Studies on Circulating Plasminogen Activator

1975 ◽  
Author(s):  
D. Ogston ◽  
B. Bennett ◽  
M. Mackie
Keyword(s):  
Plasminogen Activator ◽  
Phosphate Buffer ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Chloromethyl Ketone ◽  
Α2 Macroglobulin ◽  
Sulphonyl Fluoride

A plasminogen activator was prepared in crude form from postvenous occlusion plasma by gel filtration of the plasma on Sephadex G-200 in 0.005 M phosphate buffer (pH 7.5) containing 0.15 M NaCl followed by further gel filtration of the fractions with maximal activator activity on Sephadex G-200 in 0.005 M phosphate buffer containing 1 M NaCl. The preparations were free of plasminogen, α1-antitrypsin, α2-macroglobulin, CI inactivator and antithrombin III as assessed by immunological techniques. The activity of the partially purified plasma activator preparations was markedly more stable at 37° C than the original plasma, was stable at 4° C for several days, and withstood heating at 56° C for 1 hour. The activity was inhibited by exposure to phenylmethyl sulphonyl-fluoride, but not by tosyl-L-lysine chloromethyl ketone or iodoacetamide.

Neutralization of Human Extrinsic (Tissue-Type) Plasminogen Activator in Human Plasma: No Evidence for a Specific Inhibitor

1981 ◽  
Vol 46 (03) ◽  
pp. 662-665 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Rate Constant ◽  
Active Site ◽  
Gel Filtration ◽  
Specific Inhibitor ◽  
Culture Fluid ◽  
Void Volume ◽  
Tissue Type ◽  
Α2 Macroglobulin

SummaryHuman extrinsic plasminogen activator (EPA), highly purified from a melanoma cell culture fluid is inactivated in human plasma with a half-life (t ½) of 90–105 min. Gel filtration on Ultrogel AcA 34 of mixtures of 125I-labeled EPA and human plasma, incubated at 37°C, revealed the progressive formation of two radioactive components, one with an apparent Mr of 150,000 and one eluting at the void volume. The component with an Mr of 150,000 was identified as consisting at least in part of EPA-α2-antiplasmin complex since: 1) it reacted with antibodies against α2-antiplasmin, but not with antibodies against the other known plasma protease inhibitors, and 2) formation of this component was strongly reduced in plasma specifically depleted in α2-antiplasmin or when the active site of EPA was blocked. The component eluting at the void volume was identified as consisting at least in part of EPA-α2-macroglobulin complex since: 1) it only reacted with antibodies against these two proteins and 2) was not formed in plasma depleted in α2-macroglobulin or when the active site of EPA was blocked.In purified systems α2-antiplasmin inhibited one-chain EPA with a rate constant of 60 M-1s-1 and two-chain EPA with a rate constant of 130 M-1s-1, which corresponds to a t ½ in plasma of 180 min or 90 min, respectively. α2-Macroglobulin inhibited one-chain EPA with a rate constant of 15 M-1s-1 and two-chain EPA with a rate constant of 30 M-1s-1, which corresponds to a t ½ plasma of 4 or 2 hrs.All these findings taken together indicate that EPA is slowly neutralized in human plasma primarily by α2-antiplasmin and to a lesser extent by α2-macroglobulin. There appears to be no specific inhibitor in human plasma, which would inactivate EPA either rapidly or to a significant extent.

scholarly journals SUBSTRATES OF HAGEMAN FACTOR

1974 ◽  
Vol 140 (6) ◽  
pp. 1615-1630 ◽  
Author(s):  
Louis W. Heck ◽  
Allen P. Kaplan
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Lima Bean ◽  
Hageman Factor ◽  
Α2 Macroglobulin ◽  
Coagulant Activity ◽  
Bean Trypsin Inhibitor

