Tissue-type Plasminogen Activator Activity Research Articles

Overexpression of tissue-type plasminogen activator in atherosclerotic human coronary arteries

The plasminogen activator (PA)/plasmin system is involved in various pathological processes that are considered important features of atherogenesis and atherothrombosis. These include the proteolysis of fibrin deposits and extracellular matrix components as well as the induction of cell migration and mitogenesis. Tissue-type PA (TPA) is a key enzyme mediating plasminogen to plasmin conversion. TPA plasma concentrations are elevated in patients with advanced atherosclerosis and correlate with an increased risk for myocardial infarction and stroke. In this study, we have analysed the content and expression of TPA in human coronary arteries and their relation to the presence and severity of atherosclerotic lesions. Methods: Segments of coronary arteries obtained from heart explants ( n=15) were classified by the presence and types of atherosclerotic lesions. TPA was quantitatively determined in protein extracts of intimal and medial layers. In situ hybridization and immunohistochemical analyses were performed on serial sections of representative tissue specimens. Results: PA activity entirely attributable to the presence of active TPA was consistently detected in the protein extracts. Extractable TPA antigen and activity showed a significant graded increase in relation to the presence and severity of atherosclerotic lesions. The ratios of active over total TPA were increased several-fold in extracts of advanced lesions despite a concomitant threefold increase in TPA complexed to its inhibitor PAI-1. In macroscopically normal arterial segments and in early lesions, TPA was expressed in the endothelium and in colocalization with vascular smooth muscle cells (VSMCs). In advanced plaques, TPA mRNA was mainly detected in the lateral regions of the fibrous caps in association with migrating VSMCs and in the vicinity of the core areas infiltrated by CD68-positive macrophages. Conclusions: TPA content and expression is consistently increased in relation to the severity of the lesions in atherosclerotic coronary arteries. This may contribute to plaque destabilization and disruption. Conversely, the increased intramural TPA activity may counteract mural fibrin deposition.

Fibrinolytic activity is similar in physically active men with and without a history of myocardial infarction.

The purpose of this study was to evaluate fibrinolytic potential at rest and after a fibrinolytic stressor in men with a history of myocardial infarction (MI) compared with an age- and activity-matched group of men without coronary artery disease (CAD). All men were currently enrolled in exercise programs. Tissue-type plasminogen activator (TPA) and plasminogen activator inhibitor 1 (PAI-1) activity and antigen levels were measured at rest and after a maximal exercise test. A 2 x 2 (group x time) ANOVA with repeated measures was used to evaluate fibrinolytic potential. Bivariate regressions were conducted to evaluate relations between fibrinolytic potential and maximal oxygen uptake (VO2max). Age was similar between groups (CAD, 57.5 +/- 6.6; non-CAD, 58.1 +/- 7.3 years); however, VO2max was higher in non-CAD subjects (36.2 +/- 6.2 vs 27.5 +/- 5.9 mL.kg-1.min-1). Mean +/- SEM resting TPA and PAI-1 activities were similar between CAD and non-CAD subjects (TPA, 2.8 +/- 0.2 vs 2.8 +/- 0.2 IU/mL; PAI-1, 15.9 +/- 3.1 vs 13.1 +/- 4.1 AU/mL). Both groups showed similar significant increases in TPA activity with exercise (P < .05), and postexercise TPA activity was also similar (CAD, 9.1 +/- 2.0 IU/mL; non-CAD, 11.7 +/- 2.6 IU/mL). Both groups also showed similar significant decreases in PAI-1 activity with exercise (P < .05) and no differences in postexercise PAI-1 activity (CAD, 13.2 +/- 2.5 AU/mL; non-CAD, 10.4 +/- 3.6 AU/mL). Significantly higher resting TPA antigen levels were seen in CAD (14.8 ng/mL) than non-CAD (10.2 ng/mL) subjects (P < .05), but neither group showed significant changes with exercise (CAD, 12.9 ng/mL; non-CAD, 11.8 ng/mL). Resting PAI-1 antigen was similar in the two groups (CAD, 71.4 ng/mL; non-CAD, 74.2 ng/mL) and did not significantly change with exercise (CAD, 77.9 ng/mL; non-CAD, 72.3 ng/mL). VO2max was positively correlated with postexercise TPA activity (r = .52, P < .05) and negatively correlated with resting TPA antigen (r = -.43, P < .05). Resting TPA antigen was also directly correlated with body mass index (r = .63, P < .05). The finding that functional fibrinolytic activity was not different in physically active men with and without CAD contrasts with previous reports. This suggests that matching subjects on the bases of age and habitual physical activity status and controlling exercise intensity are important factors to consider when evaluating fibrinolytic potential.

