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PubMed2005

Basal release of urokinase plasminogen activator, plasminogen activator inhibitor-1, and soluble plasminogen activator receptor from separated and cultured endometriotic and endometrial stromal and epithelial cells.

OBJECTIVE: To investigate whether separated and cultured endometriotic and endometrial stromal and epithelial cells release urokinase plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and soluble plasminogen activator receptor (suPAR) antigens in vitro. DESIGN: In vitro study. SETTING: University hospital clinic. PATIENT(S): Regularly menstruating women with and without endometriosis. INTERVENTION(S): Tissue samples were collected at surgery performed for clinical reasons. MAIN OUTCOME MEASURE(S): The antigen concentrations of uPA, PAI-1, and suPAR in culture medium were assayed by enzyme-linked immunosorbent assay. RESULT(S): Both stromal and epithelial cells from endometriotic and endometrial tissue released the three types of antigens, but the release of PAI-1 was significantly higher from stromal cells in the three types of tissue than from epithelial cells. Furthermore, the release of PAI-1 was significantly higher from endometriotic cells than from endometrial stromal cells. CONCLUSION(S): This study has demonstrated the basic capacity of separated epithelial and stromal cells from all three types of tissue to release uPA, PAI-1, and suPAR without any paracrine influence, as in vivo. The higher release of PAI-1 from endometriotic stromal cells might have importance for the invasive growth.

Adult↗

PubMed1998

Physiological and pathological changes of plasma urokinase-type plasminogen activator, plasminogen activator inhibitor-1, and urokinase-type plasminogen activator receptor levels in healthy females and breast cancer patients.

The plasma urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and urokinase-type plasminogen activator receptor (uPAR) levels were measured in healthy volunteers and breast cancer patients. In pre-menopause healthy females, blood was sampled weekly during one menstruation cycle and menstruation phases (follicular, ovulatory, luteal) were determined by FSH/LH levels. uPA, PAI-1, and uPAR levels were at the nadir during ovulatory phase. uPA level was highest at follicular phase while PAI-1 level was highest at luteal phase. In comparison between pre- and post-menopause states, uPA and uPAR levels were higher in post-menopause state while PAI-1 level was higher in pre-menopause state. In breast cancer patients, uPA, PAI-1, and uPAR positive rates were low when we use the menopause-state-unmatched cut-off points. As we adjusted the cut-off points by menopause states, the PAI-1 positivity increased mainly in post-menopause cancer patients. These findings suggest that there is a minor but possible sequential change of these molecules during menstruation cycle which might blur the pathological positivity in pre-menopause cancer patients. The pathological elevation of PAI-1 was well detected in post-menopause cancer patients, but this elevation did not correlate with tumor burden such as number of metastatic sites or metastatic location. In conclusion, adjustment of physiological changes of uPA, PAI-1, and uPAR is required in determining pathological elevation of the plasma levels in cancer patients, especially in females.

Adult↗

PubMedDate not supplied

Circadian variations of plasminogen activator activity, tissue-type plasminogen activator antigen, plasminogen activator inhibition and plasmin inhibition in rat aorta, heart and brain are influenced by endotoxin or aspirin or endotoxin plus aspirin.

The effect of aspirin or endotoxin or aspirin plus endotoxin on the circadian variations of plasminogen activator activity (PAA), tissue-type plasminogen activator (t-PA) antigen level, plasminogen activator inhibition (PAI) and plasmin inhibition (PI) in aorta, heart and brain of the rat was studied. In aorta the circadian variations of PAA, t-PA antigen and tissue-type PAI (t-PAI) were blunted by endotoxin, while the circadian variation of PI was reversed; t-PAI variation was blunted by aspirin plus endotoxin, while PI variation was reversed. Various disturbances of the circadian variations of PAA, t-PA antigen, PAI or PI in heart and brain were also observed. In conclusion, the circadian variations of fibrinolytic parameters in key organs are influenced by aspirin or endotoxin or aspirin plus endotoxin in a varying way or degree depending on the treatment, the parameter studied or the organ.

