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The plasminogen-activating system plays an important part in tissue proteolysis in physiological as well as pathological processes. Plasminogen activators u-PA (urokinase) and t-PA (tissue) as well as the inhibitors PAI-1 and PAI-2 are present in gingival crevicular fluid in concentrations significantly greater than in plasma. This fact, and the finding that the concentrations of t-PA and PAI-2 are higher in areas with gingival inflammation, indicate local production of these components. The present study describes, by means of in situ hybridization and immunohistochemistry, the localization of the plasminogen activators and their inhibitors in gingival tissues from patients undergoing periodontal surgery. t-PA mRNA and t-PA antigen were primarily found in the epithelial tissues, predominantly in the sulcular and junctional regions, although occasionally in the oral epithelium and in blood vessels of the connective tissue. u-PA and u-PA-receptor signals were seen in single cells within the junctional and sulcular epithelia and adjacent to blood vessels close to the junctional epithelium, but rarely in the oral epithelium. Similar to t-PA, the predominant location of PAI-2 mRNA was the gingival epithelia. In the junctional and sulcular epithelia, PAI-2 mRNA was seen throughout the thickness, while in the oral epithelium the strongest signals were seen in stratum granulosum and stratum spinosum. PAI-1 mRNA was invariably found in the connective tissue associated with blood vessels. The present study confirms earlier indications of local production of plasminogen activators and their inhibitors in gingival tissues. In addition, the results demonstrate that t-PA and PAI-2 in these patients are produced predominantly in the epithelial tissues. Furthermore, the presence of t-PA and PAI-2 seems to be most pronounced in the areas likely to be subjected to bacterial assault.

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cells types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6 ng/microl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/microl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Two functional assays for protein C inhibitor (PCI) were evaluated in parallel for the same plasma samples. One assay is specific for active PCI and measures its ability to form complexes with activated protein C (APC) in the presence of heparin (España et al, Thromb Res 64, 309, 1991) (Method A). After incubation of samples with heparin and an excess of APC, the amount of APC:PCI formed is measured by an ELISA. The second functional PCI assay is based on the determination of the residual amidolytic activity of the APC added to the sample. In all cases a good correlation was obtained with both methods. For 20 healthy subjects and 20 patients before surgery, there were no significant between-assay differences in the levels of functional PCI activity. On the other hand, there were significant between-assays differences in the levels of functional PCI of the 20 patients measured 1 and 3 days after surgery. In this case, method B gave higher values than method A (38% +/- 13% versus 29% +/- 11% at day 1 and 59% +/- 16% versus 43% +/- 12% at day 3) (mean +/- SD). The level of alpha 1-antitrypsin (alpha 1 AT), the second major plasma APC inhibitor, was significantly increased in patients at day 1 and day 3 (133% +/- 30% and 190% +/- 57%, respectively). Furthermore, there was a positive correlation between the level of alpha 1AT and the normalized difference between the level of functional PCI measured by method B and that measured by method A. Additionally, when normal plasma was supplemented with increasing amounts of purified alpha 1AT, the level of PCI activity measured by method B increased parallel to the increase in alpha 1AT added, but that measured by method A did not. Determination of APC:inhibitor complexes in aliquots of the final mixtures utilized in the PCI assays revealed that the level of APC:alpha 1AT complex increased parallel to the increase in the amount of alpha 1AT added, whereas the APC:PCI complex level remained unchanged. We conclude that current functional PCI assays based on the determination of APC residual activity in plasma are not specific for PCI, are influenced by the level of plasma alpha 1AT and are not reliable for the determination of functional PCI levels in patients with altered levels of alpha 1AT.(ABSTRACT TRUNCATED AT 400 WORDS)

1. Immunotrap assays that can measure the activities of urokinase-type plasminogen activator (uPA) and its inhibitors (PAIs) were characterized. 2. Both human plasma and mouse macrophages in culture were found to contain much higher inhibitor activity than uPA-like activity. 3. The balance between pro- and anti-fibrinolytic activities was quantitatively changed in the murine macrophages after the injection of thioglycollate. uPA-like materials were synthesized by the macrophages and secreted to the conditioned medium continuously, while PAI activity was unchanged during the same time period.

