bluuebunny/crossref_metadata_2025_split huggingface dataset:
A MECHANISM FOR THE POTENTIATING EFFECT OF UROKINASE (UK) OR TISSUE PLASMINOGEN ACTIVATOR (t-PA) ON CLOT LYSIS BY PRO-UROKINASE (PRO-UK)
Authors: Gurewich V , Black J , Pannell R 
Published: August 2018

Pro-UK has been shown to selectively activate fibrin-bound plasminogen (Blood 67:1215, '86). Synergism with t-PA has been postulated to be related to t-PA's rapid induction of plasmin generation which exposes new plasminogen binding sites on fibrin (JBC Harpel et al 260:4432, '85). As a result, more substrate is made available for pro-UK, thereby attenuating its lag phase and greatly potentiating its fibrinolytic effect (Thrombos. Res. 44:217, '86). This hypothesis was tested with UK, which has a rapid onset of action similar to t-PA. In vitro lysis (125I-clots suspended in citrate plasma), by UK (0.05-0.2 μg/ml) plus pro-UK (0.8-0.95 μg/ml) was compared with that of UK or pro-UK alone. Marked potentiation consistent with synergism by UK was found. For example, 0.1 μg/ml UK plus 0.9 μg/ml pro-UK induced clot lysis equivalent to that of 1.25 μg/ml pro-UK or 1.50 μg/ml UK alone using either 50% or 100% clot lysis as end points. Similar findings were obtained when clots were exposed to these concentrations of UK and pro-UK sequentially instead of simultaneously. For this, a clot suspended in plasma was incubated (37°C) for 1 h. with UK, then removed and suspended in fresh plasma (3 ml) for 1 h. after which pro-UK was added. In another set of experiments, a radiolabeled clot was incubated in buffer containing plasmin (2 μg/ml) for 20 min. ( <1% lysis), then transferred to plasma and incubated for 30 min., after which pro-UK (0.8-1.0 μg/ml) or t-PA (0.04 μg/ml) was added. Plasmin pretreatment of clots caused marked potentiation of lysis by pro-UK but did not potentiate t-PA. This potentiation of clot lysis by plasmin pretreatment was nullified by treatment of clots with carboxypeptidase-B, to remove lysine residues.It was concluded that UK potentiates pro-UK induced clot lysis by exposing plasminogen binding sites on fibrin according to the mechanism postulated above for t-PA. The potentiating effect of UK on pro-UK appears to be analogous to that of t-PA and may also be defined as being synergistic.
IN VITRO CLOT LYSIS INDUCED BY SINGLE-CHAIN UROKINASE (scu-PA) AND ACTIVATION OF THE CONTACT PHASE SYSTEM
Authors: Hauert J , Bachmann F 
Published: August 2018

Scu-PA was purified from ccaiditioned medium of cell lines (HT 1080, MG 118) by immunoaffinity chrcxnatography on anti-u-PA Sepharose and passage through benzamidine-Sepharose to remove traces of two-chain u-PA (tcu-PA). The amidolytic activity determined with S-2444 was 20'000 u-PA IU/mg before and undetectable ( <700 IU/mg) after the benzamidine-Sepharose step. Pretreatment of scu-PA by plasmin yielded a specific activity of 100'000 IU/mg. We have studied, in vitro, the lysis of plasma clots containing 125I-fibrin as tracer. To obtain 50% lysis in 90 min, 130 IU/ml of scu-PA had to be added to plasma before clotting; an equivalent rate of lysis was obtained with 45 IU/ml of tcu-PA. When, after the addition of scu-PA, the contact phase system of plasma was activated using ellagic acid and phospholipids, lysis was complete within minutes following clotting; 7 IU/ml of scu-PA induced 50% lysis in 90 min. An equivalent concentration of tcu-PA or plasmin-treated scu-PA did not bring about clot lysis after 7 h. Using plasmas deficient in prekalli-krein or in high molecular weight kininogen no lysis occured in the presence of 7 IU/ ml of scu-PA and contact phase activation; lysis reached 15% after 7 h with FXII deficient plasma and was normal with FXI deficient plasma. Clot digestion was totally abolished in the presence of a kallikrein inhibitor (SBTI 0.2 mg/ml) or antibodies directed against prekallikrein or u-PA. Hie addition of FXII inhibitor (LBTI 0.2 mg/ml) or of antibodies against FXII reduced the rate, but did not totally block the lysis. Contact dependent fibrinolysis in the presence of 7 IU/ml of scu-PA was furthermore modulated by plasminogen activator inhibitor 1 (PAI-1). A positive exponential correlation between the level of PAI-1 activity and 50% lysis time was observed (r2=0.83, n=12).We have previously reported that kallikrein efficiently converts scu-PA into tcu-PA. The present study shows that, in normal human plasma, the fibrinolytic effect of scu-PA is twenty fold potentiated through contact activation and suggests an important physiological role for scu-PA counteracting excessive clot formation in situations associated with contact activation of the coagulation system.
SENSITIVE AND QUANTITATIVE ASSAYS OF FUNCTIONALLY ACTIVE PLASMINOGEN ACTIVATORS IN PLASMA
Authors: Mahmoud M , Gaffney P J 
Published: August 2018

