor, accompanied by a decrease in free PA-inhibitor level. We failed to detect a significant rise in plasma urokinase activity. The concentrations of fibrinogen, plasminogen, alpha 2-antiplasmin, antithrombin III and coeruloplasmin did not change either. Renal production of t-PA under basal conditions was inferred from a negative arterio-venous (A-V) difference in t-PA-activity and in t-PA-antigen levels but this could not be confirmed by orthogonal regression analysis of the same data. A-V differences of other fibrinolytic factors were negligible. In the hepatic vessels a significant positive A-V difference of t-PA-activity and of t-PA-antigen levels was a uniform finding. After DDAVP, when plasma levels were elevated, the mean A-V difference was proportionally higher, consistent with a constant fractional elimination rate. Free PA-inhibitor was virtually absent from arterial blood after DDAVP, but appeared in hepatic vein blood, indicating either production of the inhibitor by the liver or dissociation of a circulating complex of t-PA and its inhibitor in the liver. The blood levels of the other investigated components did not show any change upon passage through the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

Cellular and extracellular plasminogen activator and inhibitor in an experimental tumour.

We determined plasminogen activator (PA) and PA inhibitor (PAI) activities in the intra- and extracellular compartments of an experimental pancreatic ascites tumour with indirect and direct functional assays, and partially characterized these activities on SDS-polyacrylamide gels coupled with fibrin and reverse fibrin autography. Intact tumour cells caused lysis of plasminogen-rich but not plasminogen-free fibrin clots, and the extent of lysis of the former was related to tumour cell count. Direct assay of PA with a synthetic substrate yielded an equivalent of 109 urokinase units per 10(9) tumour cells. No PAI activity was demonstrated in tumour cells with functional assays. Contrary to tumour cells, cell-free ascitic fluids caused no lysis of fibrin clots. Instead, it inhibited tumour cell- and urokinase-induced, but not plasmin-induced, clot lysis in a dose-dependent fashion. Although functional assays failed to demonstrate PA in ascitic fluid and PAI in tumour cells, both activities were detected in electrophoresed samples of cell lysates and fluids by fibrin and reverse fibrin autography. In tumour cells, a mixture of tissue-type PA (tPA) and urokinase-type PA (uPA) were present. In the fluid, uPA together with two other PAs with greater molecular weights than tPA were detected.

Animals↗

Levels of fibrinolytic activators and inhibitors in plasma after severe trauma.

Levels of tissue plasminogen activator antigen (t-PA:ag), tissue plasminogen activator inhibitor 1 antigen (PAI-1:ag) and tissue plasminogen activator inhibitor activity (PAI activity), and alpha-2-antiplasmin (AAP) were measured in plasma from 19 patients with severe trauma on admission and on days 1, 2, 3 and 7 after the incident. In all patients the Injury Severity Score (ISS) and the number of blood transfusions were recorded. The development of post-traumatic pulmonary dysfunction was observed in four patients. Levels of t-PA:ag, PAI-1:ag and PAI activity were increased, and levels of AAP were low immediately after trauma. Levels of t-PA:ag normalized during the first week, whereas PAI-1:ag levels decreased gradually from day 1 to day 3. Thereafter a secondary increase was observed. A similar trend was observed in levels of PAI activity. Levels of APP increased significantly during the first week. t-PA:ag, PAI-1:ag or PAI activity levels were not correlated with the ISS on any day, but levels of AAP showed a weak correlation with the ISS on day 7. Post-traumatic levels of t-PA:ag and PAI-1:ag were higher in patients who had 6 or more units of blood transfusions for resuscitation. The fibrinolytic markers were not significantly different in patients who had pulmonary dysfunction compared with patients without.

Adolescent↗

The operative peritoneal fibrinolytic response to abdominal operation.

