Effect of plasmin, plasminogen activators and a plasmin inhibitor on bovine in vitro embryo production

2008 ◽  
Vol 20 (2) ◽  
pp. 320 ◽  
Author(s):  
Thomas Papanikolaou ◽  
Georgios S. Amiridis ◽  
Ioannis Dimitriadis ◽  
Emmanuel Vainas ◽  
Constantinos A. Rekkas
Keyword(s):  
Plasminogen Activator ◽  
Embryo Development ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Embryo Production ◽  
Development Rates ◽  
Type Plasminogen Activator ◽  
In Vitro Embryo Production ◽  
Plasmin Inhibitor

In the present study, four experiments were conducted to investigate the possible effects of plasminogen activators (urokinase-type plasminogen activator (u-PA) and tissue-type plasminogen activator (t-PA)), plasmin, and a plasmin inhibitor (epsilon-aminocaproic acid (ϵ-ACA)) on different stages of bovine in vitro embryo production (IVP). The concentrations of these modifiers in IVP media were conditioned according to the plasminogen activator activity of bovine preovulatory follicular fluid. Media were modified in a single phase of IVP with an 18 h or 24 h incubation for in vitro maturation (IVM) and a 24 h or 48 h incubation for the IVF or in vitro culture (IVC), respectively. After IVM the oocytes were either fixed and stained or underwent IVF and IVC. The main findings were: (1) plasmin added to the 18 h IVM medium increased maturation rate without affecting fertilisation or embryo development rates; (2) t-PA added to the IVF medium significantly increased cleavage; (3) u-PA added to the IVC medium significantly increased embryo development rates; (4) the efficiency of all phases of IVP was reduced after the addition of ϵ-ACA; and (5) plasminogen addition had no effect in any IVP phase tested. We conclude that the members of the plasminogen activator–plasmin system contribute in different ways to bovine IVM, IVF and IVC.

Assay of Functional Plasminogen in Rat Plasma Applicable to Experimental Studies of Thrombolysis

2000 ◽  
Vol 84 (08) ◽  
pp. 299-306 ◽  
Author(s):  
Kristian Bangert ◽  
Sixtus Thorsen
Keyword(s):  
Plasminogen Activator ◽  
Experimental Studies ◽  
Fold Increase ◽  
Rat Plasma ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryAn improved sensitive, specific, precise and accurate assay of plasminogen in rat plasma was developed. It is performed in 96-well microtiter plates and can be completed within one hour. The assay is based on activation of plasminogen by human urokinase-type plasminogen activator (uPA) and simultaneous measurement of generated plasmin with the specific plasmin substrate H-D-Val-Phe-Lys-4-nitroanilide (S-2390), using purified native rat plasminogen for calibration. The concentration of S-2390 in the final reaction mixture during the whole reaction period is much greater than the K m value (≈20 µM) for rat plasmin-cleavage of S-2390 ensuring that hydrolysis of substrate follows zero order kinetics and that the substrate produces a 20-35 fold decrease in rate of inhibition of plasmin by its target inhibitors in plasma. Analogous to the human system the target plasma inhibitors of rat plasmin are shown to be plasmin inhibitor and α-macroglobulins. Tranexamic acid (0.8 mM) is incorporated in the reaction mixture resulting in a 19-fold increase in the rate of plasminogen activation and presumably an about 50-fold decrease in the rate of inhibition of generated plasmin by plasmin inhibitor. The assay is suitable for accurate measurement of plasminogen in samples obtained from animals containing pharmacological concentrations of uPA or tissue-type plasminogen activator (tPA) in their plasma when in vitro plasminogen activation is blocked at pH 5 by collecting blood in acidic anticoagulant. Judged from in vitro experiments formation of catalytic active plasmin-α-macroglobulin complexes during massive activation of plasminogen in vivo does not interfere with the assay.

Monitoring of Hemostasis Parameters During Coronary Thrombolysis with Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 59 (02) ◽  
pp. 133-137 ◽  
Author(s):  
D C Stump ◽  
E J Topol ◽  
A B Chen ◽  
A Hopkins ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Fibrinogen Level ◽  
Tissue Type ◽  
Poor Correlation ◽  
Blood Collection ◽  
Tissue Type Plasminogen Activator ◽  
Optimal Monitoring ◽  
Type Plasminogen Activator ◽  
Plasmin Inhibitor