Unactivated partial thromboplastin antecedent (PTA) has been purified by sequential chromatography of plasma on quaternary aminoethyl Sephadex, sulphoprophyl Sephadex, Sephadex G-150, and passage over an anti-IgG immunoadsorbant. The preparation gave a single band after alkaline disc gel electrophoresis, sodium dodecyl sulfate (SDS) gel electrophoresis and isoelectric focusing in acrylamide gels and was found to have a mol wt of 175,000 by gel filtration, 163,000 by SDS gel electrophoresis, and an isoelectric point of 8.8–9.4 (peak 9.0–9.1). Pre-PTA was activated directly by activated Hageman factor or by Hageman factor prealbumin fragments. Its coagulant activity was inhibited by DFP, soybean trypsin inhibitor and trasylol but not by lima bean trypsin inhibitor or ovomucoid trypsin inhibitor indicating that activated PTA possesses the same inhibition profile utilizing these reagents as does plasma kallikrein. A major plasma inhibitor of activated PTA was found to be a 65,000 mol wt α-globulin which was isolated free of α1-chymotrypsin inhibitor, inter α-trypsin inhibitor, α2-macroglobulin, and the other known inhibitors of activated PTA, the activated first component of complement (C1 INH), and antithrombin III. Its physicochemical properties were identical to α1-antitrypsin, and it was absent in α1-antitrypsin-deficient plasma thereby identifying this PTA inhibitor as α1-antitrypsin.

Inhibition Of Human Tissue Plasminogen Activator By Human Plasma : No Evidence For A Specific Antiactivator

1981 ◽  
Author(s):  
C Korninger ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Rate Constant ◽  
Human Tissue ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Α2 Macroglobulin ◽  
Tissue Plasminogen ◽  
The One

Human tissue plasminogen activator (TPA) was highly purified (one-chain or two-chain form) from the culture fluid of a melanoma cell line and labeled with 125 Gel filtration of mixtures of human plasma with trace amounts of labeled and 10 to 1,000 units/ml of unlabeled TPA revealed formation of two radioactive complexes with apparent Mr of 150,000 and 800,000, which were devoid of lytic activity of fibrin plates. These radioactive complexes were precipitated (90 and 63 percent) by antisera against α2 antiplasmin (α2AP) and α2-macroglobulin (α2M) respectively, but not by antisera against other known plasma protease inhibitors. In plasma specifically depleted in α2AP formation of the complex with Mr ≃ 150,000 did not occur, whereas removal of α2M abolished formation of the complex with Mr ≃ 800,000. The initial rates of formation of these two complexes in plasma were very similar to those obtained with mixtures of TPA and 1 μM α2AP or 3.5 μM α2M respectively. Complex formation was completely abolished by blocking the active site serine of TPA.In purified systems TPA was inhibited by α2AP with a rate constant of 140 M-1s-1 and by α2M with a rate constant of 30 M-1s-1. These rate constants correlate well with the rate of formation in plasma of the Mr ≃ 150,000 complex and the Mr ≃ 800,000 complex respectively.All these data indicate that TPA is slowly inhibited in human plasma by α2AP (tl/2 ≃ 60') and by α2M (tl/2 ≃ 120'). We found no evidence for the existence of another significant inhibitor in plasma. Both the one-chain and two-chain forms of TPA behaved very similarly in all experiments.

Separation of Plasminogen Activators from Human Uterine Tissue and a Comparison with Activators from Human Urine and Porcine Tissue

1979 ◽  
Vol 41 (04) ◽  
pp. 718-733 ◽  
Author(s):  
Preben Kok
Keyword(s):  
Plasminogen Activator ◽  
Human Urine ◽  
Gel Filtration ◽  
Ammonium Thiocyanate ◽  
Plasminogen Activators ◽  
Particle Sizes ◽  
Uterine Tissue ◽  
Porcine Tissue ◽  
Major Activity ◽  
Proliferative Phase

