Human tumor cell urokinase-type plasminogen activator (uPA): degradation of the proenzyme form (pro-uPA) by granulocyte elastase prevents subsequent activation by plasmin.

When human granulocytes were stimulated with the chemotactic peptide FNLPNTL (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tyrosinyl- lysin; in the presence of cytochalasin B), proteolytic enzymes were released which prevented activation of tumor-cell derived pro-uPA by plasmin. Elastase was identified by use of eglin C (elastase inhibitor) and an inhibitory monoclonal antibody to elastase as the functional proteolytic enzyme in these granulocyte supernatants. Purified human granulocyte elastase cleaves pro-uPA at amino acid position lle159-lle160 thus generating an enzymatically inactive two-chain form of uPA, as judged by N-terminal amino acid sequence analysis. An additional minor elastase-mediated cleavage site was detected at position Thr165-Thr166. This form of uPA was indistinguishable by SDS-PAGE from plasmin-generated enzymatically active HMW-uPA. Action of plasmin on the proenzyme form of uPA (pro-uPA) generates an enzymatically active uPA-molecule (high molecular weight form; HMW-uPA) which is cleaved at amino acid position Lys158-lle159 (Mr = 33,000 (B-chain) and 22,000 (A-chain). Thus elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. Enzymatically active HMW-uPA, however, was not affected by elastase. Elastase-containing granulocytes were identified by immunohistochemical staining of elastase in breast cancer tissue. Granulocytes were located close to the tumor cells and also in the tumor stroma surrounding the tumor nests. These tumor cells contain pro-uPA. Evidently, the conversion of tumor cell pro-uPA into enzymatically active HMW-uPA is controlled by elastase released from granulocytes into the tumor tissue.

Role of plasmin in the degradation of the stroma-derived fibrin in human ovarian carcinoma.

The aim of this study was to evaluate the type of enzymes involved in tumor-associated fibrinolysis of the stroma component fibrin in ovarian cancer patients. For this purpose, the high-molecular-mass fibrin degradation products (HMM-XDP) were isolated from malignant ascitic fluid by protamine sulfate precipitation and further purified by gel filtration and acid precipitation. After reduction with 2-mercaptoethanol, the peptide chain components were separated by reverse-phase high-performance liquid chromatography (RP-HPLC). The nature of these components was elucidated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequence analysis and compared with fibrin-derived fragments formed in vitro. The results indicate that plasmin is the essential protease involved in the degradation of the stroma-derived fibrin portion found in ovarian cancer ascites.

Elastase released from human granulocytes stimulated with N-formyl-chemotactic peptide prevents activation of tumor cell prourokinase (pro-uPA).

Proteolytic enzymes released from granulocytes upon stimulation with the chemotactic N-formyl peptide FNLPNTL (in the presence of cytochalasin B) prevented activation of tumor cell single-chain urokinase-type plasminogen activator (pro-uPA) by plasmin. Elastase was identified by the use of eglin C (elastase inhibitor) and a monoclonal antibody to elastase as the functional proteolytic enzyme in granulocyte supernatants. Action of purified granulocyte elastase on pro-uPA generated enzymatically inactive two-chain uPA linked by disulfide bridges which was indistinguishable by SDS-PAGE from plasmin-generated HMW-uPA. The major elastase cleavage site in pro-uPA was located between Ile159 and Ile160. a minor one between Thr165 and Thr166. Elastase cannot substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. However, when pro-uPA was first activated by plasmin to form enzymatically active HMW-uPA, this enzymatic activity was not impaired by subsequent elastase treatment.

[Genital infections and the course of pregnancy: a prospective study]. / Genitale Infektionen und Schwangerschaftsverlauf: Eine prospektive Studie.

