Euglobulin Fraction Research Articles

Lipoprotein(a) levels and fibrinolytic activity in patients with nephrotic syndrome

Recently there has been a renewed interest in the possibility that lipoprotein(a)--Lp(a)--may be important in the pathogenesis of thrombosis-related disease. In nephrotic syndrome, hyperlipidemia is a common finding, and thrombosis is a major complication. With this regard, if Lp(a) levels increase concomitantly with low-density lipoprotein and/or very-low-density lipoprotein levels in nephrotic syndrome, this may be considered a thrombogenic factor. To probe this possibility and to corroborate the relationship between Lp(a) and fibrinolytic profiles, we measured the Lp(a) levels in patients with nephrotic syndrome (n = 43), in patients with chronic glomerulonephritis with less proteinuria than in nephrotic syndrome (n = 28), and in healthy controls (n = 50) and observed the relation between Lp(a) levels and tissue-type plasminogen activator (t-PA) activity, euglobulin fibrinolytic activity, and t-PA antigen. The Lp(a) levels were significantly higher in the patients with nephrotic syndrome as compared with both patients with chronic glomerulonephritis and healthy controls (p < 0.001). There was a direct correlation with serum cholesterol level (r = 0.780; p = 0.0001), triglyceride level (r = 0.445; p = 0.0001), and urine protein level (r = 0.675; p = 0.0001) and a reverse correlation with serum albumin levels (r = 0.566; p = 0.0001). The Lp(a) levels showed a reverse correlation with t-pA activity (r = 0.627; p = 0.0001), total fibrinolytic activity in euglobulin fraction (r = 0.458; p = 0.0001), and t-PA activity divided by the t-PA antigen (r = 0.567; p = 0.0001), but no correlation with t-PA antigen.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthetic dipeptide, N-stearoyl-D-Ser-L-Pro-OEt, induces release of tissue-type plasminogen activator in cultured cells and in experimental animals.

The tissue-type plasminogen activator (t-PA)-releasing action of synthetic dipeptides containing Gly, Ser or Pro was investigated. Among 10 dipeptides, Boc-L-Ser-L-Pro-OH and H-L-Ser-L-Pro-OH induced t-PA release in vitro, but the others were inactive. Since Boc-L-Ser-L-Pro-OH was more effective than H-L-Ser-L-Pro-OH, 7 related dipeptides with N-acylation were synthesized. Five of them enhanced the release of t-PA; N-stearoyl-L-Ser-L-Pro-OH (FK-5) had the greatest effect. Four compounds were further examined for activity to enhance the release of t-PA in rats. FK-5 produced a two-fold increase in fibrinolytic activity, and N-palmitoyl-L-Ser-L-Pro-OH (FK-4) also markedly enhanced the release of t-PA. Since FK-5 caused severe hemolysis, 7 analogues of FK-5 were synthesized. All of them enhanced the release of t-PA from melanoma (Bowes) cells. In rats, FK-5, N-stearoyl-D-Ser-L-Pro-OH (FK-8) and N-stearoyl-D-Ser-L-Pro-OEt (FK-10) enhanced the fibrinolytic activity two-fold. FK-5 and FK-8 also exhibited strong hemolytic activity, but FK-10 did not induce hemolysis. Therefore, FK-10 was examined in rabbits. After the injection of this compound, the fibrinolytic activity in the euglobulin fraction was markedly enhanced without accompanying hemolysis. Thus, FK-10 potently enhances fibrinolytic activity both in vitro and in vivo.

Characterization of serum immunoglobulins in a chondrostean fish, Acipenser baeri

The euglobulin fraction of sturgeon ( Acipenser baeri) serum was analyzed using electrophoretic and immunoblotting techniques. The major protein of this fraction is an IgM-like molecule composed of equimolar 70-kDa glycosylated H chains and 26–30 kDa L chains. In the absence of a reducing agent, the L and H polypeptides may form (μ2L2)n high molecular weight polymers, μ2L2 170-kDa units or L2 dimers. These different bonding patterns suggest some structural heterogeneity in the distribution of cysteine residues along the sturgeon Ig chains. The H chain N-terminal sequence indicates significant homologies with the conserved V HIII subgroup. Heavy chains antigenically different from the 70-kDa H chain were not detected, suggesting that IgM is the only Ig class synthesized by this sturgeon species.