A previously described (Mahmoud, M. and Gaffney, P.J., Thrombos.Haemostas. 53: 356-9, 1985) bioimmunoassay for tissue plasminogen activator (t-PA) has been modified in terms of catcher antibody concentration and conditions of incubation of various of the steps in this assay to achieve a sensitivity range of 5 - 500 x 10-3 iu/ml of t-PA. A similar assay has been developed for urokinase (UK) achieving a sensitivity range of 5500 x 10-4 lu/mi. The sensitive t-PA assay has been demonstrated to be a valuable replacement of the traditional euglobulin clot lysis time (ECLT) assay and has the added advantage of avoiding loss of t-PA during formation of a euglobulin fraction and of calibration in terms of an International Standard for t-PA. Using both these assays it has been demonstrated that the enhanced fibrinolytic activity observed following exercise is due to increased levels of t-PA while the level of UK in plasma remains unaltered. This was further confirmed by quenching experiments using specific antibodies to t-PA and UK. In contradiction to an earlier report from this laboratory, free t-PA (10 - 60 x 10-3 iu/ml) has been demonstrated in resting plasma, while the level or free UK activity was 50 - 100 x 10-3.These sensitive t-PA and UK assays suggest that physiological and drug induced (heparin fraction CY 222 from Laboratoire Choay, Paris, and SP54 from Benechemie, Munich) enhancement of fibrinolytic potential m the human circulation is mainly mediated by t-PA. Venous occlusion of matched groups of subjects with and without deep venous thrombosis (DVT) showed a wide variation in response m t-PA levels in each group and no change in u-PA levels. There was no difference between the DVT and non DVT groups and this suggests that the notion of the 'non-responder' being more prone to thrombosis needs re-examination. Further experimentation with these sensitive assays may help to elaborate whether the levels of UK in plasma play any role in the haemostatic balance m man.
SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA
Authors: Grøndahl-HANSEN J , Agerlin N , Nielsen L S , Danø K 
Published: August 2018

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.
INTRAVENOUS PRESSURE AND t-PA ANTIGEN RELEASE DURING VENOUS OCCLUSION OF UPPER AND LOWER LIMBS
Authors: Haaland K A , SkjØnsberg H O , Thaulow E , GjØnnes G , Godal C H 
Published: August 2018

