In Vitro Studies on the Fibrinolytic, Thrombolytic and Fibrinogenolytic Properties of a Tissue Plasminogen Activator from Guinea Pig Keratocytes

1985 ◽  
Vol 53 (02) ◽  
pp. 200-203 ◽  
Author(s):  
A Electricwala ◽  
R J Ling ◽  
P M Sutton ◽  
B Griffiths ◽  
P A Riley ◽  
...  
Keyword(s):  
Guinea Pig ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Thrombolytic Activity ◽  
Tissue Plasminogen ◽  
In Vitro Systems

SummaryThe fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37° C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (<50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3–6 hr with the concentration of GPK activator in the range of 1–5 μg/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.

scholarly journals Preparation of polyethylene glycol-tissue plasminogen activator adducts that retain functional activity: characteristics and behavior in three animal species

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1641-1647 ◽  
Author(s):  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Polyethylene Glycol ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Functional Activity ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
Thrombolytic Activity ◽  
Wide Range ◽  
Tissue Plasminogen

Abstract Conditions were defined for the derivatization of recombinant tissue plasminogen activator (rt-PA) with polyethylene glycol (PEG) so as to retain functional activity as a possible means of producing a t-PA species with a prolonged circulating lifetime. Derivatives with a wide range of retention of activities were prepared by varying the concentration and species of activated PEG. The specific activities of the PEG-rt-PA derivatives were dependent on the method of assay. Assays using preformed fibrin gave higher estimates of retention of activity than assays using soluble components. Plasma elimination studies in mice and rats indicated prolonged circulating lifetimes for the radiolabeled PEG-rt-PA derivatives after a rapid clearance and distribution phase; however, the disappearance of functional activity was much more rapid than the disappearance of radiolabeled material. The PEG-rt-PA derivatives appeared to accumulate in tissues above their interstitial fluid concentrations and were rapidly inactivated, apparently by reaction with the plasma protease inhibitors. These results were consistent with the inactivation of the PEG-rt-PA derivatives in rat plasma in vitro. A somewhat longer half-life (t1/2) of the one derivative studied was observed in dogs (t1/2, 16 minutes) as compared with the rat (t1/2, five minutes). This was sufficient to confer thrombolytic activity upon the derivative (administered by bolus injection) in contrast to native rt-PA. The potential of PEG-modified rt-PA as a long-lived thrombolytic agent in humans will depend, however, on whether there will be a further extension of the t1/2 because of a reduction in clearance and/or a reduction in the rate of inactivation.

scholarly journals Preparation of Peptide and Recombinant Tissue Plasminogen Activator Conjugated Poly(Lactic-Co-Glycolic Acid) (PLGA) Magnetic Nanoparticles for Dual Targeted Thrombolytic Therapy

2020 ◽  
Vol 21 (8) ◽  
pp. 2690 ◽  
Author(s):  
Huai-An Chen ◽  
Yunn-Hwa Ma ◽  
Tzu-Yuan Hsu ◽  
Jyh-Ping Chen
Keyword(s):  
Magnetic Nanoparticles ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Glycolic Acid ◽  
Blood Clot ◽  
Recombinant Tissue Plasminogen Activator ◽  
Clot Lysis ◽  
High Dose ◽  
Tissue Plasminogen