The reported study investigates the relationship of genital infections, pathobiochemical findings and demographic data to preterm labor, premature rupture of membranes (PROM) and premature delivery. The predictability of chorioamnionitis, puerperal and neonatal infections by these parameters was evaluated concurrently. 301 patients were included in this study between July 1985 and June 1986. 147 of these patients were studied longitudinally during pregnancy, delivery and puerperium (longitudinal group). A second group consisted of 154 women who presented themselves on start of labor to the labor and delivery unit of our Department (peripartal group). The incidence of preterm labor and of PROM was 26%. The incidence of premature delivery, chorioamnionitis, puerperal and neonatal infection was 11.4%, 5.5%, 7.6% and 3% respectively. Cervical colonization with Mycoplasma hominis correlated positively with PROM (relative risk 2.2), premature delivery (3.9) and neonatal infection (6.9). Chorioamnionitis, premature delivery and puerperal infection were also significantly increased in patients with positive vaginal Ureaplasma urealyticum cultures during pregnancy and delivery. Premature delivery (2.8) and puerperal infection (4.0) were associated with a vaginal group B-Streptococci (GBS) colonization during pregnancy, as was a positive GBS culture during delivery associated with puerperal infection. Bacterial vaginosis also correlated positively with premature delivery (5.6) and puerperal infection. Preterm labor correlated negatively with the socioeconomic level, PROM correlated negatively with the marital status, positively with age, a history of cervical cerclage, conization or PROM during former pregnancies. Sexual intercourse more often than once weekly during the last month of pregnancy was also associated with an increased number of PROM. Gardnerella vaginalis, Candida and Trichomoniasis during pregnancy and delivery were associated with preterm labor and puerperal infections. Levels of maternal plasma fibrinogen concentrations in patients with PROM were elevated 48 hours after delivery in accordance to the characteristics of acute phase proteins. In contrast, the maternal PMN-granulocyte-elastase concentration was significantly elevated at time of delivery and 24 hours thereafter in those patients who developed puerperal infections. The derived positive predictive value was 26%, the negative 94.7%, respectively. The overall accuracy of the prediction was 83.1%. Six out of seven mothers with neonates treated because of neonatal infection showed significantly elevated plasma concentration of PMN-granulocyte-elastase.(ABSTRACT TRUNCATED AT 400 WORDS)

Fibrin-fibronectin compounds in human ovarian tumor ascites and their possible relation to the tumor stroma.

Covalently linked heterogeneous fibrin-fibronectin compounds were detected in ascitic fluid of 31 patients with advanced ovarian cystadenocarcinoma by means of enzyme-linked immunosorbent assay techniques, immunoaffinity chromatography, and Western blot analysis. Deposition of fibrin and fibronectin could also be demonstrated immunohistochemically in Carnoy-fixed tissue sections. Fibrin and fibronectin were found in the tumor stroma within tumor nests and more prominently in stroma surrounding the tumor nests. The association of fibrin and fibronectin was especially pronounced in the stroma surrounding the tumor islands. Fibronectin was also found to be associated with stroma cells. Areas within the tumor stroma showed superimposed staining for both fibrin and fibronectin supporting the assumption that the covalently linked fibrin-fibronectin conjugates found in ascitic fluid may stem from the provisional tumor stroma by proteolytic release.

The role of coagulation, fibrinogenolysis and fibrinolysis in the development of fluid and clotted cadaver plasma.

Parameters for coagulation, fibrinogenolysis and fibrinolysis were measured in order to understand the formation of fluid or clotted cadaver plasma. All values found were distinctly elevated without significant differences between fluid and clotted material. The electrophoretic banding pattern of the fibrinogen and fibrin material also proved extensive coagulation and fibrinolysis in all cases. In vitro experiments in which similar electrophoretic banding patterns were obtained suggested that the development of fluid cadaver plasma depends on the activation of fibrinolysis prior to complete coagulation, and is enhanced by persistence of circulation as well as a physiological pH (7.4). In correspondence to post mortal time only an increase in elastase values was found.

Fibrinolysis in ascitic fluid: isolation and characterization of fibrin derivatives, their interaction with albumin.

A method for the preparative isolation of high molecular weight fibrin degradation products (XDP) from ascitic fluid of patients with advanced ovarian cancer is described. By non-reduced and reduced PAA-Gel electrophoresis we could demonstrate that high molecular weight fibrin derivatives exist as E-complexes. A similarity of the polypeptide chain composition from in vitro samples and ascitic fluid could be shown. Immunoadsorption with anti-albumin followed by Westernblotting with anti-fibrinogen-demonstrated the existence of XDP/albumin-associates in ascitic fluid.

[Detection of fibrinolysis in chronic subdural hematoma]. / Nachweis der Fibrinolyse im chronischen subduralen Hämatom.