Animals↗

PubMed2004

[The plasma levels of urokinase plasminogen activator and urokinase plasminogen activator receptor and plasminogen activator inhibitor-1 in patients with different stages of liver cirrhosis following chronic hepatitis B].

OBJECTIVES: To measure the plasma levels of urokinase plasminogen activator (uPA), urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1), and study the relationship between the plasma levels of uPA, PAI-1 and the serum albumin (Alb), collagen type IV (CIV), the serum hyaluronic acid (HA), prothrombin time (PT) and prothrombin activity (PTA) in patients with different stages of liver cirrhosis following chronic hepatitis B. METHODS: 72 cases with liver cirrhosis of different stages were classified according to child-pugh's categories A, B, C, in which there were 23 cases in child A, 29 cases in child B, and 20 cases in child C. The plasma levels of uPA, uPAR, PAI-1 and the serum levels of HA, CIV were detected by ELISA. The serum PCIII concentration was determined by radioimmunoassay. RESULTS: With the progression of hepatic fibrosis, the plasma levels of uPA, uPAR and PAI-1 were (1.36+/-0.43) microg/L, (3.03+/-1.48) microg/L and (24.09+/-7.14) microg/L respectively in group A, (1.79+/-0.62) microg/L, (4.80+/-2.22) microg/L and (41.40+/-17.52) microg/L respectively in group B. The highest levels were in child C, whose levels were (1.88+/-0.64) microg/L, (4.82+/-2.02) microg/L and (52.60+/-16.87) microg/L respectively, compared with group A and group B, t value were from 2.81 to 7.38, all of P value were less than 0.01. There was negative correlation between the plasma levels of uPA and the serum PCIII (r=-0.4785, P<0.05) in child A, but, positive correlation between the plasma PAI-1 and the serum HA (r=0.5447, P<0.01) in child C. The value of PAI-1/uPA was significantly decreased in child A, but increased in child B and child C. CONCLUSION: In the late of liver cirrhosis, increased PAI-1 together with uPA, uPAR are associated with overall inhibition of matrix degradation. The plasma levels of uPA and PAI-1 were correlation to the progression of liver cirrhosis.

Female↗

PubMed1988

Activation of Val442-plasminogen (mini-plasminogen) by urokinase, streptokinase and tissue plasminogen activator.

Glu-plasminogen (Glu-plg), Lys-plg and Val442-plg (mini-plg) were activated by urokinase (UK), streptokinase (SK) or tissue plasminogen activator (t-PA). Their activation rates were kinetically analyzed. UK activated Lys-plg with smaller Km and nearly identical Vmax as Glu-plg. Mini-plg was activated by UK with higher Vmax but with the same Km as Glu-plg. On the other hand, t-PA activated Glu-plg, Lys-plg and mini-plg with the same Vmax, but Km was in the order of Glu-plg greater than mini-plg greater than Lys-plg. Fibrin hardly enhanced the UK activation of mini-plg, and fibrinogen, fragment D or E did not enhance the activation of mini-plg by UK. Only D domain, but not E domain, complexed with K5 of mini-plg and SK hydrolysed S-2251 faster than a dimolecular complex of mini-plg and SK, thus a trimolecular mixture of D, SK and mini-plg having a better activator activity than SK-mini-plg complex. In the t-PA activation of mini-plg, fibrin and fibrinogen enhanced the activation, but fragment D or E was ineffective, suggesting that a molecule containing more than one domain was required for the enhanced activation of mini-plg by t-PA.

Fibrin↗

PubMed2001

Conversion of Glu-plasminogen to Lys-plasminogen is necessary for optimal stimulation of plasminogen activation on the endothelial cell surface.