SummaryThree types of plasminogen activator could be distinguished in extracts from human uterine tissue. The activators differed in thermostability or in mode of inhibition by EACA.All the extracts contained stable as well as labile activators. The saline extracts were uniformly inhibited by increasing concentrations of EACA. Extracts made with 2 M ammonium thiocyanate were either uniformly inhibited by EACA or showed deflections indicating contamination with an activator, which was inhibited in a biphasic manner. It was possible to distinguish between: (1) An activator, abundantly present in the tissue, which was uniformly inhibited and stable. (2) Another uniformly inhibited activator, which was labile. (3) An activator, inhibited in a biphasic manner, similar to urokinase, which was present in varying amounts in uteri with the endometrium in the proliferative phase.Gel filtration of the uterine extracts showed two major activity peaks corresponding to particle sizes of 60,000 dalton and about 10,000 dalton.Antiserum to purified plasminogen activator, prepared from porcine ovaries, inhibited the activity of the human uterine extracts, but not the activities of human urokinase or urine. Urokinase antiserum in a concentration completely inhibiting human urine or urokinase, inhibited only 10% or less of the activities of human uterine extracts.

Immunoreactivity of Tissue Plasminogen Activator and of Its Inhibitor Complexes

1989 ◽  
Vol 61 (03) ◽  
pp. 409-414 ◽  
Author(s):  
M Rånby ◽  
G Nguyen ◽  
P Y Scarabin ◽  
M Samama
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Degradation Products ◽  
Gel Filtration ◽  
Free Form ◽  
Enzyme Linked Immunosorbent Assay ◽  
Plasma Samples ◽  
Significant Difference ◽  
Tissue Plasminogen ◽  
Pai 1

SummaryAn enzyme linked immunosorbent assay (ELISA) based on goat polyclonal antibodies against human tissue plasminogen activator (tPA) was evaluated. The relative immunoreactivity of tPA in free form and tPA in complex with inhibitors was estimated by ELISA and found to be 100, 74, 94, 92 and 8l% for free tPA and tPA in complex with PAI-1, PAI-2, α2-antiplasmin and C1-inhibitor, respectively. Addition of tPA to PAI-1 rich plasma resulted in rapid and total loss of tPA activity without detectable loss of ELISA response, indicating an immunoreactivity of tPA in tPA/PAI-1 complex of about l00%. Three different treatments of citrated plasma samples (acidification/reneutralization, addition of 5 mM EDTA or of 0.5 M lysine) prior to determination by ELISA all resulted in increased tPA levels. The fact that the increase was equally large in all three cases along with good analytical recovery of tPA added to plasffi, supported the notion that all tPA antigen present in plasma samples is measured by the ELISA. Analysis by ELISA of fractions obtained by gel filtration of plasma from a patient undergoing tPA treatment identified tPA/inhibitor complexes and free tPA but no low molecular weight degradation products of tPA. Determinations of tPA antigen were made at seven French clinical laboratories on coded and randomized plasma samples with known tPA antigen content. For undiluted samples there was no significant difference between the tPA levels found and those known to be present. The between-assay coefficient of variation was 7 to 10%. In conclusion, the ELISA appeared suited for determination of total tPA antigen in human plasma samples.

Isolation and Characterization of Plasminogen Activator from Pig Leucocytes

1974 ◽  
Vol 31 (01) ◽  
pp. 072-085 ◽  
Author(s):  
M Kopitar ◽  
M Stegnar ◽  
B Accetto ◽  
D Lebez
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Ph Optimum ◽  
Isolation And Characterization ◽  
Ph Range ◽  
Inhibition Studies ◽  
Final Purification

SummaryPlasminogen activator was isolated from disrupted pig leucocytes by the aid of DEAE chromatography, gel filtration on Sephadex G-100 and final purification on CM cellulose, or by preparative gel electrophoresis.Isolated plasminogen activator corresponds No. 3 band of the starting sample of leucocyte cells (that is composed from 10 gel electrophoretic bands).pH optimum was found to be in pH range 8.0–8.5 and the highest pH stability is between pH range 5.0–8.0.Inhibition studies of isolated plasminogen activator were performed with EACA, AMCHA, PAMBA and Trasylol, using Anson and Astrup method. By Astrup method 100% inhibition was found with EACA and Trasylol and 30% with AMCHA. PAMBA gave 60% inhibition already at concentration 10–3 M/ml. Molecular weight of plasminogen activator was determined by gel filtration on Sephadex G-100. The value obtained from 4 different samples was found to be 28000–30500.

