Organ cultures of human fetal hepatocytes in the study of extra-and intracellular alpha1-antitrypsin.

The rate of synthesis of alpha 1-antitrypsin has been studied in organ cultures of fetal human liver. By de novo synthesis, alpha 1-antitrypsin of the same electrophoretic mobility and molecular size as plasma alpha 1-antitrypsin was produced. Synthetic rate was comparable to in vivo conditions and was suppressed by cycloheximide, colchicine and neuraminidase. By increasing alpha 1-antitrypsin levels in cultre medium, suppression of alpha 1-antitrypsin release from the intra-to the extracellular site was achieved, i.e., synthesis does not proceed autonomously. This suppression was preceded by a temporary enhancement of synthesis. Both effects were found to be independent of degree of sialylation of add-d alpha 1-antitrypsin. In contrast to alpha 1-antitrypsin released in tissue culture, the intracellular protein, as analyzed by crossed immunoelectrophoresis of Triton X-100 extracts from fetal liver, was found to occur partly as slowly moving peaks. Whether these peaks represent proforms or incompletely glycosylated precursors of export alpha 1-antitrypsin or complexes with proteases remains unsettled. A variety of other plasma proteins are released in organ cultures making the system suitable for study of factors regulating plasma protein synthesis.

Purification of urokinase by affinity chromatography.

Commercially available urokinase (EC 3.4.99.26), though highly active, is still contaminated with unrelated proteins and degradation fragments of urokinase. Further purification of a urokinase preparation by chromatography on benzamidine-Sepharose is described. The final preparation consisted of two molecular forms of urokinase with molecular weights of respectively 31 000 and 54 000. The 54 000-dalton urokinase appears to be composed of two protein chains, one of which is the 31 000-dalton urokinase. A monospecific antiserum against urokinase was raised.

An inhibitor from placenta specifically binds urokinase and inhibits plasminogen activator released from ovarian carcinoma in tissue culture.

An inhibitor present in placenta and released in placental tissue culture forms specific complexes with each of two molecular forms of urokinase. Autoradiography demonstrated that the inhibitor shifted the electrophoretic position of 125I-labelled urokinase. It did not change the migration of diisopropyl-fluorophosphate-inactivated 125I-labelled urokinase, thereby indicating complex formation dependent on active serine site in urokinase. The inhibitor had a strong neutralizing effect on the plasminogen activators released from human ovarian carcinoma in tissue culture. The placental inhibitor might prove useful in inhibiting the fibrinolytic process necessary for proliferation of tumour vessels.

Production of protein HC by human fetal liver explants.

Human fetal lever explants were found to secrete protein HC into the medium in molar amounts comparable to those of albumin, alpha 1-antitrypsin and orosomucoid. Incorporation of a radioactive amino acid from the medium into the secreted protein HC demonstrated de novo synthesis. The secreted protein HC had the same size and electrophoretic mobility as protein HC of plasma and urine and gave a reaction of immunochemical identity with the protein in these biological fluids.

Identity between the placental protein PP10 and the specific plasminogen activator inhibitor of placental type PAI-2.

The highly specific plasminogen activator inhibitor of placental type, PAI-2, occurs in the placenta in a low molecular mass form of 46.6 kDa, and in pregnancy plasma in a (possibly glycosylated) high molecular mass form of 60 kDa. Extensive knowledge is available about the functional properties of PAI-2 as a plasminogen activator inhibitor and about its molecular biology and regulation. Of the several placenta proteins (PP) isolated, one of them, PP10, has a molecular mass of 48 kDa and its occurrence in malignancy and in complications during pregnancy has been the topic of a number of studies, though its properties and physiological significance are unknown. The present findings constitute evidence of immunological identity between PP10 and PAI-2. The sections of the amino acid sequence of PP10 analysed here were found to have identical counterparts in the sequence of the low molecular mass form of PA1-2, but in several preparations PP10 was found to occur in an inactive two-chain form due to cleavage of an Arg-Thr bond, the two peptide chains being linked to each other by a disulphide bridge. The cleavage site is identical to that observed in the reaction between PAI-2 and urokinase. The results make it possible to coordinate and correlate the findings of many separate studies and our own observations on PP10 and PAI-2.

Urokinase binds to a plasminogen activator inhibitor type-2-like molecule in placental microvillous membranes.