When Glu-plasminogen is bound to cells, plasmin (Pm) formation by plasminogen (Pg) activators is markedly enhanced compared with the reaction in solution. It is not known whether the direct activation of Glu-Pg by Pg activators is promoted on the cell surface or whether plasminolytic conversion of Glu-Pg to the more readily activated Lys-Pg is necessary for enhanced Pm formation on the cell surface. To distinguish between these potential mechanisms, we tested whether Pm formation on the cell surface could be stimulated in the absence of conversion of Glu-Pg to Lys-Pg. Rates of activation of Glu-Pg, Lys-Pg, and a mutant Glu-Pg, [D646E]Glu-Pg, by either tissue Pg activator (t-PA) or urokinase (u-PA) were compared when these Pg forms were either bound to human umbilical vein endothelial cells (HUVEC) or in solution. ([D646E]Glu-Pg can be cleaved at the Arg(561)-Val(562) bond by Pg activators but does not possess Pm activity subsequent to this cleavage because of the mutation of Asp(646) of the serine protease catalytic triad.) Glu-Pg activation by t-PA was enhanced on HUVEC compared with the solution phase by 13-fold. In contrast, much less enhancement of Pg activation was observed with [D646E]Glu-Pg ( approximately 2-fold). Although the extent of activation of Lys-Pg on cells was similar to that of Glu-Pg, the cells afforded minimal enhancement of Lys-Pg activation compared with the solution phase (1.3-fold). Similar results were obtained when u-PA was used as activator. When Glu-Pg was bound to the cell in the presence of either t-PA or u-PA, conversion to Lys-Pg was observed, but conversion of ([D646E]Glu-Pg to ([D646E]Lys-Pg was not detected, consistent with the conversion of Glu-Pg to Lys-Pg being necessary for optimal enhancement of Pg activation on cell surfaces. Furthermore, we found that conversion of [D646E]Glu-Pg to [D646E]Lys-Pg by exogenous Pm was markedly enhanced ( approximately 20-fold) on the HUVEC surface, suggesting that the stimulation of the conversion of Glu-Pg to Lys-Pg is a key mechanism by which cells enhance Pg activation.

Cell Membrane↗

PubMed2005

Expression of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitors in patients with renal cell carcinoma: correlation with tumor associated macrophage and prognosis.

PURPOSE: Urokinase-type plasminogen activator (uPA) has an important role in tumor progression through the degradation of extracellular matrix. In addition, uPA receptor (uPAR) and plasminogen activator inhibitors (PAIs), composed of PAI-1 and 2, are also known to affect such activities. Tumor associated macrophage (TAM) is an important regulator of tumor progression that is associated with the uPA system in various cancers. However, to our knowledge the clinical significance of PAI-2 and the relationship between the uPA system and TAM in human renal cell carcinoma (RCC) tissues have not been investigated. We investigated and clarified these issues. MATERIALS AND METHODS: The subjects of the current study were 106 consecutive surgically resected specimens from patients with RCC. The expression of uPA, uPAR, PAI-1 and PAI-2 was determined by immunohistochemistry. We also examined the relationships among these molecules, survival and TAM. RESULTS: The mean immunoreactive scores (range 0 to 6) of uPA, uPAR, PAI-1 and PAI-2 were 3.09, 2.22, 1.99 and 0.56, respectively. These scores correlated with the grade and presence of metastasis. The expression of uPA, uPAR and PAI-1 but not PAI-2 correlated negatively with cause specific survival. Of uPA family members multivariate analysis showed that PAI-1 independently influenced cause specific survival. TAM counts correlated with PAI-1 only (p <0.001). CONCLUSIONS: Our results suggest that PAI-1 is an important regulator of tumor progression and survival, and PAI-1 may modulate them via TAM. On the other hand, PAI-2 has a minimum role in survival. Our results may help discussions of treatment strategy in patients with RCC.

Aged↗

PubMed1991

Effects of N-terminal peptide of Glu-plasminogen on the activation of Glu-plasminogen and its conversion to Lys-plasminogen.

In order to analyze the mechanism of the intramolecular binding of the N-terminal peptide of Glu-plasminogen (Glu-plg) to its kringles, which results in its tight conformation, we synthesized peptides of the N-terminal portion of Glu-plg molecules and analyzed their effects on the activation of Glu-plg and its conversion to Lys-plasminogen (Lys-plg) by plasmin. Three peptides of Ala44-Lys50, Ala44-Glu51 and Ala44-Ser49 were synthesized in order to examine the effect of lysine residue in the peptide. Ala44-Lys50 and Ala44-Glu51 enhanced the activation of Glu-plg by urokinase, whereas the activation of Lys-plasminogen (Lys-plg) was slightly inhibited. The conversion of Glu-plg to Lys-plg by plasmin was also enhanced by these peptides. The results suggest that Ala44-Lys50 and Ala44-Glu51 worked on Glu-plg in a similar manner as lysine analogues by making its conformation looser. The third peptide Ala44-Ser49 did not have any effect on the activation of Glu-plg by urokinase or the conversion of Glu-plg to Lys-plg by plasmin. Ala44-Lys50 residue of Glu-plg is, therefore, strongly implicated as a candidate for the responsible site of the intramolecular binding in Glu-plg.