Venous occlusion by means of a sphygmomanometer cuff is a well-established part of the procedure for assessment of fibrinolytic capacity. It has been suggested that the resultant intravenous pressure is responsible for plasminogen activator release.In this present study we wanted to compare i .v. pressure in the upper and lower limbs, during venous occlusion, and measure t-PA ag release. 8 male volunteers participated. A VenfIon(O.D. 1.2 mm) was inserted in a distal, superficial vein. Blood samples, discarding the first 2-3 ml. were drawn at 4 min. intervals and pressure recordings (Hewlett-Packard)made every min. during the first 8 min. of venous occlusion and thereafter at 4 min. intervals. The cuff was inflated to a pressure midway between systolic and diastolic values after separate measurements of blood pressures of the upper and lower extremities. The occlusion lasted for 20 min.Blood pressure measurements in the lower limbs gave consistently higher values than for the upper limbs. Hence, the resultant cuff pressure was on the average 20 mmHg higher in the legs.There was no significant difference in i.v. pressure, evaluated as total area beneath a pressure/time graph, between the upper and lower extremities. In the arms the i.v. pressure reached a plateau phase after 2-4 min. The pressure buildup was slower in the legs.t-PA values for the upper limbs after 20 min. of venous occlusion: median 15 ng/ml (range 9.4-26.0) and for the lower limbs: 8.15 ng/ml (range 4.8-13.3). This difference is significant, p = 0.035. There was no sifnificant difference between resting t-PA ag' levels prior to occlusion.We conclude that the fibrinolytic response, measured as t-PA ag, after venous occlusion, is twice as high in the upper as in the lower limbs and not 4-times as high as previously claimed. A possible explanation for the lower fibrinolytic response, could be the slower buildup of i.v. pressure during venous occlusion in the lower extremities.
ISOLATION OF THE GENE CODING FOR HUMAN PLASMINOGEN ACTIVATOR INHIBITOR TYPE 1 (PAI-1)
Authors: Bosma P J , van den Berg E A , Kooistra T 
Published: August 2018

A human placenta genomic DNA cosmid library was screened for the presence of the PAI-1 gene using a cDNA probe coding for PAI-1. Two overlapping recombinant cosmids were obtained that contain human DNA spanning 55 kb. The cosmids were mapped using 3' and 5' end probes isolated from an almost full-length cDNA clone of 2.5 kb. The two cosmids were found to contain the entire structural PAI-1 gene (approximately 15 kb) and also included 25 kb 5' flanking sequences. The transcription initiation site was identified by SI nuclease protection experiments and the promotor region was sequenced. Further experiments will be directed at characterizing the regulatory elements of the PAI-1 gene.In order to determine the chromosomal localization of the PAI-1 gene we have hybridized our genomic clones in situ to metaphase chromosomes of a human blood cell culture. Preliminary experiments show a specific hybridization signal which will enable us to sublocalize the chromosomal position of the PAI-1 gene.
INTERACTION OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (tPA)-INHIBITOR COMPLEX WITH FIBRIN
Authors: Okada M , Sakata Y , Matsuda M 
Published: August 2018

Tissue type plasminogen activator (tPA) binds to fibrin resulting in marked enhancement of its activity, but also binds to its some specific fast acting inhibitors (PAIs). The ternary interaction of tPA, PAI and fibrin has not been fully elucidated, however. We studied the interaction of the tPA-PAI complex with fibrin using PAI-1 derived from human umbilical vein endothelial cells (ECs), either as a denaturant activated purified form (dPAI) or a native form (nPAI).When ECs were incubated with125I-tPA, nPAI formed a complex with 125I-tPA (125I-complex) in dose and time dependent manners. Resultant 125I-complex was immunopurified to give a single HOKd protein and examined for its binding to fibrin under various conditions. Mixtures of fibrinogen and 125I-complex were clotted with thrombin and aprotinin, and the clots formed were separated by synersis, washed with Tris-buffer containing albumin and aprotinin. The radioactivity was counted in the clots and the clots liquor, and the %binding of the 125I-complex was caluculated. We found that the 125 I-complex bound to fibrin and that the binding was inhibited by excess amounts of tPA competitively , but by neither purified latent PAI nor dPAI. Inthe presence of 50 mM EACA, 125I-comolex did not bind to fibrin. However, 50% of 125I-tPA bound to fibrin in the presence of excess amounts of EACA. Moreover, binding of 125I-complex to fibrin decreased in accordance with decreasing amounts of fibrinogen clotted. Since tPA has been reported to bind to fibrin through the finger domain at low concentrations of fibrinogen and also through kringle-2 domain at high concentrations of fibrinogen, respectively, our data indicate that the complex binds to fibrin rather specifically via tPA part of the complex, probably via its kringle-2 domain and the finger domain might be masked by PAI complexed with tPA.As denaturant activated purified PAI a Tone found to bind to fibrin, the binding of tPA-dPAI complex may be rather differently including binding mediated by the dPAI part of the complex.
QUANTIFICATION OF ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) ANTIGEN BY AN ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)
Authors: MacGregor I R , Booth N A , Hunter N R , Bennet B 
Published: August 2018