OBJECTIVE: To measure changes in the fibrinolytic properties of human peritoneum during operation. DESIGN: Open study. SETTING: University hospital, UK. SUBJECTS: 20 patients undergoing elective operations for non-inflammatory disease. INTERVENTIONS: Peritoneum was biopsied at the beginning and end of operation. MAIN OUTCOME MEASURES: Peritoneal plasminogen activating activity (PAA) and the concentrations of tissue plasminogen activator (t-PA), urokinase, and plasminogen activator inhibitors 1 and 2 were measured at both time points. RESULTS: Peritoneal PAA was reduced over the time of the operation (p < 0.05) as was the concentration of t-PA (p < 0.05). The urokinase concentration rose significantly (p < 0.05), but plasminogen activator inhibitors 1 and 2 were not detected. CONCLUSIONS: Elective abdominal operation caused an immediate reduction in peritoneal PAA which seemed to be secondary to a reduced concentration of t-PA. Such a reduction in peritoneal fibrinolytic activity allows the early deposition of fibrinous deposits within the peritoneal cavity.

Abdomen↗

[Tissue-type plasminogen activator in patients with lupus nephritis].

ELISA was used to measure tissue plasminogen activator antigen in blood plasma and urine of 42 patients with active lupus nephritis (16 with isolated urinary syndrome, 13 with nephrotic syndrome, 13 with rapidly progressive LN). Control groups consisted of 17 patients with inactive LN and 15 healthy subjects. Inhibition of fibrinolysis in vascular system correlating with the disease severity was found in patients with active LN. This may result from both defective synthesis of tissue plasminogen activator and neutralizing action of antiactivator. Urinary fibrinolytic activity was inhibited only in patients with rapidly progressive LN because of hyperactive synthesis of antiactivator. The above changes occur as endothelial cell dysfunction due to activating and damaging action of immune complexes, antibodies, cytokines on vascular endothelium. Zero activator activity of plasma and urine in patients with active LN reflects severe dysfunction of endothelial cell caused by its structural disorganization.

Adolescent↗

[Epidemiological study of blood stasis and plasma tissue plasminogen activator and its inhibitor activity in healthy aged].

According to the blood stasis survey of 77 healthy subjects in presenile geriatric period, in comparison with 35 healthy young people, it was found the 51.79% of healthy subjects had blood stasis. The detectable rate were significantly different among various age groups (P < 0.005), and the rate increase was age-dependent. Chromogenic peptide substrate analytical method was used to detect plasma tissue plasminogen activator (t-PA), tissue plasminogen activator inhibitor (PAI) activity and ratio of P/t. The result revealed that the activity of plasma t-PA in presenile geriatric period subjects was significantly higher than that of young people (P < 0.01), but the activity of PAI had no difference in various age groups, and the ratio of P/t was markedly decreased. It showed that this was the physiological compensatory reaction of aging organism against hypercoagulability, and it was an effective way to maintain the physiological balance of the aged.

Age Factors↗

Effects of fish oil on plasma fibrinolysis in patients with mild rheumatoid arthritis.

As rheumatoid arthritis (RA) patients have been shown to have impaired plasma fibrinolysis and fish oil has been suggested to be useful for RA, this study investigated the effects of fish oil on fibrinolytic parameters in patients with RA. Forty-five RA patients were randomised to receive either fish oil (1.7 gm eicosapentaenoic acid and 1.1 gm docosahexaenoic acid/day) or placebo treatment for at least 6 months. Plasma levels of fibrinogen, tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI) activity were measured at 3-month intervals. In the fish oil treatment group, plasma levels of fibrinogen and t-PA activity were reduced at 6 months when compared with baseline [fibrinogen: 3.2 (2.85-3.53) milligrams vs 3.89 (3.56-4.22) milligrams, mean (95% confidence intervals for mean), p < 0.02; t-PA activity 1.4 (1.01-1.78) units/ml vs 1.94 (1.55-2.33) units/ml, p < 0.01]. No significant changes in plasma PAI activity were seen during the treatment period in these patients. Placebo treatment did not significantly alter the plasma levels of fibrinogen or t-PA and PAI activity. In conclusion, fish oil supplementation does not appear to produce an improvement in plasma fibrinolysis. Indeed, plasma fibrinogen levels and t-PA activity were reduced. This could be due to an effect of fish oil on acute phase protein production. Alternatively, as t-PA is produced on an "on demand" basis, its reduction may be related to the lowering of fibrinogen levels following fish oil therapy.

Adult↗