Impairment of fibrinolysis during the growth of two murine mammary adenocarcinomas.

The fibrinolytic activity present in the euglobulin (EU) fraction of BALB/c mice before and during the growth of M3 and MM3 murine mammary adenocarcinomas was characterized. The main plasminogen activator (PA) form contained in EUs from control mice was defined as murine urokinase-type PA (uPA). Overall fibrinolytic activity decreased significantly during tumor development. Zymographies showed that this fall was associated with a reduction in the free uPA band (47 kDa) and to the detection of a tissue-type PA (tPA) complexed band (117 kDa). Western blotting showed free tPA protein (68 kDa) in control mice, that disappeared in M3 tumor-bearing mice. In this model, high subcutaneous tumor burden induces a severe impairment in the circulating fibrinolytic system.

Favorable long-term effect of a low-fat/high-fiber diet on human blood coagulation and fibrinolysis.

In an 8-month strictly controlled dietary study of 16 healthy young men, the long-term effect of a low-fat (26% of energy) high-fiber (4.5 g/MJ) diet on cardiovascular risk markers of the hemostatic system was assessed. Fasting blood sampling was performed during a 4-week baseline period and then monthly during the intervention. A matched control group of 16 men on habitual diets was also monitored. Median fibrinolytic activity of tissue-type plasminogen activator (t-PA) in plasma was significantly elevated (twofold to fourfold) by the experimental diet. A significant increase in the systemic fibrinolytic activity of the euglobulin fraction of plasma was also observed. Median plasma factor VII coagulant activity (F VIIc) was depressed by 5-10% during the first 2 months and the last month of the study period. The dietary change did not significantly affect plasma levels of fibrinogen, t-PA antigen, or plasminogen activator inhibitor type I antigen. In conclusion, young men who were switched from a typical Danish diet high in saturated fat to a low-fat/high-fiber diet showed a permanent increase in plasma fibrinolytic activity and a biphasic decrease in F VIIc. The dietary change thus had a favorable effect on cardiovascular risk markers of the hemostatic system.

Simultaneous Determination of Free Tissue-Type and Free Urokinase-Type Plasminogen Activators in Biological Fluids by a Solid-Phase Immunoassay

Tissue-type and urokinase-type plasminogen activators (t-PA and u-PA) coexist in numerous biological fluids, where their fibrinolytic activities are determined by the concentration of the free, uncomplexed species. A simple, sensitive method has been developed for the simultaneous determination of free t-PA and u-PA concentrations in biological fluids using a solid-phase immunoassay. Microtiter plates were coated with polyclonal goat antibodies and incubated with PA standards or unknown samples. The absorbed PAs were then assayed by incubation with a mixture of plasminogen, poly-L-lysine, and the chromogenic substrate H-D-norleucylhexahydrotyrosyllysine- p-nitroanilide. Free t-PA and free u-PA were detectable in human plasma and urine, and in conditioned media from different endothelial cell cultures. The method is sensitive, with lower limits of quantitation being 0.76 mU/ml (1.25 pg/ml) for free t-PA and 0.16 mU/ml (2.0 pg/ml) for free u-PA. There was no cross-reaction between the two PA species and the recovery in plasma was greater than 95% for both. The intra- and interassay coefficients of variation for t-PA and u-PA were 3.5-12.2 and 3.2-11.3%, and 2.4-11.8 and 1.6-10.4%, respectively. The presence of PA-inhibitor complexes and PA inhibitors in biological fluids did not interfere with the assay. Application of the assay has demonstrated that free u-PA levels are several fold higher than free t-PA levels in human plasma and in conditioned media of human vascular endothelial cells. Free t-PA and free u-PA in human plasma obtained from five resting, healthy male volunteers ranged from 18 to 72 mU/ml (0.03 to 0.12 ng/ml) and from 54 to 100 mU/ml (0.68 to 1.25 ng/ml), respectively. The results of the present investigation indicate that free t-PA and free u-PA can be quantitated in a straightforward manner without acidification of plasma and preparation of euglobulin fractions during sample processing.