Alanine↗

PubMed2003

Nipple aspirate fluid expression of urokinase-type plasminogen activator, plasminogen activator inhibitor-1, and urokinase-type plasminogen activator receptor predicts breast cancer diagnosis and advanced disease.

BACKGROUND: Tumor expression of urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR) are breast cancer prognostic factors. Less is known about their usefulness in breast cancer diagnosis. Nipple aspirate fluid (NAF) is secreted into the breast duct and collected noninvasively, making it potentially useful both in breast cancer diagnosis and prognosis. We determined the association of uPA, PAI-1, and uPAR levels in NAF with breast cancer (1) detection and (2) advanced disease. METHODS: A total of 88 NAF specimens were collected from women with or without breast cancer, and uPA, PAI-1, and uPAR expression were measured by enzyme-linked immunosorbent assay. RESULTS: uPA and uPAR were independent predictors of cancer presence; uPAR was also an independent predictor of advanced disease stage. Higher PAI-1 expression in breast cancer that was found with univariate analysis was not observed after logistic regression was applied. CONCLUSIONS: NAF evaluation of uPA, uPAR, and, perhaps, PAI-1 (significant only in univariate analysis) may provide useful breast cancer diagnostic and prognostic information.

Adult↗

PubMed1992

A comparison of the plasminogen activator activity units of urinary-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA).

The earliest tissue plasminogen activator (t-PA) preparations prepared from melanoma cells were expressed in urinary-type plasminogen activator (u-PA) units of activity using the u-PA International Standard (66/46). This report describes a comparison between u-PA and t-PA units by various types of fibrin plate procedure using both human and bovine fibrin. Within the biometric limits of this procedure it was found that the potency ratio of u-PA/t-PA was 3.5 (human fibrin), 5.29 (enriched bovine fibrin) and 4.6 (crude bovine fibrin). Specific activity figures of approximately 100,000 IU/mg for pure t-PA using the urokinase standard (66/46) would convert to approximately 350,000-530,000 IU/mg using the International Standards for t-PA (83/517 and 87/670). This latter figure is compatible with the reported specific activity for purified preparations of recombinant t-PA.

Animals↗

PubMed2000

Tissue plasminogen activator, plasminogen activator inhibitor-1, and tissue plasminogen activator/plasminogen activator inhibitor-1 complex as risk factors for the development of a first stroke.

UNLABELLED: BACKGROUND NAD PURPOSE: Abnormalities in the fibrinolytic system have been associated with an increased risk for stroke in a few studies. This study was designed to test whether plasma levels of tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), and tPA/PAI-1 complex could predict a first-ever stroke. METHODS: The study was an incident case-control study nested within the Västerbotten Intervention Program and the Northern Sweden Monitoring Trends and Determinants in Cardiovascular Disease (MONICA) cohorts. In this study 108 first-ever stroke cases were defined according to the MONICA classification, and 216 controls from the same cohort were randomly selected and matched for age, sex, sampling time, and geographic region. RESULTS: Stroke occurred on average 30 months after the blood sampling date. The mean plasma concentration of tPA/PAI-1 complex was higher for the stroke cases than for the controls (3.9 versus 3.0 microgram/L). In univariate regression analysis, significantly higher odds ratios were found for the tPA/PAI-1 complex as continuous variable. When divided into quartiles, the odds ratio was 2.74 for the highest quartile compared with the lowest. In the multivariate model, the tPA/PAI-1 complex remained an independent predictor for stroke. Additionally, tPA mass concentration quartiles 3 and 4 showed a significant association with all stroke as outcome. No association was found, however, for PAI-1. In subgroup analysis of cerebral hemorrhage (n=18), the mean tPA/PAI-1 complex level was higher for the cases than for the controls (4.8 versus 3.0 microgram/L), and in multivariate analysis including all controls (n=216), only tPA/PAI-1 complex remained significant. CONCLUSIONS: This prospective study shows that tPA/PAI-1 complex, a novel fibrinolytic marker, is independently associated with the development of a first-ever stroke, especially hemorrhagic stroke. This finding supports the hypothesis that disturbances in fibrinolysis precede a cerebrovascular event.