Various biochemical processes in hematoma fluid arising from chronic subdural bleeding have not yet been clearly explained. We examined the question whether formation of hematoma fluid after onset of bleeding is preceded by complete clotting with fibrin formation and subsequent lysis of the clot. 22 samples of hematoma fluid taken during operation were used. The protein content was 5.7 +/- 9.8 g/l (mean +/- SD), and coagulable fibrinogen could not be demonstrated whereas 0.99 +/- 0.45 mg/ml fibrin(ogen) break-down products were found immunologically. The high levels of crosslinked D-D fibrin derivatives (0.32 +/- 0.26 mg/ml) were conspicuous: there was a correspondingly high degree of crosslinking (86.2% x/- 12.2%) of the fibrin(ogen) break-down products (calculated from the proportion of dimeric to monomeric gamma-chains). Monoclonal antibodies specific for D-D were employed for the first time to answer the question. The characterization of the crosslinked fibrin derivatives together with their submolecular sub-units and the quantification of the D-D content serve to prove that fibrin is both formed and broken down in chronic subdural hematomas.

[A test for the detection of fibrin in the plasma]. / Ein Test zum Nachweis von Fibrin im Plasma.

The article reports on measurements of D dimer, a terminal plasmic lysis product of crosslinked fibrin, with an enzyme immunoassay (ELISA) employing recently developed specific monoclonal antibodies. Due to its sensitivity the test can be used on plasma samples. The D dimer concentrations in patients with deep vein thrombosis diagnosed by laboratory apparatus were significantly increased compared to a control group; in one patient with additional pulmonary embolism, the level was even higher. Moderately elevated concentrations of D dimer were observed in the hypercoagulable state of pregnancy, puerperium and during the postoperative course. This reduces the specificity of the test with regard to the recognition of thromboembolic episodes under these conditions. Obstetric patients with disseminated intravascular coagulation (DIC) showed excessively increased levels of D dimer. Hence, a marker function with regard to the recognition of thromboembolic disease can be attributed to the D dimer; the diagnosis of DIC can be confirmed if very high concentrations are detected.

[Tumor-associated antigens and fibrin derivatives as reaction products of ovarian cancer]. / Tumorassoziierte Antigene und Fibrinderivate als Reaktionsprodukte des Ovarialkarzinoms.

The detection of tumour-associated antigens in blood, ascites fluid, and tissue by the use of monoclonal antibodies establishes new aspects for the course control of ovarian cancer therapy and the evaluation of tumour-associated fibrinolysis. Monoclonal antibodies to the cross-linking site of fibrin derivatives in an enzyme immunoassay (D-dimer-ELISA) allow to compare quantitative results of fibrinolysis products with different tumour markers and the clinical tumour situation. In a prospective follow-up-study in 102 patients with epithelial ovarian carcinoma Ca 125, D-dimer, TPA, Ca 19-9, and CEA were investigated as tumour markers. Specificity (control group 61 gynaecologic patients with benign diseases) and sensitivity were compared in sensitivity-specificity diagrams (receiver operating characteristic curves) for different clinical tumour situations (preoperative, S0-S3). Applying a limit value of normal range, indicated in parentheses, the specificity of Ca 125 (greater than 65 mu/ml) amounts to 100%, D-dimer (greater than 700 ng/ml) 100%, TPA (greater than 120 mu/ml) 90%, CEA (greater than 5 ng/ml) 96%, Ca 19-9 (greater than 37 mu/ml) 98%. In the same sequence, sensitivity values came to 91%, 82%, 65%, 8% and 19% in manifest tumour situations. Antigen-concentration levels and frequency of pathological values depend on the degree of tumour disease. The results of seroimmunodiagnostic methods correlate with the clinical tumour situation: In 11 of 12 cases clinical tumour progression or relapse were accompanied by increasing concentrations of Ca 125 and D-dimer. In 21 cases of second-look laparotomies, tumour marker values in the normal range were often found not to be conclusive for diagnosing a tumour-free situation. The predictive value for true negative results amounted to only 18%. Immunohistochemical investigations and plasma-ascites diffusion ratios of antigens indicate that enrichment occurs in ascites. Fibrin deposition often surrounds tumour plugs and is found in interstitional tissue, whereas Ca 125 is expressed mainly at tumour cell surface. The histochemical image reflects fibrin as a tumour surrounding antigen and Ca 125 as a tumour-originated antigen. Clinical relevant serodiagnostic methods for ovarian cancer are brought about by the monoclonal detection of the investigated antigens.(ABSTRACT TRUNCATED AT 400 WORDS)

[Simultaneous determination of Ca 125 and D-dimer in plasma and ascites in ovarian cancer]. / Die simultane Bestimmung von Ca 125 und D-Dimer im Plasma und Aszites beim Ovarialkarzinom.