Recombinant tissue plasminogen activator (rtPA) is the only thrombolytic agent that has been approved by the FDA for treatment of ischemic stroke. However, a high dose intravenous infusion is required to maintain effective drug concentration, owing to the short half-life of the thrombolytic drug, whereas a momentous limitation is the risk of bleeding. We envision a dual targeted strategy for rtPA delivery will be feasible to minimize the required dose of rtPA for treatment. For this purpose, rtPA and fibrin-avid peptide were co-immobilized to poly(lactic-co-glycolic acid) (PLGA) magnetic nanoparticles (PMNP) to prepare peptide/rtPA conjugated PMNPs (pPMNP-rtPA). During preparation, PMNP was first surface modified with avidin, which could interact with biotin. This is followed by binding PMNP-avidin with biotin-PEG-rtPA (or biotin-PEG-peptide), which was prepared beforehand by binding rtPA (or peptide) to biotin-PEG-maleimide while using click chemistry between maleimide and the single –SH group in rtPA (or peptide). The physicochemical property characterization indicated the successful preparation of the magnetic nanoparticles with full retention of rtPA fibrinolysis activity, while biological response studies underlined the high biocompatibility of all magnetic nanoparticles from cytotoxicity and hemolysis assays in vitro. The magnetic guidance and fibrin binding effects were also confirmed, which led to a higher thrombolysis rate in vitro using PMNP-rtPA or pPMNP-rtPA when compared to free rtPA after static or dynamic incubation with blood clots. Using pressure-dependent clot lysis model in a flow system, dual targeted pPMNP-rtPA could reduce the clot lysis time for reperfusion by 40% when compared to free rtPA at the same drug dosage. From in vivo targeted thrombolysis in a rat embolic model, pPMNP-rtPA was used at 20% of free rtPA dosage to restore the iliac blood flow in vascular thrombus that was created by injecting a blood clot to the hind limb area.

The effect of 40 kHz ultrasound on tissue plasminogen activator-induced clot lysis in three in vitro models

2004 ◽  
Vol 30 (11) ◽  
pp. 1545-1552 ◽  
Author(s):  
Marlien Pieters ◽  
Rob T. Hekkenberg ◽  
Marrie Barrett-Bergshoeff ◽  
Dingeman C. Rijken
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
In Vitro Models ◽  
Clot Lysis ◽  
Tissue Plasminogen

scholarly journals Physico-Chemical and Biological Properties of Biosimilar and Reference Tissue Plasminogen Activator Products

2019 ◽  
Vol 19 (1) ◽  
pp. 39-49
Author(s):  
V. D. Gusarova ◽  
M. S. Pantyushenko ◽  
V. M. Simonov ◽  
R. R. Shukurov ◽  
R. A. Khamitov ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Specific Activity ◽  
Reference Product ◽  
Fibrin Clot ◽  
Biological Properties ◽  
Clot Lysis ◽  
Physico Chemical ◽  
Tissue Plasminogen

Recombinant tissue plasminogen activator (international nonproprietary name — alteplase) which was developed by «GENERIUM» (Russia) and received a marketing authorisation in Russia is completely analogous to Actilyse® which is used to treat medical conditions accompanied by thrombosis, such as acute myocardial infarction, pulmonary embolism, and ischemic stroke. The aim of the study was to carry out a comprehensive comparison of physico-chemical and biological properties of Revelyse® and the reference product Actilyse® in order to assess their biosimilarity. Materials and Methods: comparative peptide mapping and determination of comparability of chromatographic profiles of tryptic hydrolysates was performed using RP-HPLC and massspectrometry; the molecular weight distribution was determined by mass-spectrometry and polyacrylamide gel electrophoresis (Laemmli method). The purity and homogeneity of products as well as the content of related impurities (oligomers and fragments) were determined using gel filtration; N-glycosylation profile was analysed by hydrophilic HPLC, total sialic acid was quantified by the Svennerholm resorcinol method. Protein binding to fibrin and human fibrinogen was assessed by surface plasmon resonance, and the specific activity was compared by fibrin clot lysis. Results: the research demonstrated a complete overlap of the products’ peptide maps, which indicates the identity of аlteplase amino acid sequences in the two medicines being compared. The authors of the study also determined the molecular weight and the content of the intact single-stranded form of the protein, and quantified post-translational modifications, the content of sialic acids and neutral sugars. The analysis of the N-glycosylation profile revealed insignificant differences in the percentage of multiantenna complex glycans. The specificity of alteplase was evaluated by analysing the formation of protein complexes with natural alteplase ligands – fibrin and plasminogen activator inhibitor-1, but no significant differences were found. The comparison of specific activation of plasminogen fibrinolytic activity was performed based on the results of the assay analysing the fibrin clot lysis rate, and it demonstrated comparability of Revelyse® and Actilyse®. Conclusions: comparative experimental studies have shown no differences in the structure, charge distribution heterogeneity, impurities content, and specific activity of alteplase as a component of Revelyse® and the reference product Actilyse®, which leads to the conclusion that they are similar in terms of physicochemical and biological properties.