Case-Control Studies↗

PubMed1989

Inhibition of tissue plasminogen activator activity by aspirin in vivo and its relationship to levels of tissue plasminogen activator inhibitor antigen, plasminogen activator and their complexes.

The observation that aspirin inhibits the increment in tissue plasminogen activator (t-PA) activity induced by venous occlusion of the forearm became controversial with the publication of several nonconfirmatory studies. The current study was performed to confirm the original observation and determine the mechanism by which aspirin suppresses the incremental t-PA activity induced by venous occlusion. Aspirin (650 mg/d X 2) caused no change in resting levels of t-PA antigen (t-PA:Ag) or activity, plasminogen activator inhibitor 1 antigen (PAI-1:Ag), or activity or t-PA-PAI-1 complexes. In contrast, aspirin reduced the increments induced by venous occlusion as follows: t-PA:Ag by 45% (P = .001); t-PA activity (euglobulin lysis time, ELT) by 43% (P = .006); and t-PA activity (alpha 2-plasmin inhibitor-plasmin complexes, PIPC) by 41% (P = .003). The inhibition of incremental t-PA activity measured as ELT or PIPC was linearly correlated with the inhibition of incremental t-PA:Ag (respectively, r = .75, P less than .02; r = .67, P less than .05). Aspirin had no effect on the increment in PAI-1:Ag induced by venous occlusion, but similar to the effect on t-PA:Ag, aspirin induced a 51% inhibition of the increment in t-PA-PAI-1 complex formation. Aspirin did not alter the ability of alpha 2-plasmin inhibitor to bind plasmin, nor the ability of plasma to support the fibrin-catalyzed generation of plasmin by t-PA, nor the subsequent formation of PIPC. Aspirin inhibits the t-PA activity induced by venous occlusion primarily by inhibiting the release of t-PA antigen.

Antifibrinolytic Agents↗

PubMed1997

Adenovirus-mediated transfer of tissue-type plasminogen activator augments thrombolysis in tissue-type plasminogen activator-deficient and plasminogen activator inhibitor-1-overexpressing mice.

Impaired fibrinolysis, resulting from increased plasminogen activator inhibitor-1 (PAI-1) or reduced tissue-type plasminogen activator (t-PA) plasma levels, may predispose the individual to subacute thrombosis in sepsis and inflammation. The objective of these studies was to show that adenovirus-mediated gene transfer could increase systemic plasma t-PA levels and thrombolytic capacity in animal model systems. Recombinant adenovirus vectors were constructed that express either human wild type or PAI-1-resistant t-PA from the cytomegalovirus (CMV) promoter. Both t-PA-deficient (t-PA(-/-)) and PAI-1-overexpressing transgenic mice were infected by intravenous injection of these viruses. Intravenous injection of recombinant adenovirus resulted in liver gene transfer, t-PA synthesis, and secretion into the plasma. Virus dose, human t-PA antigen, and activity concentrations in plasma and extent of lysis of a 125I-fibrin-labeled pulmonary embolism were all closely correlated. Plasma t-PA antigen and activity were increased approximately 1,000-fold above normal levels. Clot lysis was significantly increased in mice injected with a t-PA-expressing virus, but not in mice injected with saline or an irrelevant adenovirus. Comparable levels of enzyme activity and clot lysis were obtained with wild type and inhibitor-resistant t-PA viruses. Adenovirus-mediated t-PA gene transfer was found to augment clot lysis as early as 4 hours after infection, but expression levels subsided within 7 days. Adenovirus-mediated transfer of a t-PA gene can effectively increase plasma fibrinolytic activity and either restore (in t-PA-deficient mice) or augment (in PAI-1-overexpressing mice) the thrombolytic capacity in simple animal models of defective fibrinolysis.