The plasminogen activator (PA) activity in various cell lines is suppressed by glucocorticoids. These phenomena are attributed to either a suppression of PA biosynthesis, to an increase of PA inhibitor or to a combination of both. The regulation of urokinase (UK) production in a human pre-B cell lymphoma line, RC-K8, by dexamethasone (Dex) and phorbol myristate acetate (PMA) was investigated. RC-K8 is a cell line which is consistently producing a high molecular weight UK in the conditioned medium (Kubonishi, I., et al: Jpn. J. Cancer Res. 76, 12-15, 1985). The cells were cultured in RPMI-1640 with Dex or PMA for 1-4 days. UK activity was measured using a chromogenic substrate S-2444 and the antigen by an ELISA kit. PAI-1 and PAI-2 antigens were also measured by ELISA kits and the complex between PA and PAI was examined by SDS-PAGE fibrin-zymography. The UK secretion in RC-K8 cells was inhibited by cycloheximide and actinomycin D. PMA at 0.16-1.6 uM up-regulated the UK activity approximately two-fold, parallel with the antigen, whereas Dex at 1-10 uM decreased the UK expression approximately half. These were verified by SDS-PAGE fibrin-zymography. Neither PAI-1, PAI-2 nor PA/PAI complex was detected in the conditioned medium and in the cell lysate. These data suggest that PMA up-regulates the UK secretion without inducing PAIs and the down-regulation of the UK secretion by Dex results from the inhibition of the expression of UK itself but not from the induction of PAIs.

Decreased fibrinolytic activity (FA) is considered a risk factor in the development of deep venous thrombosis (DVT). The purpose of this study was to test the hypothesis that an abnormally low FA is more likely in idiopathic DVT as opposed to DVT in the presence of other predisposing risk factors. 156 patients were studied three months after an acute DVT confirmed by venography. In 82 patients a predisposing factor such as trauma, surgery or malignancy was present. In 74, DVT was idiopathic. Fibrinolytic activity was measured in all patients using the following tests: 1. Euglobulin Lysis Time (ELT, in minutes). 2. Global Fibrinolytic activity (FA in mg fibrin lysed per hour). ELT measures plasminogen activator activity (PAA) while FA which is a more global test measures the net effect of the PAA, the plasminogen activator inhibitor (PAI) and the clot structure. PAI was measured in 67 patients from both groups using an Enzyme-Linked immunosorbent assay (ELISA). The results obtained show a significant increase in the incidence of abnormal FA and ELT in the idiopathic group. This supports the hypothesis that low FA is an aetiological factor in the development of idiopathic DVT. The results also demonstrate the importance of associating the FA test to the classical tests such as ELT, PA and PAI for the detection of hypofibrinolysis.

Five criteria for poor response to a 20 min venous occlusion test were applied to 58 patients 3 months or more after acute deep vein thrombosis (DVT). The criteria were arbitrarily defined as the last 5 percentiles of response distributions in an age- and sex-matched healthy control group of 51 subjects. The criteria were: 1. euglobulin clot lysis time after venous occlusion greater than or equal to 140 min; 2. t-PA activity after venous occlusion less than or equal to 0.04 IU/ml; 3. increase in t-PA antigen above resting value less than or equal to 2-fold; 4. ratio between t-PA antigen increase and resting PAI activity less than or equal to 0.5 ng/IU; 5. PAI activity after venous occlusion greater than or equal to 6 IU/ml. The last criterion of poor response was the only one that was significantly more frequently reached by patients than by controls: 28% (p less than 0.005) of all DVT patients and 35% (p less than 0.005) of the subgroup with idiopathic DVT (N = 34) were found to be poor responders. The percentage of poor responders according to the other four criteria was 7-11% in all patients and 9-15% in the subgroup with idiopathic DVT and thus was not significantly higher than in controls (5% by definition). It was concluded that residual PAI activity after venous occlusion might be a useful criterion for prospective studies on recurrence of DVT.

Impaired fibrinolysis due to high plasminogen activator inhibitor levels is present in patients with a variety of thrombotic diseases. Plasminogen activator inhibitor levels correlate with triglyceride levels and are elevated in patients with hypertriglyceridemia. Treatment of asymptomatic hypertriglyceridemic patients with diet or medication results in improvement of fibrinolytic parameters. To determine if similar improvements in fibrinolysis occur during treatment of hypertriglyceridemia in patients with a history of venous thrombosis and impaired fibrinolysis due to high plasminogen activator inhibitor levels, 5 patients were given gemfibrozil. Triglyceride levels and fibrinolytic response to venous occlusion were determined before and after 30 days of treatment. Gemfibrozil therapy resulted in a predictable decrease in triglyceride levels in all patients, a reduction that was statistically significant (p = 0.04). There was no significant change in fibrinolytic variables during gemfibrozil therapy. Independent factors appear to control the plasminogen activator inhibitor and triglyceride levels in patients with a history of venous thrombosis.