Comparison of the Relative Fibrinogenolytic, Fibrinolytic and Thrombolytic Properties of Tissue Plasminogen Activator and Urokinase in Vitro

1981 ◽  
Vol 45 (03) ◽  
pp. 225-229 ◽  
Author(s):  
O Matsuo ◽  
D C Rijken ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Human Tissue ◽  
Thrombolytic Agent ◽  
Blood Clot ◽  
Culture Fluid ◽  
Fibrinolytic System ◽  
Tissue Plasminogen

SummaryThe relative fibrinogenolytic, fibrinolytic and thrombolytic properties of human tissue plasminogen activator and human urokinase were compared in purified systems, in whole human plasma and in a system composed of a radioactive human blood clot (<sup>125</sup>I-fibrinogen) hanging in circulating human plasma. The human tissue plasminogen activator was highly purified from the culture fluid of a human melanoma cell line.In purified systems composed of fibrinogen or fibrin, plasminogen and α<sub>2</sub>-antiplasmin as well as in whole plasma, tissue plasminogen activator digested fibrin without degrading fibrinogen significantly. Urokinase did not have this specific fibrinolytic effect.In the circulating plasma system, the degree of fibrinolysis was proportional to the amount of activator added, tissue plasminogen activator being about 10 times more efficient than urokinase. In addition, tissue plasminogen activator appeared to cause negligible fibrinogen degradation. Tissue plasminogen activator still induced significant thrombolysis at a concentration of 10 IU per ml whereas no effect of urokinase was observed at 20 IU per ml. Infusion of 100 IU (1 (μg) of tissue plasminogen activator per ml resulted in moderate activation of the fibrinolytic system as judged from a decrease of plasminogen and α<sub>2</sub>-antiplasmin to 40-50 percent. Nevertheless, extensive fibrinolysis (50 to 80 percent of radioactivity released after 12 hrs) and only very limited fibrinogenolysis were observed. An equivalent amount of urokinase (100 IU per ml) only induced approximately 15 percent lysis in 12 hrs. At higher concentrations of urokinase (260 IU per ml or more) extensive activation of the fibrinolytic system was obtained as evidenced by a depletion of plasminogen, α<sub>2</sub>-antiplasmin and fibrinogen. This was associated with extensive fibrinolysis (approximately 60 percent after 12 hrs). It is concluded that human tissue plasminogen activator is a more specific and effective fibrinolytic-thrombolytic agent than human urokinase.

Substrate Composition and the Effect of ε-Aminocaproic Acid on Tissue Plasminogen Activator and Urokinase-induced Fibrinolysis

1974 ◽  
Vol 32 (02/03) ◽  
pp. 306-324 ◽  
Author(s):  
Sixtus Thorsen ◽  
Tage Astrup
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Aminocaproic Acid ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Substrate Composition ◽  
Lysis Time ◽  
Biphasic Effect ◽  
Tissue Plasminogen ◽  
Inhibition Curve

SummaryThe influence of variations in substrate composition on the biphasic pattern of inhibition by e-aminocaproic acid (EACA) of urokinase-induced fibrinolysis, first observed on bovine plasminogen-rich fibrin, was studied using a fibrin clot lysis time assay. Different species of fibrinogen and plasminogen or plasmin were used. Preparations of different degrees of purification were compared at varying concentrations. The behavior of urokinase was compared with that of a porcine tissue plasminogen activator. Variations in the conditions of the assay greatly influenced the results. The concentration of fibrinogen had a particularly marked influence. At increased fibrinogen concentrations the biphasic response of urokinase was enhanced and a weak biphasic effect was also produced by the tissue activator which usually yields a uniformly increasing inhibition curve. The phase of fibrinolysis enhancement produced by urokinase with genuine plasminogen substrates occurs at substrate concentrations and EACA levels present in patients treated with EACA. The observed variations may explain discordant findings reported by various investigators.