An ELISA to measure PAI-1 antigen has been developed using PAI-1 purified from human endothelial cell conditioned medium and a monospecific antiserum raised against it in the rabbit. Test and standard samples diluted in assay buffer containing non-immune rabbit serum were incubated in microplate wells coated with anti-PAI-1 IgG. Then biotinylated anti-PAI-1 IgG was added to the wells followed by a streptavidin-biotinylated horseradish peroxidase complex. Tetramethylbenzidine was used as substrate and the optimised ELISA had a detection limit of 0.2 ng PAI-1 ml-1 sample, using purified PAI-1 of known concentration as a standard for the assay.PAI-1 antigen was readily detectable in human plasmas and higher concentrations were invariably detected in the corresponding whole blood sera.α2antiplasmin and antithrombin III which have been shown to have high degrees of sequence homology with PAI-J were undetectable in the ELISA at concentrations of 10 ug ml-1 . Sera from baboon and Rhesus and Cynomologus monkeys exhibited partial cross reactivity in the ELISA while no crossreactivities were observed with a wide range of non-primate sera that included dog, goat, cow, horse, pig and rat.A variety of human cell cultures were assayed for PAI-1 antigen. Endothelial cells from umbilical cord veins and arteries and from adult sapgenous veins secreted, typically,1-2 ug PAI-1 24-1 hr per 10 cells. This represented approximately 2-4% of the total secreted protein. Detectable but considerably lower levels of PAI-1 were secreted by keratinocytes and fibroblasts as well as a lung carcinoma cell line A549.No PAI-1 was detected in media conditioned by two melanoma cell lines, a breast carcinoma cell line MCF7 or a monocytic leukaemia JIII. The ELISA proved suitable for monitoring altered cellular PAI-1 metabolism, and in conjunction with functional assays, for investigating changes in PAI-1 specific activity.
PARALLEL VARIATIONS OF PLASMA INSULIN, TRIGLYCERIDE AND PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI 1) LEVELS IN OBESE NON DIABETIC SUBJECTS
Authors: Juhan-Vague I , Vague P , Alessi M C , Atlan C , Valadier J , Badier C 
Published: August 2018

We had shown in non diabetic healthy subjects with Body Mass Index (BMI) varying largely, a significant correlation (r) between PAI activity levels and BMI : (r=0.66) and insulinemia (r=0.52).PAI levels were then studied in non diabetic Obese women (0)(n=50) and in age matched healthy women with normal BMI (N) (m ± SEM) = 0 versus N : BMI : 33.4± 0.8/ 20.2± 0.8 ; plasma insulin (Ins -uu/ml) : 22.7±1.5/12±1; Triglyceride(TG-mmol/1):1.240.09/0.8±0.1.The low value of euglobulin fibrinolytic activity (EFA) in Obese was due to high levels of PAI 1 : 0 versus N : EFA (mm) : 5.2 ±0.3/ 9 ±0.3 ; PAI activity (PAIact.- Verheijen's method-u/ml) :14 ± 2.1/5.04 ±0.6 -(p 0.01). In 10 0 and 10 N PAIact and PAI 1 antigen (PAI 1 Ag- Kruithof's method-ng/ml) were determined in parallel : 0 versus N : PAIact.: 29.4±3.8/ 5.8±0.9 ; PAI 1 Ag : 100±14.2/19.7 ±1.7, (p0.01). In Obesegroup correlations between, PAIact. and BMI (r=0.51), and insulin (r=0.69) and Triglyceride (r=0.48) were significant (p 0.01).When in Obese subjects, insulinemia was decreased by 24 hours Fast (n=10) or by 15 days Metformin treatment (1.7g/day) (n=9), PAI activity (measured on fibrin plates)decreased significantly in a parallel way : compared to initial values -after 24 hours Fast =Ins : 75 %, PAIact. 73% -after Metformin treatment=Ins :75%, PAIact. :57%, TG :73%.From these results, a direct action of insulin on the synthesis cells of PAI 1 could be evoked. An effect of Triglyceride levels cannot be excluded, the variation of Triglyceride, insulin, PAI levels being parallel.As hyperinsulinemia, hypertriglyceridemia are risk factors for atherothrombosis, as there is a link between insulin, triglyceride, PA Inhibitor 1 levels, the pathogenic role of hyperinsulinemia and hypertriglyceridemia in the development of atherothrombosis could be in part mediated by plasma hypofibrinolysis due to high level of PAI 1.
A PLASMINOGEN ACTIVATOR INHIBITOR (PAI-2) CIRCULATES IN TWO HIGH MOLECULAR WEIGHT FORMS IN PREGNANCY
Authors: Booth N A , Reith A , Bennett B 
Published: August 2018