Fibrinolytic Activity in End-Stage Renal Disease

Recently, we described that in the physiologic state, urokinase concentration is higher in the renal vein than in the renal artery which suggests that the kidney is the essential organ providing urokinase to the systemic circulation. In end-stage renal disease (ESRD) patients, urokinase provided by the kidney may be decreased, and this might be the cause of decreased systemic fibrinolytic activity seen in ESRD. To study this possibility, we measured fibrinolytic profiles including urokinase, tissue plasminogen activator (t-PA) and fibrinolytic activity in the euglobulin fraction of ESRD patients and compared the results with those of two control groups consisting of renal transplantation patients and age- and sex-matched healthy controls. The euglobulin fibrinolytic activity was 92.2 +/- 14.8 BAU in the ESRD group, 108.7 +/- 13.5 BAU in the kidney transplantation (KT) group and 101.5 +/- 8.0 BAU in the healthy control group. Although it was lower in the ESRD than in the KT (p < 0.005) and the healthy control group (p < 0.005), there was no difference between the KT and the healthy control group. t-PA antigen was 1.2 +/- 1.3 ng/ml in the ESRD group, 3.5 +/- 1.7 ng/ml in the KT group and 3.1 +/- 2.1 ng/ml in the healthy control group. It was lower in the ESRD compared to the KT (p < 0.001) and the healthy control group (p < 0.001) but there was no difference between the KT and the healthy control group.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dilution, pH and ionic strength of plasma on t-PA precipitation in euglobulin fraction.

In order to evaluate the influence of dilution, pH and ionic strength on the precipitation of t-PA and PAI-1 during euglobulin precipitation, we measured t-PA Ag, PAI-1 Ag and fibrinolytic activity in the euglobulin fraction made of pooled plasma from liver cirrhosis patients, under various conditions by changing pH, ionic strength and degree of dilution. The precipitation of t-PA Ag in the euglobulin fraction was enhanced by decreasing the ionic strength and greatest at pH 6.0. The fibrinolytic activity in the euglobulin fraction showed consistent changes with t-PA Ag under varying pH and ionic strength. The precipitation of t-PA Ag was not influenced by the dilution factor but the larger the dilution factor, the greater the PAI-1 and the smaller the fibrinolytic activity in the euglobulin fraction. PAI Ag in euglobulin fraction showed consistent changes with t-PA Ag in the euglobulin fraction regardless of the changes in ionic strength and pH. The amount of precipitation of t-PA and PAI-1 was increased by the presence of dextran sulfate, under varying pH, ionic strength and dilution conditions. Our results show theat the currently used conditions for standard euglobulin precipitation are the most favorable for t-PA precipitation into the euglobulin fraction. The fibrinolytic activity exerted in the euglobulin fraction seems to depend on the amout of t-PA-PAI-1 complex rather than minimized protease inhibitor in the euglobulin fraction.

Euglobulin clot lysis induced by tissue-type plasminogen activator is reduced in subjects with increased levels of lipoprotein(a)

Several reports have evaluated the in vitro effect of lipoprotein(a) [Lp(a)] levels on the fibrinolytic system, suggesting that high Lp(a) levels may inhibit fibrinolysis by competing for plasminogen binding in different systems. We have studied plasminogen activation induced by tissue-type plasminogen activator (t-PA), as well as other fibrinolytic parameters, in 25 subjects with Lp(a) levels > 30 mg/dl and the results were compared with those found in 23 subjects with Lp(a) < 30 mg/dl. Both groups were similar in age, sex distribution, living habits and lipid pattern. Plasminogen activation, when measured by t-PA-induced euglobulin clot lysis, was significantly decreased in the group with elevated Lp(a) levels (lysis time, 16.7±3.3 min) compared with the group with low Lp(a) levels (11.8±2.0 min), although 8 of the 25 subjects with high Lp(a) levels showed plasminogen activation within the range of the control group. A positive significant correlation between Lp(a) levels and t-PA-induced euglobulin clot lysis time was found. No statistical differences were demonstrated between groups for the other fibrinolytic parameters studied. Addition of purified Lp(a) to the euglobulin fraction or to plasma resulted in a decrease in euglobulin clot lysis. The present study shows that t-PA induced plasminogen activation is decreased in individuals with high circulating levels of Lp(a) supporting the hypothesis that Lp(a) may interfere with the physiological functions of plasminogen.