Placental microvillous membranes exhibited saturable binding of urokinase-type plasminogen activator with plateau achieved by 30 min at 4 degrees C and 10 min at 37 degrees C. The binding was essentially irreversible. The capacity was about 8 pmol urokinase per mg membrane protein. Half-maximal displacement of 125I-labelled urokinase was achieved with about 1.0 nM unlabelled urokinase when using 75 micrograms membrane protein/ml. 125I-labelled urokinase did not bind when treated with diisopropylfluorophosphate to block the catalytic activity. Single-chain urokinase (prourokinase), devoid of catalytic activity, did not bind. Catalytically active tissue-type plasminogen activator did compete with 125I-labelled urokinase for binding although less efficiently than urokinase. Binding activity remained in the 100,000 x g pellet after treatment of the membranes with 3 M KCl, alkaline stripping at pH 12 or extraction by the detergent Triton X-100. The binding was essentially blocked by antibodies against plasminogen activator inhibitor-type-2 (PAI-2). Sodium dodecyl sulfate polyacrylamide gel electrophoresis of solubilized membranes with bound 125I-labelled urokinase showed that the urokinase-PAI-2 complexes largely migrated in fractions corresponding to a very large Mr although no clearly defined peaks were observed. It is suggested that PAI-2 occurs in a form anchored to syncytiotrophoblast microvilli, possibly to the cytoskeleton.

Prognostic significance of birth of large infant for subsequent development of maternal non-insulin-dependent diabetes mellitus: a prospective study over 20-27 years.

In a prospective study, 270 women who gave birth to infants weighing greater than or equal to 4500 g (large baby, LB) underwent an oral glucose tolerance test (OGTT) within the first week of the puerperium. Of these women, 179 (66.3%) were retested 3-10 yr later, and 236 (87.4%) were also evaluated 20-27 yr later. The frequency of pathologic OGTTs increased with time, but the tests were of little prognostic significance for the individual patient. Women who had borne LBs developed non-insulin-dependent diabetes mellitus (NIDDM) six times more often than did a control group of women matched for age and parity and with the same period of follow-up. However, patients who developed NIDDM were also very obese, of high parity, and had a positive family history for diabetes mellitus in a high percentage of cases. Women with LBs as the only risk factor did not develop NIDDM in our study. We conclude that the birth of one LB is of minor, if any, importance in the subsequent development of NIDDM.

Epidermal plasminogen activator inhibitor (PAI) is immunologically identical to placental-type PAI-2.

Plasminogen activator inhibitor (PAI) purified from human epidermis [(1986) FEBS Lett. 408, 273-277] was immunologically identified as placental-type PAI-2. In both fibrinolytic and synthetic substrate assays inhibitory activity of epidermal PAI was neutralized by anti-PAI-2, but not by anti-endothelial type PAI-1. Immunoblotting technique confirmed that the purified epidermal PAI is reactive with anti-PAI-2, but not with anti-PAI-1. Consequently PAI in human epidermis was demonstrable by immunohistochemical technique.

Determination of intermediates, products and cleavage site in the reaction between plasminogen activator inhibitor type-2 and urokinases.

Several specific inhibitors for plasminogen activators have been isolated from various organs and cell lines, those from human placenta and the human monocyte-like cell line U-937 being virtually identical. The reaction between this type of inhibitor, designated as type-2, and high-Mr and low-Mr urokinase-type plasminogen activators was followed by reversed-phase high-performance liquid chromatography and gel electrophoresis. The components, their stable complexes and their dissociation and cleavage products could be clearly identified in both systems. The amino acid sequence of the inhibitor at the cleavage site was determined to be -Met-Thr-Gly-Arg decreases Thr-Gly-His-Gly-. A 35-residue carboxy-terminal fragment was found to be released.

Human fetal dopamine neurons grafted into the striatum in two patients with severe Parkinson's disease. A detailed account of methodology and a 6-month follow-up.

By using stereotaxic surgical techniques, ventral mesencephalic tissues from aborted human fetuses of 8 to 10 weeks' gestational age were implanted unilaterally into the striata in two patients with advanced Parkinson's disease. The patients were treated with a cyclosporine, azathioprine, and steroid regimen to minimize the risk for graft rejection. They were examined for 6 months preoperatively and 6 months postoperatively and continued to receive the same doses of antiparkinsonian medication. There were no significant postoperative complications. No major therapeutic effect from the operation was observed. However, in the clinical tests, both patients showed small but significant increases of movement speed for repeated pronation-supination, fist clenching, and foot lifting. The rate of walking also increased in the one patient tested. For both patients, there was an initial worsening postoperatively, followed by improvement vs preoperative performance at 1 to 3 months. Both patients also showed significant improvement in the magnitude of response to a single dose of levodopa (L-dopa), but there was no increase in the duration of drug action. The motor readiness potential increased in both patients postoperatively, primarily over the operated hemisphere. Neurophysiological measurements also showed a more rapid performance of simple and complex arm and hand movements on the side contralateral to transplantation in one patient at 5 months postoperatively. Positron emission tomography demonstrated no increased uptake of 6-L-(18F)-fluorodopa in the transplanted striatum at 5 and 6 months. Taken together, these results suggest that the fetal nigral implants may have provided a modest improvement in motor function, consistent with the presence of small surviving grafts. Although our results support further scientific experimentation with transplantation in Parkinson's disease, widespread clinical trials with this procedure are probably not warranted at this time.