COVALENT Mr ∼92,000 HYBRID PLASMINOGEN ACTIVATOR DERIVED FROM THE PLASMIN FIBRIN-BINDING DOMAIN AND THE TISSUE PLASMINOGEN ACTIVATOR CATALYTIC DOMAIN

1987 ◽  
Author(s):  
K C Robbins ◽  
I Boreisha
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Catalytic Domain ◽  
Specific Activity ◽  
Fibrin Clot ◽  
Binding Domain ◽  
Molar Ratio ◽  
Clot Lysis ◽  
Chromatography Method ◽  
Tissue Plasminogen

A covalent hybrid plasminogen activator was prepared from the sulfhydryl forms of the NH2-terminal A chain of human plasmin (Pln^) containing the fibrin-binding domain, and the COOH-terminal B chain of tissue plasminogen activator (t-PAB) containing the catalytic domain. The PlnA (SH)2 and t-PAB(SH) chains were mixed in a 1:1 molar ratio, and hybridization was allowed to proceed at 4 °C for 6 days. The covalent PlnA-t-PAB hybrid activator was isolated from the mixture by a two-step affinity chromatography method, with L-lysine-substituted Sepharose and Zn-chelated agarose. The protein yield of purified hybrid was 10% with a major component (77%) of Mr ∼92,000. The covalent PlnA-t-PAB hybrid activator, contained 1 mol of each chain; after reduction, it gave the two parent chains, PlnA and t-PAA, also shown to be present by double immunodiffusion. The specific plasminogen activator activity, with soluble fibrin, and the specific amidolytic activity, of the purified covalent hybrid activator was determined to be 200,000 IU/mg of protein, about 40% of the specific activity of the parent t-PA. In a fibrin clot lysis assay, the covalent hybrid activator and t-PA have similar specific fibrinolytic activities, 500,000 IU/mg of protein; however, the clot lysis time curves were not parallel. The binding of the covalent PlnA-t-PAB hybrid activator and t-PA to forming fibrin were found to be similar; at physiological fibrinogen concentrations, binding of both activators to forming fibrin was about 90%.

scholarly journals Preparation of polyethylene glycol-tissue plasminogen activator adducts that retain functional activity: characteristics and behavior in three animal species

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1641-1647
Author(s):  
H Jr Berger ◽  
SV Pizzo
Keyword(s):  
Polyethylene Glycol ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Functional Activity ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
Thrombolytic Activity ◽  
Wide Range ◽  
Tissue Plasminogen

Conditions were defined for the derivatization of recombinant tissue plasminogen activator (rt-PA) with polyethylene glycol (PEG) so as to retain functional activity as a possible means of producing a t-PA species with a prolonged circulating lifetime. Derivatives with a wide range of retention of activities were prepared by varying the concentration and species of activated PEG. The specific activities of the PEG-rt-PA derivatives were dependent on the method of assay. Assays using preformed fibrin gave higher estimates of retention of activity than assays using soluble components. Plasma elimination studies in mice and rats indicated prolonged circulating lifetimes for the radiolabeled PEG-rt-PA derivatives after a rapid clearance and distribution phase; however, the disappearance of functional activity was much more rapid than the disappearance of radiolabeled material. The PEG-rt-PA derivatives appeared to accumulate in tissues above their interstitial fluid concentrations and were rapidly inactivated, apparently by reaction with the plasma protease inhibitors. These results were consistent with the inactivation of the PEG-rt-PA derivatives in rat plasma in vitro. A somewhat longer half-life (t1/2) of the one derivative studied was observed in dogs (t1/2, 16 minutes) as compared with the rat (t1/2, five minutes). This was sufficient to confer thrombolytic activity upon the derivative (administered by bolus injection) in contrast to native rt-PA. The potential of PEG-modified rt-PA as a long-lived thrombolytic agent in humans will depend, however, on whether there will be a further extension of the t1/2 because of a reduction in clearance and/or a reduction in the rate of inactivation.

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