Orthotopic liver transplantation (OLT) is frequently associated with systemic fibrinogenolysis and diffuse bleeding. At present antifibrinolytic treatment has not been initiated routinely in OLT. Therefore the influence of high dose aprotinin in OLT (2 million kallikrein inactivator units (KIU) given after induction of anesthesia followed by a 500,000 KIU/h infusion throughout the operation) on intraoperative blood loss and fibrinolysis was studied in 25 patients compared to 25 patients without aprotinin. The incidence of fibrinolysis shown in thrombelastography was 72% in the control group versus 16% in the aprotinin group. Oozing after reperfusion of the graft caused by severe fibrinolysis defined as a clot lysis index below 15% was only observed in the control group (42.8%). In contrast no significant difference was found between the groups in the course of fibrin and fibrinogen degradation product levels (FbDP, FgDP) although the mean concentrations of both parameters were evidently lower in the aprotinin treated patients. Levels of tissue-type plasminogen activator (t-PA) activity were initially high in both groups and peaked during and after the anhepatic period. After aprotinin there was a trend of lower t-PA levels which reached significance at the time of reperfusion (p less than 0.02). In both groups the course of thrombin antithrombin complex was in line with the variations of FbDP and FgDP. No correlation between thrombin formation and t-PA activity was found. Mean homologous blood requirement was reduced by 50% (5.6 +/- 4.0 vs. 11.2 +/- 8.6 units, p less than 0.005). The blood saving effect was more pronounced in the postanhepatic period (p less than 0.000001). In conclusion high dose aprotinin inhibits hyperfibrinolysis and reduces intraoperative homologous blood requirement. Therefore its routine use in OLT is recommended.

Eight healthy volunteers were given single subcutaneous doses of dermatan sulfate (DS, 100, 200 and 400 mg), heparin (5,000 IU) and placebo in random order. Wash-out between treatments was greater than or equal to 10 days. Serial blood samples were taken before and up to 24 hours after treatment to measure coagulation and fibrinolytic parameters. Thrombin generation was significantly inhibited by DS and heparin as compared to placebo. The effect of DS was dose-dependent. Peak inhibition after 200 mg DS was comparable to that of 5,000 IU heparin, but lasted longer. A small, bordeline significant prolongation of APTT was observed after 400 mg DS and heparin. The changes in PAI and fibrinolytic activities were those of the circadian variation. No changes were seen in the other parameters tested. In conclusion, single s.c. doses of DS (200, or 400 mg) inhibit ex vivo thrombin generation equally or more than 5,000 IU heparin and for a longer time. The effect of both treatments on fibrinolysis is negligible.

Cardiopulmonary By-Pass (CPB) Surgery may at times induce a haemostatic defect, at present not too well understood, causing severe bleeding from the operative site and chest tube drain. We present here some data on antigen increase in tissue Plasminogen Activator (tPA) and D 2 Dimer (D2D) detected during CPB and apparently not compensated by enhanced Plasminogen Activator Inhibitor type 1 (PAI 1) activity. tPA concentration (antigenic) ranged around 6.15 ng/ml (SD 5.6) before thoracotomy and 5.8 g/ml (SD 4.74) 5-10 minutes after a heparin 250 IU/Kg bolus injection. During CPB, tPA increased to 20.34 ng/ml (SD 9.17) before protamine infusion, and 16.93 ng/ml (SD 8.13) after heparin neutralization. As the D2D went up to 2000-4000 ng/ml (before/after protamine) and it was not correlated by fibrinogen consumption or FDP production, we find these observations suggestive of fibrin-dependent fibrinolytic activity, as an acquired haemostatic defect developed during CPB.

Thirty-four patients envenomed by Bothrops jararaca in Brazil were studied. Of these, 20 (59%) had incoagulable blood associated with local and/or systemic bleeding and 10 of the 20 were thrombocytopenic. Among 14 patients with coagulable blood, 6 (43%) had bleeding symptoms and 3 (21%) were thrombocytopenic. High levels of von Willebrand factor (vWF), plasminogen activator inhibitor type 1 (PAI-1) and tissue type plasminogen activator (t-PA) antigens were also recorded in some patients with systemic bleeding with or without incoagulable blood. These substances may have been released from endothelial cells. Admission serum venom antigen levels were similar in both groups. The study indicated that systemic haemorrhage may occur in patients with coagulable blood and thrombocytopenia and that coagulopathy is not therefore the primary cause of haemorrhage.