Effect of nerve block on t-PA release by venous occlusion

In order to investigate the influence of neural blockade on tissue plasminogen activator (t-PA) release by venous occlusion, we compared the increase in t-PA and fibrinolytic activity in the euglobulin fraction initiated by cubital venous occlusion (100 mmHg for 10 min), in pre-neural block and post-neural block states, in 7 cases. In all patients, the supra-clavicular approach was used to obtain a brachial plexus block and venous occlusion was achieved at the cubital levels on the ipsilateral arm. The euglobulin fibrinolytic activity before venous occlusion was 100.9±27.5 BAU in the pre-nerve block state and 102.7±29.4 BAU in the post-nerve block state (p>0.5). The t-PA antigen level before venous occlusion was 3.5±1.2ng/ml in the pre-nerve block state and 4.0±1.0 ng/ml in the post-nerve block state (p>0.05). The increase in the euglobulin fibrinolytic activity after venous occlusion was 18.0±16.7 BAU in the pre-nerve block state, and 18.3±15.6 BAU in the post-nerve block state (p>0.5). The increase in the t-PA antigen levels after venous occlusion was 3.0±2.0 ng/ml in the pre-nerve block state and 3.0±2.1ng/ml in the post-nerve block state (p>0.5). These findings suggest that the peripheral nervous system does not exert any influence on t-PA release during venous occlusion.

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.

Bioimmunoassay for tissue-type plasminogen activator (t-PA) in human plasma: Evaluation of blood sampling and handling procedures and comparison with other t-PA activity methods

The blood sampling and handling procedure for a recently developed BioImmunoAssay (BIA) kit method has been evaluated. It was observed that blood collection at acid pH (pH 6; Stabilyte® tubes) yielded higher t-PA activity values (median 317 mIU/ml; 95% CI: 127–424) than collection in citrate (median 89 mIU/ml; 95% CI: 6–135) (n = 25 healthy volunteers). The BIA results in citrated plasma are comparable to results with other methods: t-PA activity in normal euglobulin fractions recorded with a chromogenic assay (median 5 mIU/ml; 95% CI: 5–36) or on the fibrin plate method (median 195 mIU/ml; 95% CI: 85–405). Further acid treatment of Stabilyte plasma yielded again higher values in the BIA (median 386 mIU/ml; 95% CI: 225–518); the increase in t-PA activity due to additional acid treatment correlated with the plasminogen activator inhibitor (PAI) activity in the plasma. Comparison between the t-PA activity obtained with the BIA method and our own in-house chromogenic assay method using in both cases acid treated plasma showed a strong correlation (r = 0.962, P < 0.0001, n = 25). We concluded that measurement of t-PA activity with the BIA can reliably be done provided blood is collected in acid milieu and plasma is subjected to additional acid treatment to inactivate PAL

On the Fibrinolytic System in Aged Rats, and Its Reactivity to Endotoxin and Cytokines

Aged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats. To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin. Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity. PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart. Rats were treated with either a low dose (1 microgram/kg) or a high dose (10 mg/kg) of endotoxin. Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity. Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor. Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin. The aged rats also responded to an injection of interleukin-1 beta or tumor necrosis factor-alpha with a larger increase of PAI activity than did the younger rats. Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines.

Modified analysis for C1q as protein-bound hydroxyproline in the euglobulin fraction of serum

Journal Article Modified analysis for C1q as protein-bound hydroxyproline in the euglobulin fraction of serum Get access J A Hutter, J A Hutter Search for other works by this author on: Oxford Academic Google Scholar D Simmons-Perry, D Simmons-Perry Search for other works by this author on: Oxford Academic Google Scholar B E Haskell B E Haskell Search for other works by this author on: Oxford Academic Google Scholar Clinical Chemistry, Volume 37, Issue 11, 1 November 1991, Pages 2013–2014, https://doi.org/10.1093/clinchem/37.11.2013a Published: 01 November 1991

Time dependent release of tissue-type plasminogen activator and plasminogen activator inhibitor into the circulation of pigs during shock