SummaryThe monitoring of changes in the blood coagulation and fibrinolytic systems during thrombolytic therapy with recombinant tissue-type plasminogen activator (rt-PA) may be complicated by artifacts due to in vitro activation after blood collection and to interference of other agents (e. g., heparin) in the assays. In 106 patients with early acute myocardial infarction, infused with 150 mg of rt-PA (G11044) intravenously over 5 to 8 hours, blood samples were collected into liquid citrate supplemented with the plasmin inhibitor aprotinin (200 KlU/ml plasma) or on a lyophilized mixture of acidified citrate and the synthetic t-PA inhibitor D-Phe-Pro-Arg-CH2Cl (PPACK). A good correlation between precipitable (sulphite) and functional (clotting rate) fibrinogen levels was observed in plasma collected on citrate before therapy (r = 0.76) and in samples collected after 3 hours on either aprotinin (r = 0.87) or PPACK (r = 0.82). Precipitable fibrinogen levels were approximately 10% higher than functional level, in baseline samples collected on citrate alone and approximately 20% higher in 3 hour samples collected on either PPACK or aprotinin. Fibrinogen levels measured with both assays correlated well, but were somewhat higher in samples collected on PPACK than on aprotinin. rt-PA antigen levels assayed in plasma collected in either inhibitor correlated well (r = 0.90) but were 10-20% higher in PPACK containing samples. Addition of heparin up to 9 units/ml to plasma had no effect on the functional fibrinogen assay.Even with these precautions for assay artifact, a very poor correlation (r =-0.15) was observed between the plasma rt-PA level and the residual functional fibrinogen level, both after 3 hours and towards the end of the rt-PA infusion. A decrease of the fibrinogen level at the end of the infusion to below 1 g/l was observed in 36% of the patients and to below 0.5 g/l in 11%. Optimal monitoring of hemostasis during rt-PA infusion is achieved by fibrinogen assays with a clotting rate method on samples collected on either PPACK or aprotinin. Heparin at therapeutic levels does not interfere with this assay.

scholarly journals Urokinase-type plasminogen activator does not affect in vitro bovine embryo development and quality

2015 ◽  
Vol 63 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Fotini Krania ◽  
Eleni Dovolou ◽  
Constantinos A. Rekkas ◽  
Sonia Heras ◽  
Ioannis Pappas ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Embryo Development ◽  
Culture Media ◽  
Cell Monolayer ◽  
Cumulus Cell ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The effects of modification of the in vitro embryo culture media (IVC) with the addition of urokinase-type plasminogen activator (u-PA) on the yield and/or quality of bovine embryos were examined in two experiments. In Experiment 1, denuded embryos were cultured in semi-defined synthetic oviductal fluid (SOF) for seven days, while in Experiment 2 embryos were co-cultured with cumulus cell monolayer in a serum-containing SOF medium. Plasminogen activator activity (PAA) and plasminogen activator inhibition (PAI) were determined in all spent IVC media. At the activity used (5 IU/ml), u-PA had no effect either on in vitro embryo production rates or on embryo quality as revealed by gene expression analysis of 10 important mRNA transcripts related to apoptosis, oxidation, implantation and metabolism. PAA and PAI analysis indicated the need for wellbalanced plasminogen activators and inhibitors as a culture environment for embryo development. However, more research is needed to unveil the mechanism by which u-PA is involved in in vitro embryo production systems.

Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 247-250 ◽  
Author(s):  
H R Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rate Constants ◽  
Plasminogen Activators ◽  
Second Order ◽  
Tissue Type ◽  
Single Chain ◽  
Chain Molecules ◽  
Order Rate ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

Pharmacokinetics and Haemostatic Status During Consecutive Infusions of Recom binant Tissue-Type Plasminogen Activator in Patients with Acute Myocardial Infarction

1989 ◽  
Vol 61 (03) ◽  
pp. 497-501 ◽  
Author(s):  
E Seifried ◽  
P Tanswell ◽  
D Ellbrück ◽  
W Haerer ◽  
A Schmidt
Keyword(s):  
Myocardial Infarction ◽  
Acute Myocardial Infarction ◽  
Plasminogen Activator ◽  
Peak Plasma ◽  
Peak Plasma Concentration ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Precise Control ◽  
Type Plasminogen Activator

SummaryPharmacokinetics and systemic effects of recombinant tissue type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 12 acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in 2 minutes resulting in a peak plasma concentration (mean ±SD) of 3310±950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of. 2210±470 nglml and 930±200 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380±74 ml/min, t,½α was 3.6±0.9 min and t,½β was 16±5.4 min.After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 52%, α2-antiplasmin to 25%, α2-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin D-dimer concentrations increased from 0.40 to 2.7 μg/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

1993 ◽  
Vol 70 (05) ◽  
pp. 867-872 ◽  
Author(s):  
Dingeman C Rijken ◽  
Gerard A W de Munk ◽  
Annie F H Jie
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Single Chain ◽  
Physiological Conditions ◽  
Amino Terminal ◽  
Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

1993 ◽  
Vol 69 (05) ◽  
pp. 466-472 ◽  
Author(s):  
M Colucci ◽  
L G Cavallo ◽  
G Agnelli ◽  
A Mele ◽  
R Bürgi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Cleavage Site ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood

1983 ◽  
Vol 50 (02) ◽  
pp. 518-523 ◽  
Author(s):  
C Kluft ◽  
A F H Jie ◽  
R A Allen
Keyword(s):  
Plasminogen Activator ◽  
Venous Occlusion ◽  
Tissue Type ◽  
Functional Assay ◽  
Uterine Tissue ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Baseline Values

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

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