Isolation and characterization of tissue-type plasminogen activator- binding proteoglycans from human umbilical vein endothelial cells.

We analyzed the tissue-type plasminogen activator (TPA)-binding proteoglycans (PGs) on human umbilical vein endothelial cells (HUVECs), which were metabolically labeled with [35S]NA2SO4. Cell extracts were then prepared and subjected to affinity chromatography on diisopropyl fluorophosphate (DFP)-inactivated TPA-Sepharose 4B. Approximately 6% of the incorporated 35S radioactivity bound to DFP-treated TPA-Sepharose 4B and was eluted with 2 mol/L NaCl. In addition to NaCl, heparin, arginine, and lysine but not glycine, epsilon-amino-n-caproic acid or aspartic acid inhibited this binding and eluted the bound 35S radioactivity. Urea-containing polyacrylamide gel electrophoresis of the eluted material consistently revealed two main signals of 35S radioactivity (one with an M(r) between 600,000 and 750,000 [PGA] and the other with an M(r) between 120,000 and 180,000 [PGC]). Occasionally a less intense signal with an M(r) between 340,000 and 440,000 (PGB) was seen. Heparitinase treatment markedly decreased the intensities of both 35S signals (PGA and PGB), and chondroitinases AC and ABC abolished the 35S signal of PGC, indicating that most of the HUVEC-incorporated radioactivity with an affinity for TPA could be attributed to heparan sulfate- and chondroitin sulfate-like structures. Reductive elimination, which was performed to separate the possible glycosaminoglycan moieties from the core proteins, confirmed the PG-like nature of this material and again revealed heparan sulfate and chondroitin sulfate as the major glycosaminoglycan components. We therefore conclude that HUVECs synthesize TPA-binding, heparan sulfate- and chondroitin sulfate-containing PGs. In vivo, similar PGs may play a role in TPA binding to endothelial cells and thereby possibly influence TPA activity and/or provide an intravascular storage pool of TPA.

T-Cell Lymphokines, Interleukin-4 and Gamma Interferon, Modulate the Induction of Vascular Smooth Muscle Cell Tissue Plasminogen Activator and Migration by Serum and Platelet-Derived Growth Factor

Platelet-derived growth factor (PDGF)-induced smooth muscle cell (SMC) fibrinolysis is necessary for SMC migration. In order to determine whether the T-cell lymphokines interleukin-4 (IL-4) and gamma interferon (gamma-IFN) affect SMC fibrinolysis and migration, we examined the effects of human recombinant IL-4 and gamma-IFN on human aortic SMC tissue-type plasminogen activator (TPA), urokinase-type plasminogen activator (UPA), and plasminogen activator inhibitor type-1 (PAI-1) antigen production, as determined by enzyme-linked immunosorbent assays. Although IL-4 had no direct effect on SMC TPA antigen, IL-4 potentiated SMC TPA antigen levels and activity in conditioned media and cellular lysates in media containing 2% fetal bovine serum but did not change UPA or PAI-1 production. gamma-IFN attenuated IL-4 augmentation of SMC TPA antigen production in conditioned media, although gamma-IFN itself had no direct effects on SMC TPA and PAI-1 antigen production. IL-4 augmented PDGF induction of SMC TPA antigen. gamma-IFN inhibited PDGF induction of SMC TPA antigen and IL-4 potentiation of this process. gamma-IFN diminished the promigratory effects of both IL-4 and PDGF on in vitro SMC migration. Tranexamic acid, a plasmin inhibitor, abrogated the stimulation of SMC migration by IL-4. Therefore, IL-4 and gamma-IFN modulate the induction of SMC TPA and SMC migration by 2% fetal bovine serum and PDGF.

Tissue-type plasminogen activator and its inhibitor in rat aorta. Effect of endotoxin.