Hormonal regulation in the production of a plasminogen activator (PA) was studied in rat hepatocytes in primary culture. Insulin and epidermal growth factor had no effect on the hepatic PA activity. However, glucagon and epinephrine augmented the activity, whereas dexamethasone suppressed it by lowering the production of hepatic PA rather than by inducing plasmin inhibitors or a plasminogen activator inhibitor (PAI). Dibutyryl cAMP, an analogue of cAMP, also augmented hepatic PA activity. The augmented activity level was lowered by either H-8, cycloheximide, or actinomycin D, suggesting that A-kinase and protein biosynthesis are closely associated with the augmentation. Glucocorticoid and hormones that act to raise the intracellular cAMP level may participate in hepatic PA production by the liver.

Extravascular coagulation and fibrinolysis is an integral part of inflammatory reactions. Disordered expression of procoagulant and profibrinolytic factors by mononuclear phagocytes of the lung (i.e. lung alveolar macrophages (LAM) and interstitial macrophages) may have important bearings on inflammatory lung tissue destruction and repair. Based on this hypothesis we have measured the presence of trigger molecules and activation products of the coagulation and fibrinolytic system in cell-free bronchoalveolar lavage fluid and in bronchoalveolar cells. Patient groups with chronic obstructive disease (COLD) (n = 76), idiopathic pulmonary fibrosis (IPF) (n = 29), sarcoidosis (n = 22), lung cancer (n = 36), pneumonia (n = 39), acquired immunodeficiency syndrome (AIDS) (n = 17) and a control group (n = 60) were studied by bronchoalveolar lavage (BAL). In all patient groups tissue thromboplastin (TPL) and fibrinopeptide A (FPA) were significantly increased compared to controls. Plasminogen activator (PA) activity was significantly lower in patients than in normals, and usually associated with high levels of antifibrinolytic activity. The level of PA inhibitor (PAI-2) was not significantly higher in any patient group compared to controls. The sensitivity of the method for fibrin degradation products (FDP) analysis was not high enough to detect FDP in BAL fluid of control individuals, whereas such products could be demonstrated in 25-53% of patients in various categories. We conclude that disordered expression of procoagulant and plasminogen activator activities in bronchoalveolar lavage fluid may reflect a milieu that favours accumulation of fibrin in inflammatory lung tissue and form the basis for the development of pulmonary fibrosis.

The concentration of tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) and the complex of t-PA and PAI-1 (t-PA-PAI-1 complex) were measured using an enzyme immunoassay, where the first antibody was monoclonal antibody against PAI-1 and the second one was polyclonal anti-t-PA Fab' antibody. Plasma levels of t-PA and t-PA-PAI-1 complex increased with increase in age in the total population of males and females, but there was no age related change in free and total PAI-1. Plasma levels of t-PA antigen were higher in males than in females, and those in females gradually increased with increase in age, reaching the same levels as those of males at their 60s. Plasma levels of t-PA-PAI-1 complex were also higher in males than in females, but the difference was low at their 50s and no difference was observed at their 60s. Plasma levels of free PAI-1 were higher in males than in females, but those of males declined at age group higher than their 50s. At their 60s plasma levels of free PAI-1 were higher in females than in males. These results indicate that fibrinolytic parameters such as t-PA and PAI-1 were balanced at higher levels in plasma in males than in females.

In a retrospective study of the 47 patients examined for thrombotic defects, 34 had prolonged euglobulin lysis times (ELT) with 27 of these also having elevated tissue-plasminogen activator inhibitor (t-PAI) activity. Twenty three of the 57 patients examined for possible bleeding problems had prolonged ELT with 15 of this group also having raised levels of t-PAI. There was a statistically greater (P less than 0.005) incidence of elevated t-PAI in the thrombotic group. The incidence of 40% patients with prolonged ELT in the haemostasis group was surprising and perhaps represents some compensatory mechanism to combat bleeding. Thus in vitro tests showed that a reduced capacity for fibrinolysis was present in diametrically opposed groups.