Cell membrane receptors for urokinase plasminogen activator are increased in malignant ovarian tumours.

The binding of 125I-labelled urokinase plasminogen activator (uPA) to cell membranes of ovarian tumours was characterised. Binding was fast, specific to HMW-uPA, and saturated at low concentration [1.5 (range 1.2-1.6) nmol/l]. Scatchard analysis suggested a single class of binding sites with Kd 1.1 (0.9-1.3) nmol/l. These data indicate the presence of a specific cell membrane receptor for uPA in ovarian tumours, whose characteristics are similar to those reported for uPA receptors in other tissues. Endogenously occupied receptors were uncovered by exposing the membranes to acid conditions (pH 2) before assay, thereby allowing quantitation of the total amount of receptor. uPA receptors were assayed in 10 malignant and 6 benign epithelial ovarian tumours. The total number of receptors was higher in the malignant tumours. This was secondary to increases of both free and occupied receptors. We conclude that this reflects the biological function of cell surface bound plasminogen activation in tumour growth and spread.

Plasminogen activators and plasminogen activator inhibitors in blood and tumour fluids of patients with ovarian cancer.

We quantitated urokinase and tissue plasminogen activator (u-PA, t-PA), plasminogen activator inhibitor 1 and 2 (PAI-1, PAI-2), and fibrinolytic activity in peripheral blood (PB), tumour blood (TB), peritoneal/ascitic fluid (PAF) and cystic fluid (CF) from 104 patients with benign and 36 patients with malignant ovarian tumours, and in peripheral blood from 62 healthy controls. PB levels of u-PA were higher in patients with benign and malignant tumours than in controls. High concentrations of u-PA were found in CF, but not in TB, suggesting that u-PA is released by the tumour tissue, but not by the tumour vasculature. PB levels of t-PA were higher in both tumour groups than in controls. Increased levels of t-PA were found in TB, but not in CF, indicating that t-PA is released by the tumour vasculature, but not by the tumour tissue. PB levels of PAI-1 were higher in patients with both benign and malignant tumours than in controls. High levels of PAI-1 were present in both TB and CF from malignant tumours, suggesting that PAI-1 is released from the tumour vasculature as well as the tumour tissue. Elevated concentrations of PAI-2 were found in CF, but not in TB, indicating release from the tumour tissue, but not from the vasculature. High levels of t-PA, PAI-1 and PAI-2 were found in PAF of malignant tumours, and resorption from this compartment may explain elevated PB levels in patients with ascites. None of the PAs/PAIs proved useful as a PB marker for detection of early stage ovarian cancer. However, an index based on PAF levels of t-PA and PAI-1 discriminated between malignant and benign ovarian cysts in the absence of ascites. In addition, our study stresses the importance of including patients with benign tumours as well as healthy controls when markers for malignant tumours are evaluated.

Treatment with tranexamic acid during pregnancy, and the risk of thrombo-embolic complications.

Tranexamic acid (AMCA) is an inhibitor of fibrinolysis used to treat fibrinolytic bleeding (e.g., menorrhagia and gastro-intestinal haemorrhage), and to prevent bleeding at surgery, in cases of abruptio placentae and general haemorrhage. As AMCA stabilises preformed clots and prolongs their dissolution, it has been debated whether treatment with AMCA might predispose to thrombosis by depressing the fibrinolytic system. Pregnant women constitute a group with low fibrinolytic capacity and an increased frequency of thrombosis further increased after Caesarean section, and are thus more likely to be susceptible to antifibrinolytic therapy. We therefore carried out a retrospective analysis of the case records of 2,102 patients with various bleeding disorders during pregnancy. Of the 256 patients treated with AMCA (mean duration of treatment, 46 days), 169 were delivered by Caesarean section. Of the remaining 1,846 patients (i.e., controls), 443 were delivered by Caesarean section. The relationship between the use of AMCA and the occurrence of thrombo-embolism was calculated with 95% confidence limits. Of the AMCA treated group (n = 256), two patients--one of whom belonged to the Caesarean section subgroup (n = 168)--had pulmonary embolism. Of the controls (n = 1,846), three patients had deep vein thrombosis and one had pulmonary embolism, all four cases belonging to the Caesarean section subgroup (n = 443). Thus, the findings in this high risk group of women with complicated pregnancies, frequently entailing delivery by Caesarean section, provided no evidence of any thrombogenic effect of AMCA.

Cellular localisation in placenta of placental type plasminogen activator inhibitor.