Plasminogen activator (PA) and PA inhibitor (PAI) antigen, activity, and mRNA were analyzed in the three layers of rat aorta, and the effect of endotoxin on PA and PAI was studied. All PA activity in aorta was identified as tissue-type PA (TPA) activity; no urokinase-type PA was detected. In the tunica adventitia TPA activity, TPA antigen, and TPA mRNA were detected, whereas in the tunica media TPA antigen and TPA mRNA, but no TPA activity, were found. PAI activity was detected in the tunica media, explaining the absence of TPA activity in this layer. Removal of the endothelial cells had no effect on TPA antigen and PAI activity in intima-media preparations. Also, similar amounts of PAI-1 mRNA were found in intima-media preparations, irrespective of the presence or absence of the intima. Immunohistochemical staining showed that TPA immunoreactivity was present in all three layers of the aorta, whereas PAI-1 immunoreactivity was found in medial smooth muscle cells but not in endothelial cells. After endotoxin treatment, TPA activity was decreased in extracts of the total aorta and of the adventitia, although TPA antigen and TPA mRNA were unchanged. PAI-1 mRNA was strongly increased in the tunica adventitia and in the tunica media, as was PAI activity in the tunica media. Thus, endotoxin decreased TPA activity by increasing the synthesis of PAI-1; TPA was unaffected. Our observations in rat aorta differ from observations in mouse aorta and in rat carotid artery, and they caution against extrapolation from one tissue (or species) to another.

Effect of notoginsenoside R1 on the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in cultured human umbilical vein endothelial cells.

Among other Chinese herb drugs, Panax notoginseng is used to treat cardiovascular diseases. To elucidate any possible effects of this drug on the hemostatic system in vitro, we analyzed the influence of one of its major active constituents on fibrinolytic parameters of cultured human umbilical vein endothelial cells (HUVECs). When confluent cultures of HUVECs (passages 2 to 3) were conditioned with purified notoginsenoside R1 (NR1), a dose- (0.01 to 100 micrograms NR1/mL) and time-dependent increase in tissue-type plasminogen activator (TPA) synthesis was observed, which was significant from 0.1 microgram NR1/mL and from 6 hours of incubation with 100 micrograms NR1/mL on. TPA antigen increased from 3.9 +/- 0.2 ng per 10(5) cells per 24 hours to 8.0 +/- 0.5 ng per 10(5) cells per 24 hours on addition of 100 micrograms NR1/mL. In contrast, no change in urokinase-type plasminogen activator and plasminogen activator inhibitor-1 (PAI-1) antigen synthesis was seen. There was also no effect of NR1 on PAI-1 deposition in the extracellular matrix. As judged from fibrin autography and reverse fibrin autography, TPA activity and TPA-PAI-1 complexes reached a maximal stimulation of more than threefold and twofold, respectively, at a concentration of 100 micrograms NR1/mL in conditioned media. On the contrary, NR1 induced a more than fivefold decrease in PAI-1 activity at the same concentration of NR1 in conditioned media. On Northern blot analysis of RNA obtained from NR1-stimulated and control HUVECs, NR1 induced a significant increase in TPA mRNA (192% of control value at 100 micrograms NR1/mL) while PAI-1 mRNA remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro production of plasminogen activator by human granulosa cells.

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.

Abundant urokinase activity on the surface of mononuclear cells from blood and bone marrow of acute leukemia patients

We have examined the mononuclear cell fraction from 35 individuals, 18 with hematologic malignancies and 17 healthy controls for the presence of cell surface-associated plasminogen activator (PA) activity. PA activity was found on the cell surface of 10 out of 12 samples from patients with acute leukemia. In addition to active urokinase (uPA) found on the cell surface in four out of five acute myeloid leukemia patients, tissue-type PA activity was detected in the same samples (3 of 5). Two out of four samples from acute lymphoid leukemia displayed only uPA activity and three out of three samples from biphenotypic leukemia were also clearly uPA-positive. Plasmin activity was not detected in any of the samples. PA activity was not found on the surface of mononuclear cells from either patients with chronic lymphoid leukemia or healthy controls and, in this respect, the cell surface- bound uPA activity behaved as a marker for acute leukemia. The finding of PA activity on the cell surface in acute leukemia suggests that there may be continuous generation of plasmin with consequent consumption of plasma plasmin inhibitors.