In 37 patients with ovarian carcinoma stages Ic to IV the values of preoperatively determined tumour markers and the values of the follow-ups were examined in a trial to observe the course of the disease. The following sensitivity was recorded: Ca 125 92%, D-dimer 100%, TPA 76%, CEA 29%, CA 19-9 15%. Ca 125, D-dimer and TPA are unsuitable as follow-ups. They are also detectable in ascites.

[Changes in blood coagulation during hormonal contraception]. / Veränderungen der Blutgerinnung bei hormonaler Kontrazeption.

PIP: A relationship between use of oral contraceptives and thromboembolic disease has been suspected for years. Recent experimental studies indicate that venous thromboses can be triggered by the interaction of stasis and coagulation activation, also called hypercoagulability. Stasis is promoted by widening of the veins, a change known from pregnancy, although it is not clear whether this can be definitively related to estrogen or progestagen release. In addition, estrogens cause an increase in fibrinogen neosynthesis and increased whole blood viscosity. At low flow rates this causes early occurrence of erythrocyte aggregates. Although an increase in coagulation factors observed by many authors among oral contraceptive users cannot be unequivocally equated to an increased thromboembolic risk, detection of activated coagulation factors is a sensitive indicator of coagulation activation. Thus it was shown by the level of circulating fibrin in plasma that varied coagulation activation occurred in users of 30 and 50 micrograms ethinyl estradiol. In this study of a 3 stage contraceptive (0.5 mg norethisterone, 0.035 mg ethinyl estradiol; 0.75 mg norethisterone, 0.035 mg ethinyl estradiol; 1 mg norethisterone, 0.35 mg ethinyl estradiol) with 22 patients, no significant change in fibrinogen level was found. The same applied to other hypercoagulability parameters fibrinopeptide A and antithrombin III. The constant blood level of early fibrinolysis product F-CB3 also indicated absence of relevant thrombin induced coagulation activation. This is apparently linked to low estrogen dose.^ieng

Measurement of crosslinked fibrin derivatives in plasma and ascitic fluid with monoclonal antibodies against D dimer using EIA and latex test.

D dimer and related crosslinked fibrin derivatives were measured in plasma of normal subjects and in patients with various disorders. In 23 healthy, young females low plasma levels were found (mean 47 ng/ml). In 12 patients with deep vein thrombosis, moderately elevated levels (mean 593 ng/ml) were seen. Higher levels were found in 6 patients with pulmonary embolism (mean 3,337 ng/ml). The highest values occurred in 4 patients with severe intravascular coagulation (31,000 to 390,000 ng/ml). In 22 patients with ovarian cancer and in 21 patients with other gynecologic carcinoma, normal to highly elevated levels of D dimer like material were found. These values corresponded well to concentrations of tumor marker CA 125, measured in the same samples, and to tumor activities staged in these patients based on clinical examinations. Very high values of crosslinked fibrin derivatives (75,000-525,000 ng/ml) were determined in ascitic fluid of patients with ovarian cancer.

Crosslinked fibrin derivatives and fibronectin in ascitic fluid from patients with ovarian cancer compared to ascitic fluid in liver cirrhosis.

Ascitic fluid from patients with ovarian cancer and from patients with alcohol induced liver cirrhosis were compared in respect to hematological and related parameters (content of fibrinogen and fibrin (ogen) degradation products, fibrinopeptide A, F-CB3 related antigen, ratio of crosslinked fibrin to non crosslinked fibrin (ogen), fibronectin and total protein). In tumor ascites all parameters except FPA were significantly elevated compared with cirrhosis ascites. Tumor ascites contains a six-fold higher level of fibrin (ogen) degradation products. A considerable portion of it constitute crosslinked (factor XIII induced) high molecular weight fibrin derivatives. Their content is approx. 10 times higher in tumor ascites than in cirrhosis ascites. Characterization of the crosslinked fibrin derivatives revealed the presence of fragments DD, DY, and some other fragments compatible with XY, DXD, DXY, YXY and DXX. The fibronectin content is also significantly higher in tumor ascites compared with cirrhosis ascites. The value ranges showed no overlap. The findings suggest a turnover of fibrinogen in both ascitic fluids via coagulation and fibrinolysis. In tumor ascites however, fibrinogen seems to be catabolized to a higher degree via the degradation of crosslinked fibrin.