A specific plasminogen activator inhibitor is known to occur in placenta and in pregnancy plasma. Immunohistochemical methods with polyclonal and monoclonal antibodies against the inhibitor were used for its localisation in term placentas. Immunoreactive material was found in the trophoblastic epithelium. It was absent in the stroma of the chorion villi.

Tissue plasminogen activator and urokinase in normal, dysplastic and cancerous squamous epithelium of the uterine cervix.

The distribution of tissue plasminogen activator (t-PA) and urokinase (u-PA) was investigated immunohistochemically in squamous epithelium of the uterine cervix, in normal and dysplastic conditions as well as in preinvasive and invasive carcinomas. Normal epithelium showed presence of both t-PA and u-PA immunoreactivity only in the superficial cellular layer, whereas in preinvasive lesions they were present in all layers. In sections of normal specimens as well as preinvasive lesions the UK immunoreactivity was weaker compared to that of t-PA. Invasive lesions showed a patchy distribution of t-PA and u-PA immunoreactivity. Furthermore, it could be demonstrated that stromal cells close to the infiltrating malignant squamous epithelium contained t-PA immunoreactivity and also u-PA immunoreactivity. Further immunohistochemical studies disclosed that these cells were macrophages.

Kinetics of the reaction between urokinase and an inhibitor of fibrinolysis from placental tissue.

The interaction of urokinase (EC 3.4.21.31) and a protein proteinase inhibitor of fibrinolysis partially purified from placental tissue, which inhibits urokinase but not plasmin, was investigated. The preparations of the inhibitor contained no other known proteinase inhibitors. It was found that a 1:1 complex is formed and that the reaction proceeds as a second order, one step process, the association rate constant of which is 4.5 10(6) M-1 S-1 at pH 8.4, 37 degrees C and 3.0 10(6) M-1 S-1 at pH 7.3, 37 degrees C. The binding is very tight, the dissociation constant of the inhibitor-urokinase complex was estimated to be Ki less than or equal to 10(-11) M.

Purification of urokinase by a beta-naphthamidine affinity column.

Urokinase was purified by affinity chromatography using 6-amino-naththamidine-(2), a new specific ligand based on the urokinase inhibitor beta-naphthamidine. Urokinase was firmly bound at pH 7.0 and could be eluted at pH 3.0. The protein which passed the column at pH 7.0 without being bound did not contain any urokinase activity. This is an important property because it can be utilized for raising a monospecific urokinase antiserum by absorbing unspecific antibodies with only a minor loss of antiserum titre.

Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator.

A monoclonal antibody of IgG2a-type was obtained against a specific fast acting plasminogen activator inhibitor found in placenta. The placental inhibitor was purified by affinity chromatography using the monoclonal antibody and additionally in a FPLC-system. A strong complex formation was found between the inhibitor and urokinase and also with the two-chain form of plasminogen activator of the tissue-type. A weaker complex was found between the placental inhibitor and the one-chain form of the tissue-type activator.

Immunohistochemical localisation of tissue plasminogen activator and urokinase in the vessel wall.

Immunoreactive plasminogen activators were studied in tissue sections using a peroxidase method and monospecific antibodies to tissue plasminogen activator produced by a melanoma. Tissue plasminogen activator reactivity was found in skin melanomas and in endothelial and smooth muscle cells of arteries and veins. Vessels of the umbilical cord showed higher reactivity than peripheral vessels. Only faint antiurokinase reactivity was found. By means of the fibrin slide technique, fibrinolytic activity could be shown in peripheral vessel walls but not in the umbilical cord, which suggests that immunoreactivity of tissue plasminogen activator bound to an inhibitor can also be demonstrated. This method may be a useful tool in further studies of tissue plasminogen activator in physiological as well as pathological processes.

Immunological comparison between human and rat plasminogen activators in blood and the vessel wall.

To evaluate the rat as an experimental model for plasminogen activator research, the ability of antibodies specific for human tissue type plasminogen activator and urokinase to suppress the plasminogen activator activity in whole plasma and in the vessel wall was studied in both rat and man. Plasminogen activator activity in plasma was assayed on fibrin plates containing plasminogen. Plasminogen activator in the vessel wall was shown by the fibrin side technique. Antibodies against human tissue type melanoma cell activator and urokinase were raised in goats and mixed into the fibrin film or the fibrin plates. In both species antibodies to melanoma cell activator were able to suppress the plasminogen activator activity completely in plasma and in the vessel wall. Anti-urokinase, however, had no suppressing effect. In rat plasma the inhibitory effect on the fibrinolytic activity was seen only with high concentrations of antibodies against melanoma cell activator, which suggests that rat plasminogen activator in plasma and vessel walls is similar to, but not identical with, human tissue type plasminogen activator.