Normal vascular endothelium and platelet α-granules contain an inhibitor of plasminogen activator (PAI-1) of about 48000 molecular weight, which is released by stimuli such as thrombin. An immunologically distinct inhibitor (PAI-2) of about 47000 molecular weight has been purified from placenta and from a histiocytic cell line U-937. The level of PA-inhibition in plasma is raised in late pregnancy and this may be due to increases in PAI-1 or in PAI-2 or in both.Using SDS-PAGE and zymography on fibrin/plasminogen /u-PA detector gels, we have found that normal plasma contains a band of inhibition of apparent molecular weight 40000, which can be neutralised by antiserum raised against PAI-1. Pregnancy plasma contained this band as well as additional inhibitor bands of apparent molecular weights 75000 and 130000. The novel high molecular weight PA-inhibitors were detectable by zymography at about 12 weeks gestation. They were specific for plasminogen activator and did not inhibit plasmin. They were inhibited by antiserum raised against PAI-2 from U-937 cells (a gift from Dr EKO Kruithof) and thus are immunologically related to PAI-2. They may represent circulating complexes of PAI-2 with another protein or aggregates of PAI-2, which retain inhibitory activity after SDS-PAGE. PAI-2 appears to represent a pregnancy associated protein that circulates in a number of different molecular weight forms.
PLASMINOGEN ACTIVATOR INHIBITORS IN NORMAL PREGNANCY AND PATIENTS WITH PREECLAMPSIA
Authors: Estellés A , Gilabert J , Aznar J , España F , Fernández J A 
Published: August 2018

Plasminogen activator inhibitor (PAI) activity and other fibrinolytic parameters have been studied in three groups of preeclamptic patients: A) severe preeclampsia, B) mild preeclampsia and C) chronic hypertension with superimposed severe preeclanpsia. These parameters were compared to those observed in a group of normal pregnant women at similar gestation age ( 3rd trimester of pregnancy, group D). The PAI activity, regardless of whether single-chain t-PA or two-chain t-PA was used, was significantly higher in cases of severe preeclanpsia than in cases of mild preeclanpsia and normal pregnancyThis increase in PAI activity normalizes in early puerperium. When PAI activity was evaluated in the platelet lysate no increase in severe preeclanpsia, as compared to the control group, was observed. Following the addition of purified two chain t-PA to plasma of severe preeclanptic patients, a complex with a molecular weight of about 115,000 was detected by a zymographic fibrin technique. When urokinase (90% of HMW and 10% of LMW) was used, two complexes of about 95,000 and 85,000 were detected. The increased level of PAI activity found in severe preeclanpsia was partially inhibited by the antisera against PAI-1 and PAI-2.
PLASMINOGEN ACTIVATOR INHIBITOR OF PLACENTAL TYPE IN PREECLAMPSIA
Authors: de Boer K , Lecander I , ten Cate J W , Borm J J J , Treffers P E 
Published: August 2018

In 24 patients with preeclampsia and in 24 normal pregnant controls, matched for gestational age, fibrinolytic parameters were determined. Aim of the study was to investigate plasminogen activator inhibitory activity (PAI activity) levels and plasminogen activator inhibitor of placental type (PAI 2) in preeclamptic and normal pregnancy and to establish the clinical relevance of these assays. PAI activity was measured by titrating the plasma with single chain t-PA. PAI 2 was measured by means of an ELISA using monoclonal and polyclonal antibodies against the placental inhibitor. Therefore we related testresubts to a predefined relevant clinical endpoint i.e. fetal outcome. The following results were obtained:PAI 2 levels below 43% had a positive predictive value of 91% for poor fetal outcome, levels above 43% had a negative predictive value of 95%. PAI activity levels didn't have a significant predictive value.. It is concluded that high levels of PAI activity in preeclampsia are not due to PAI 2 and that low levels of PAI 2 are related with a poor fetal outcome.