[Markerfunction of crosslinked fibrin derivatives in ascitic fluid from patients with ovarian cancer: a comparison with ascitic fluid in liver cirrhosis]. / Markerfunktion von quervernetzten Fibrinderivaten im Aszites beim Ovarialkarzinom: Ein Vergleich zu Aszites bei Leberzirrhose.

Ascitic fluid from patients with ovarian cancer contains high levels of fibrin(ogen) degradation products (FDP). Crosslinked fibrin derivatives were isolated by precipitation and separated by PAA-gel-electrophoresis. In order to evaluate the submolecular structure the total precipitate and single derivatives were investigated after cleavage of the disulfide bonds. The findings are related to the radioimmunological measurements of certain fibrinopeptides. Tumor ascites contains approximately ten times higher levels of crosslinked fibrin derivatives than cirrhosis ascites. The fibrinopeptide A content is similar, whereas the levels of plasmin-induced alpha-chain fragment are significantly higher in tumor ascites. The findings suggest a catabolism of fibrinogen in ascitic fluid via coagulation and fibrinolysis. In tumor ascites fibrinogen is catabolized to a significantly higher degree via the degradation of crosslinked fibrin. Crosslinked fibrin derivatives may therefore be usable as non-carcinoembryogenic tumor markers.

[Thrombocytopenia and disseminated intravascular coagulation in low-dose heparin prophylaxis]. / Thrombozytopenie und disseminierte intravasale Gerinnung bei niedrig dosierter Heparinprophylaxe.

During postoperative prophylaxis of thrombosis using subcutaneous low-dose heparin thrombocytopenia and consumption coagulopathy without manifest haemorrhage were observed in a female patient with gynaecologic problems on the tenth postoperative day. Using gel-electrophoresis, demonstration of cross-linked fibrin derivatives, which occur only after contact with thrombin, was proof that disseminated intravascular coagulation must have been present. Cancellation of heparin prophylaxis was sufficient for regression of the haemostatic disorder. Platelet function tests performed two months later showed as the only reproducible abnormality that the patient's plasma caused inhibition of desaggregation of normal platelets only when heparin was present. The remarkably close chronologic connection of heparin prophylaxis with thrombocytopenia and consumption coagulopathy suggests a possible causal connection. However, at present the possibly heterogeneous aetiology cannot be explained satisfactorily.

[Prevention of deep-vein thrombosis in patients with gynaecological cancer undergoing radiotherapy. A comparison of calcium heparin and a semisynthetic heparin analogue (author's transl)]. / Thromboembolische Komplikationen während der Strahlenbehandlung gynäkologischer Karzinome.

Patients treated for gynaecological cancer by combined external irradiation and intravaginal radium application participated in two randomized, controlled, and prospective trials. In the first trial the incidence of deep vein thrombosis (DVT) diagnosed by the 125I-fibrinogen test was 43% in the control group. The injection of 7,500 i.u. of subcutaneous calcium heparin twice daily prevented DVT in the test group. An increase of soluble fibrin monomer complexes (SFMC) and fibrinogen in the plasma of patients with DVT suggested that the patients were hypercoagulable. No correlation between AT III values and DVT was observed. In the second trial, the effect of the same dose of heparin was compared with a twice daily s.c. injection of 5,000 U of a semi-synthetic heparin analogue (SSHA). The incidence of DVT was reduced to 15% by heparin; in the patients given SSHA the incidence was 25%, which was not significantly different from the heparin-treated group.

[A comparative study of fibrinogenolysis of adult and cord vein fibrinogen (author's transl)]. / Vergleichende Untersuchungen zur Fibrinogenolyse von Erwachsenen- und Nabelschnurvenen-Fibrinogen.

The streptokinase induced fibrinogenolysis of cord vein fibrinogen follows a slower degradation rate than adult fibrinogen prepared from plasma in the same manner. However, no difference in the degradation rate could be observed when plasmin was used. It is suggested that the lower plasminogen content in the preparation of the cord vein fibrinogen is responsible for the slower degradation rate observed after streptokinase induction.