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
Author(s):  
Ulla Hedner
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Specific Adsorption ◽  
Partial Purification ◽  
Plasminogen Activation ◽  
Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

Antithrombin Activity of Intact Human Platelets

1975 ◽  
Vol 34 (01) ◽  
pp. 115-126 ◽  
Author(s):  
Kiyoake Watanabe ◽  
Francis C Chao ◽  
James L Tullis
Keyword(s):  
Antithrombin Iii ◽  
Human Platelets ◽  
Significant Loss ◽  
Red Cells ◽  
Antithrombin Activity ◽  
Α2 Macroglobulin ◽  
Rapid Reaction ◽  
Pre Treatment ◽  
Different Characteristics

SummaryAntithrombin activity has been identified in intact washed human platelets. An apparent activity was demonstrated at platelet concentrations above 0.31 × 109/ml, when platelet suspensions were incubated with 2.0 NIH units/ml of thrombin. Neither red cells nor white cells revealed antithrombin activity. No significant loss of the platelet antithrombin activity was observed after ten successive washings or after treatment of platelets with antibodies to antithrombin III or α2-macroglobulin. Almost the same amount of antithrombin activity as normal platelets was demonstrated in the platelets from an afibrinogenemic patient. Pre-treatment of platelets with trypsin, papain, and neuroaminidase reduced the activity significantly, whereas lipase was without effect. The platelet antithrombin reacted with thrombin in less than 3 seconds, and this rapid reaction of platelet antithrombin was different from that of plasma antithrombin III or fibrinogen. The thrombin-like clotting activity of ancrod was inhibited by fibrinogen but not platelets. Also, unlike plasma antithrombin III or fibrinogen, brief exposure to heat (56° C or 60° C) reduced considerable amounts of platelet antithrombin activity. These results suggest that platelets possess a specific antithrombin with different characteristics from other known antithrombins.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

Soluble Fibrin Complexes and Fibrinogen Heterogeneity in Diabetes mellitus

1980 ◽  
Vol 44 (03) ◽  
pp. 130-134 ◽  
Author(s):  
E B Tsianos ◽  
N E Stathakis
Keyword(s):  
Diabetes Mellitus ◽  
Molecular Weight ◽  
Plasma Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Polyacrylamide Gels ◽  
Soluble Fibrin ◽  
Α2 Macroglobulin ◽  
Patients With Diabetes

SummaryThe presence of soluble fibrin complexes (SFC) measured by gel filtration of plasma on 4% agarose columns, fibrinogen heterogeneity on 3.5% SDS-polyacrylamide gels and the concentrations of several plasma proteins were evaluated in 39 patients with diabetes mellitus (DM) and 19 matched control subjects. A small but significant increase of SFC was found in DM (p<0.01). On individual basis 51.2% of the patients had increased SFC (>M + 2 SD of the controls). Polyacrylamide gel electrophoresis of the SFC showed no evidence of cross-linking or proteolysis. Plasma clots formed in the presence of EDTA and trasylol were analysed in SDS-polyacrylamide gels in a normal and two lower molecular weight fibrin bands (band I, II, III). The percentage of band I fibrinogen was in diabetics (65.3 ± 4.7%) lower than that of the controls (71.8 ± 4.5%) (p < 0.01). Fibrinogen levels, antithrombin III, α1-antitrypsin, α2-macroglobulin and plasminogen were significantly increased in DM. We suggest that in DM there is an enhancement of intravascular fibrin formation and accelerated fibrinogen degradation to lower molecular weight forms.

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