Adenoviridae↗

PubMed2000

Thrombin-activable fibrinolysis inhibitor attenuates (DD)E-mediated stimulation of plasminogen activation by reducing the affinity of (DD)E for tissue plasminogen activator. A potential mechanism for enhancing the fibrin specificity of tissue plasminogen activator.

A complex of d-dimer noncovalently associated with fragment E ((DD)E), a degradation product of cross-linked fibrin that binds tissue plasminogen activator (t-PA) and plasminogen (Pg) with affinities similar to those of fibrin, compromises the fibrin specificity of t-PA by stimulating systemic Pg activation. In this study, we examined the effect of thrombin-activable fibrinolysis inhibitor (TAFI), a latent carboxypeptidase B (CPB)-like enzyme, on the stimulatory activity of (DD)E. Incubation of (DD)E with activated TAFI (TAFIa) or CPB (a) produces a 96% reduction in the capacity of (DD)E to stimulate t-PA-mediated activation of Glu- or Lys-Pg by reducing k(cat) and increasing K(m) for the reaction; (b) induces the release of 8 mol of lysine/mol of (DD)E, although most of the stimulatory activity is lost after release of only 4 mol of lysine/mol (DD)E; and (c) reduces the affinity of (DD)E for Glu-Pg, Lys-Pg, and t-PA by 2-, 4-, and 160-fold, respectively. Because TAFIa- or CPB-exposed (DD)E produces little stimulation of Glu-Pg activation by t-PA, (DD)E is not degraded into fragment E and d-dimer, the latter of which has been reported to impair fibrin polymerization. These data suggest a novel role for TAFIa. By attenuating systemic Pg activation by (DD)E, TAFIa renders t-PA more fibrin-specific.

Amino Acid Chloromethyl Ketones↗

PubMed1991

Plasminogen activation in diabetes mellitus: kinetics of plasmin formation with plasminogen and tissue plasminogen activator from diabetic donors.

Impaired function of the fibrinolytic system might be involved in the development of vascular disease and thromboembolic complications in diabetic patients. We studied kinetics of plasmin formation using t-PA and Pg purified from the plasma of three individual uncontrolled type I diabetic patients. Activation of diabetic Pg by normal t-PA in the presence of stimulating CNBr fragments of fibrinogen exhibited a prolonged lag phase (30 to 60 minutes) until maximally stimulated plasmin formation occurred (normal, 5 to 15 minutes) and substrate inhibition at Pg concentrations more than 10 to 30 nM. When normal Pg was activated by diabetic t-PA in the presence of CNBr-f, differentiation between lag phase and phase of maximal plasmin formation was not possible (activation time was 2 hours) and a high Km (7.5 microM) was calculated. After normalization of metabolic parameters in the patients studied, functional properties of t-PA and Pg improved. Km of diabetic t-PA returned to normal values (0.02 to 0.09 microM) and for diabetic Pg the prolonged lag phase was shortened, indicating that the functional abnormalities were reversible and possibly caused by metabolically induced changes (such as nonenzymatic glucosylation) in the t-PA or plasminogen molecule. We also studied the effect of in vitro glucosylation on functional properties of Pg. Similar, but less pronounced substrate inhibition as with diabetic Pg was observed when this glucosylated Pg was activated by t-PA in a system stimulated by CNBr fragments of fibrinogen. Therefore nonenzymatic glucosylation might explain, at least in part, functional abnormalities observed with Pg from diabetic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Enhanced activator activity of the mixture of streptokinase and a modified form of plasminogen (Lys-plasminogen) in the presence of fibrin: role of conformational change of plasminogen.