Abundant Urokinase Activity on the Surface of Mononuclear Cells From Blood and Bone Marrow of Acute Leukemia Patients

We have examined the mononuclear cell fraction from 35 individuals, 18 with hematologic malignancies and 17 healthy controls for the presence of cell surface-associated plasminogen activator (PA) activity. PA activity was found on the cell surface of 10 out of 12 samples from patients with acute leukemia. In addition to active urokinase (uPA) found on the cell surface in four out of five acute myeloid leukemia patients, tissue-type PA activity was detected in the same samples (3 of 5). Two out of four samples from acute lymphoid leukemia displayed only uPA activity and three out of three samples from biphenotypic leukemia were also clearly uPA-positive. Plasmin activity was not detected in any of the samples. PA activity was not found on the surface of mononuclear cells from either patients with chronic lymphoid leukemia or healthy controls and, in this respect, the cell surface-bound uPA activity behaved as a marker for acute leukemia. The finding of PA activity on the cell surface in acute leukemia suggests that there may be continuous generation of plasmin with consequent consumption of plasma plasmin inhibitors.

Modulation of tissue-type plasminogen activator by retinoids in rat plasma and tissues

In this study the effect of vitamin A status and retinoid treatment on the activity of tissue-type plasminogen activator (TPA) and its inhibitor, plasminogen activator inhibitor-type 1 (PAI-1), in plasma and in several tissues was investigated in BN/BiRij rats. Hypervitaminosis A and retinoic acid treatment increased plasma TPA activity by approximately 50%, but PAI-1 activity was not affected. The effect of retinyl palmitate treatment on plasma TPA activity was already significant after 5 days and continued for at least up to 8 wk. Isotretinoin treatment affected neither plasma TPA nor PAI-1 activity. In plasma of vitamin A-deficient rats, TPA activity was decreased by a factor of three, whereas PAI-1 activity was increased twofold. Modulation of plasma TPA activity by vitamin A status and retinoic acid treatment was associated with similar changes in tissue TPA activity. This suggests that the changes in plasma TPA activities might be the result of changes in tissue TPA synthesis.

The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays

A assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.

Plasminogen activator in mouse and rat oocytes: Induction during meiotic maturation

We have found that ovulated mouse and rat oocytes contain tissue-type plasminogen activator (PA). Primary oocytes isolated from ovaries did not contain the enzyme. During spontaneous meiotic maturation in vitro, tissue-type PA became detectable 5 hr after germinal vesicle breakdown. Induction of tissue-type PA activity was blocked by dibutyryl-cAMP or isobutyl-methyl-xanthine as well as by cycloheximide, but not by actinomycin D or α-amanitin. These results suggest that tissue-type PA mRNA is present in primary oocytes, and that translation of this mRNA is triggered upon resumption of meiotic maturation. Tissue-type PA catalyzed proteolysis around live secondary oocytes and fertilized eggs, indicating secretion of the enzyme. Unlike secondary oocytes, fertilized eggs denuded of their zona pellucida no longer contained the enzyme, suggesting that tissue-type PA production stops at or around fertilization, and that the bulk of the enzyme is secreted at this time.

Aspirin inhibits vascular plasminogen activator activity in vivo. Studies utilizing a new assay to quantify plasminogen activator activity.

Vascular or tissue-type plasminogen activator (TPA) is a key enzyme in physiologic fibrinolysis. To study the role of prostaglandins in modulating the synthesis and release of TPA in vivo, we prospectively studied the effect of aspirin (650 mg/d X 2) on TPA activity in 13 human subjects before and after 10 min of forearm venous occlusion. TPA activity was quantified by a newly developed enzyme-linked immunosorbent assay that both measures and differentiates between TPA and urokinase (UK)-like plasminogen activator activity. This assay is based on the observation that the concentration of alpha 2-plasmin inhibitor-plasmin complexes in Reptilase-clotted plasma increases linearly in proportion to the amount of activator added. Resting TPA activity was higher in women than in men (0.56 +/- 0.59 vs. 0.15 +/- 0.11 U/ml, P = 0.049). Venous occlusion induced an eightfold rise in TPA activity in women (to 4.5 U/ml, P = 0.006) and a 15-fold rise in men (to 2.28 U/ml, P = 0.004), whereas UK activity was not detected. Aspirin inhibited the rise in TPA activity after venous occlusion by 69% in men (P = 0.004) and 70% in women (P = 0.014). In contrast, aspirin had no effect on pre- or post-occlusion hematocrits or Factor VIII-related antigen levels. There was no correlation between plasma salicylate level and percentage inhibition of TPA. Neither exogenous aspirin (0-1 microgram/ml) nor salicylate (0-70 micrograms/ml) inhibited the generation of alpha 2-plasmin inhibitor-plasmin complexes by exogenous TPA or interfered with the assay system. We conclude that aspirin may have an antifibrinolytic effect in man that has not been previously described.