To investigate short-term activation and inhibition of fibrinolysis during shock, we studied plasma levels of tissue-type plasminogen activator (t-PA) and t-PA inhibition capacity (PAI) in anaesthetized pigs. t-PA in euglobulin fractions of plasma was measured by the conversion of plasminogen to plasmin in the presence of fibrin split products. Plasmin thus generated was measured in a chromogenic substrate assay. PAI was measured as plasma inhibition capacity for human melanoma t-PA. Controls (n = 8) had constant t-PA and PAI for 6 h. Lipopolysaccharide from Salmonella abortus equi in four different doses (n = 9 - 11), or live Escherichia coli (n = 3) induced a transient t-PA increase with peak values at 2 h. PAI decreased to 50% at 2 h and increased to 250% at 6 h. Phorbol myristate acetate (n = 7) induced no change of t-PA or PAI. Dextran sulphate (n = 4) produced a t-PA rise at 30 min, followed by a rapid decline. Endotoxin was an appropriate stimulus for activation and inhibition of fibrinolysis whereas phorbol ester failed to elicit this response.

Lipoprotein(a) impairs generation of plasmin by fibrin-bound tissue-type plasminogen activator. In vitro studies in a plasma milieu.

Apoprotein(a), (apo[a]), the specific antigen of lipoprotein(a) (Lp[a]), consists of structural domains (a serine protease unit, kringles 4 and 5) with marked homology to those of the corresponding domains in plasminogen. In this study, we have investigated the impact of this unique structural mimicry on the binding and activation of plasminogen by fibrin-bound tissue-type plasminogen activator at the plasma-fibrin interface. We found that the total amount of plasmin generated on the surface of fibrin was decreased in the presence of high concentrations of Lp(a): 197 +/- 65 fmol in plasmas with greater than 60 mg/dl Lp(a) versus 287 +/- 112 fmol in control plasmas. A similar effect was also apparent in the corresponding euglobulin fractions (554 +/- 169 fmol versus 754 +/- 310 fmol), the latter lacking the plasminogen-binding proteins alpha 2-antiplasmin and histidine-rich glycoprotein, but containing Lp(a). The difference between plasma samples was significant (p less than 0.05) as calculated from the percent decrease in plasmin generated from plasmas with high levels of Lp(a) relative to that generated in the paired controls with low Lp(a) levels. The involvement of Lp(a) was verified in a reconstituted system consisting of normal human plasma supplemented with 100 mg/dl of either purified Lp(a) or low density lipoprotein. Lp(a) produced a decrease of 30% in the generation of plasmin (180 fmol versus 255 fmol in plasma, and 485 fmol versus 705 fmol in the euglobulin fraction). Moreover, using a radiolabeled sheep antibody against human apo(a), we were able to demonstrate the binding of 40 fmol Lp(a) to fibrin during ongoing plasminogen activation. These results indicate that Lp(a) impairs the binding of plasminogen to fibrin and thereby decreases the generation of plasmin by occupying C-terminal lysine residues unveiled on the fibrin surface by plasmin degradation as recently reported (Circulation 1990;82[suppl III]:III-92). In consequence, impairment of fibrinolysis and accumulation of Lp(a) at sites of vascular injury may occur, factors that may be important in the development of atherosclerosis and associated thrombosis.

Effect of fish diet versus meat diet on blood lipids, coagulation and fibrinolysis in healthy young men

Twelve healthy young men followed a 10-d controlled diet that included 210 g of fatty fish d-1. The diet was repeated after 18 d, but with lean meat substituted for fish. Blood samples were collected for assessment of serum lipids and haemostatic variables in the plasma. Both experimental diets caused serum triglycerides and plasma factor VIIc to decline to the same extent. The meat diet was also associated with significant changes in plasma levels of tissue plasminogen activator (t-PA) antigen. PA inhibitor type I (PAI-1) antigen, PAI activity, and t-PA activity of the euglobulin fraction of plasma. The fish diet left these variables unchanged from initial values. Thus, in a paired comparison of the two diets, the fish diet was associated with higher levels of t-PA antigen (5.4 vs. 4.7 g ml-1), which is considered to be beneficial with regard to prevention of cardiovascular disease. However, the fish diet was concurrently associated with the putative unfavourable higher levels of PAI-1 antigen (3.0 vs. 1.2 ng ml-1) and PAI activity (6.1 vs. 3.2 IU ml-1), and lower t-PA activity (80 vs. 140 mIU ml-1). Thus it is unclear which of the two diets has the greatest potential in the prevention of cardiovascular disease.