Equimolar concentrations of Lys-plasminogen (Lys-PLG) and streptokinase (SK) were mixed with various concentrations of fibrin, fibrinogen, fragment D or E (potentiating agents). The activity of the mixture was measured by the hydrolysis of S-2251. Kinetic analyses indicated that catalytic rate constant (kcat) of the hydrolysis of S-2251 increased in the presence of increasing amounts of potentiating agents. Km did not change in their presence. Effectiveness of the enhancement of the hydrolysis of S-2251 was in the order of fibrin greater than fibrinogen greater than E greater than D. It could be concluded that a complex of SK, Lys-PLG and potentiating agent is a better enzyme, thus activator, than a complex of SK and Lys-PLG. Since Lys-PLG does not change its conformation and its activation rate upon interaction with fibrin, the enhanced activator activity of complex of SK, Lys-PLG and potentiating agent may not be due to the conformational change of Lys-PLG.

Chromogenic Compounds↗

PubMed1990

Effect of peptides on the inactivation of tissue plasminogen activator by plasminogen activator inhibitor-1 and on the binding of tissue plasminogen activator to endothelial cells.

The effectiveness of tissue plasminogen activator (tPA) in thrombolytic therapy is dependent upon the rate at which therapeutically administered tPA reaches the clot site and the proportion of that tPA which is enzymatically active. Interactions between tPA and its main plasma inhibitor (PAI-1) and between tPA and the endothelial cells lining blood vessels are two factors which may limit efficacy. In an attempt to identify the regions of the tPA molecule involved in these interactions, we have examined a series of synthetic peptides with amino acid sequences corresponding to different regions of the tPA molecule for their ability to protect tPA from inactivation by PAI-1 and for their ability to reduce the binding of tPA to endothelial cells. Three peptides were identified which were especially effective at maintaining tPA activity in the presence of PAI-1 and three others were found which had a lesser effect. These same peptides were also found to inhibit the binding of tPA to endothelial cells. This suggests that the same regions of the tPA molecule are involved in both processes. None of the peptides inhibited the binding of tPA to fibrin. These peptides may serve as models for the development of agents for enhancing the activity of both endogenous tPA and of tPA administered in thrombolytic therapy.

Amino Acid Sequence↗

PubMed1992

Lys-plasminogen is a significant intermediate in the activation of Glu-plasminogen during fibrinolysis in vitro.

Plasminogen, the zymogen form of the fibrinolytic enzyme plasmin, is known to undergo plasmin-mediated modification in vitro. The modified form, Lys-plasminogen, is superior to the native Glu-plasminogen in fibrin binding and as a substrate for activation by tissue-type plasminogen activator (t-PA). The present study was undertaken to determine the existence and significance of the Glu- to Lys-plasminogen conversion during t-PA-mediated lysis of plasma clots in vitro. When human plasma was supplemented with exogenous Lys-plasminogen and clotted, a dose-dependent shortening of lysis time was observed. Formation of Lys-plasminogen in situ during fibrinolysis was determined using 131I-Glu-plasminogen-supplemented plasma. By the time of lysis, Lys-plasminogen had accumulated to about 20% of the initial concentration of Glu-plasminogen. Quantitation of activation of both Glu- and Lys-plasminogen as well as the conversion of Glu- to Lys-plasminogen in plasma supplemented with both 131I-Glu-plasminogen and 125I-Lys-plasminogen was accomplished by determining the flux of the isotopically labeled species along three pathways: Glu-plasminogen-->Glu-plasmin, Glu-plasminogen-->Lys-plasminogen, and Lys-plasminogen-->Lys-plasmin. After a brief lag, the Glu-plasminogen activation rate was constant until lysis was achieved, at which point activation ceased. The Lys-plasminogen activation rate also was essentially constant until lysis but was not characterized by a lag phase. The rate of conversion of Glu- to Lys-plasminogen was nonlinear and correlated directly with the rate of fibrinolysis. By the time lysis had occurred, Glu-plasminogen consumption had been distributed equally between direct activation to plasmin and conversion to Lys-plasminogen, and 45% of the plasmin which had been formed was derived from Lys-plasminogen. These results demonstrate both the formation and the subsequent activation of Lys-plasminogen during fibrinolysis. As a result of improved fibrin binding and activation of Lys-plasminogen compared to Glu-plasminogen, the formation of Lys-plasminogen within a clot constitutes a positive feedback mechanism that can further stimulate the activation of plasminogen by t-PA as fibrinolysis progresses.

Fibrinolysis↗