PAI-1 Plays an Important Role in the Expression of t-PA Activity in the Euglobulin Clot Lysis by Controlling the Concentration of Free t-PA

The antigen levels of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were assayed in the plasma and in the euglobulin fraction, and their contributions to the euglobulin clot lysis time (ECLT) and t-PA activity were analyzed. Total and free PAI-1 levels in both fractions showed significant positive correlation with ECLT (p less than 0.001), whereas t-PA antigen level did not have a high correlation coefficient with ECLT. t-PA activity showed significant negative correlation with ECLT (p less than 0.001) and positive correlation with free t-PA level (p less than 0.001), which was calculated by the ratio of the concentrations of t-PA-PAI-1 complex and the free PAI-1. Thus free t-PA seems to dissolve the euglobulin clot and its concentration seems to be controlled by the concentration of free PAI-1. These findings were confirmed by the analyses of the effects of C1-inactivator and antibody against t-PA to regular ECLT and kaolin activated ECLT, the latter of which was only inhibited by the addition of C1-inactivator whereas the former was inhibited by anti-t-PA antibody.

Plasminogen Activators t-PA, u-PA and Its Inhibitor (PAI) in Normal Males and Females

We determined the plasma levels of tissue plasminogen activator (t-PA), plasminogen activator inhibitor (PAI) activity and their antigen levels including urokinase plasminogen activator (u-PA) in 33 male and 27 female normal subjects. Males had mean t-PA activity of 0.50 iu/ml which was significantly lower (p less than 0.01) than the females 0.64 iu/ml. Males had higher (p less than 0.001) mean PAI activity (15.5 AU/ml) as compared to females 10.3 AU/ml. The respective mean levels of t-PA and PAI antigen were significantly higher (p less than 0.01) in males (8.1 ng/ml and 17.6 ng/ml) than in females (6.2 ng/ml and 12.1 ng/ml). The mean u-PA level in males was 1.54 ng/ml which was significantly higher (p less than 0.01) than in females with 1.02 ng/ml. In post-venous occlusion studies, females had a greater mean response of 8.6 fold in t-PA activity as compared to males with a mean of 4.5 fold increase. The mean t-PA antigen response in males was 2.0 fold increase as compared to 2.6 fold increase in the females. No significant responses were seen in both sexes in either PAI activity or antigen levels when compared with the resting state. In zymography studies, free t-PA, its inhibitor complexes and u-PA were demonstrated in the euglobulin fractions of stored plasma. This study demonstrates that significant differences in t-PA, u-PA and PAI exist between male and female subjects which should be taken into account when determining their levels in clinical conditions.

An Analysis of the Activators of Single-Chain Urokinase-Type Plasminogen Activator (scu-PA) in the Dextran Sulphate Euglobulin Fraction of Normal Plasma and of Plasmas Deficient in Factor XII and Prekallikrein

An analysis was made of the various possible activators of single-chain urokinase-type plasminogen activator (scu-PA) in the dextran sulphate euglobulin fraction (DEF) of human plasma. scu-PA activators were detected in an assay system in which the substrate scu-PA, in physiological concentration (50 pM), was immuno-immobilized. After activation of the immobilized scu-PA for a certain period of time the activity of the generated amount of immuno-immobilized two-chain u-PA was determined with plasminogen and the chromogenic substrate S-2251. The scu-PA activator activity (scuPA-AA) in the DEF of plasmas deficient in factor XII or prekallikrein was about half of that in the DEF of normal plasma. Separation of scuPA-AA in the DEF by gel chromatography showed to major peaks, one eluting with an apparent Mr of 500,000 and the other around Mr 100,000. The former peak, which coincided with the activity peak of the kallikrein-kininogen complex, was absent in the DEF of plasma depleted of prekallikrein and therefore was identified as kallikrein. The latter peak was still present in the depleted plasma and most likely represents plasmin, because its scuPA-AA coincided with the activity peak of plasmin and could be fully inhibited by antibodies raised against human plasminogen. It is concluded that plasmin and the contact-activation factor kallikrein each contribute for about 50% to the scuPA-AA in the DEF. Compared on a molar basis, however, plasmin was found to be almost 1,000 times more effective than kallikrein, and we conclude, therefore, that in vivo plasmin is the primary activator of scu-PA and the role of the contact system is of secondary importance.