Investigation by HPLC of the Catabolism of Recombinant Tissue Plasminogen Activator in the Rat

1988 ◽  
Vol 60 (01) ◽  
pp. 107-112 ◽  
Author(s):  
Roy Harris ◽  
Louis Garcia Frade ◽  
Lesley J Creighton ◽  
Paul S Gascoine ◽  
Maher M Alexandroni ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Half Life ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
High Molecular Weight Complex ◽  
Molecular Weights ◽  
Major Activity ◽  
Tissue Plasminogen

SummaryThe catabolism of recombinant tissue plasminogen activator (rt-PA) was investigated after injection of radiolabelled material into rats. Both Iodogen and Chloramine T iodination procedures yielded similar biological activity loss in the resultant labelled rt-PA and had half lives in the rat circulation of 1 and 3 min respectively. Complex formation of rt-PA was investigated by HPLC gel exclusion (TSK G3000 SW) fractionation of rat plasma samples taken 1-2 min after 125I-rt-PA injection. A series of radiolabelled complexes of varying molecular weights were found. However, 60% of the counts were associated with a single large molecular weight complex (350–500 kDa) which was undetectable by immunologically based assays (ELISA and BIA) and showed only low activity with a functional promoter-type t-PA assay. Two major activity peaks in the HPLC fractions were associated with Tree t-PA and a complex having a molecular weight of ̴ 180 kDa. HPLC fractionation to produce these three peaks at various timed intervals after injection of 125I-rt-PA showed each to have a similar initial rate half life in the rat circulation of 4-5 min. The function of these complexes as yet is unclear but since a high proportion of rt-PA is associated with a high molecular weight complex with a short half life in the rat, we suggest that the formation of this complex may be a mechanism by which t-PA activity is initially regulated and finally cleared from the rat circulation.

CATABOLIC BEHAVIOUR OF RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR (R-t-PA) IN THE RAT

1987 ◽  
Author(s):  
R Harris ◽  
L Garcia Frade ◽  
S Poole ◽  
M Mahmoud ◽  
A D Curtis ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Tween 80 ◽  
Recombinant Tissue Plasminogen Activator ◽  
Rat Plasma ◽  
Major Activity ◽  
Thrombosis Model ◽  
Tissue Plasminogen ◽  
Chloramine T

To compliment ongoing experimentation on the use of tissue plasminogen activator (t-PA) as a thrombolytic agent in a rat thrombosis model, clearance data for labelled R-t-PA in the rat were investigated. Both Iodogen and Chloramine T labelling procedures for incorporation yielded similar biological activity in the resultant labelled t-PA, while the half-lives in the rat circulation for Iodogen- and Chloramine T-label led materials were 5 1/2 and 7 1/2 minutes respectively. Clearance data using promoter based and ELISA assays support that obtained with 125j_ labelled t-PA. Following fractionation of various timed rat plasma samples by HPLC on a gel exclusion column (TSK G-3000 SW), 90% of the labelled t-PA was distributed between two inhibitor/t-PA complex peaks m the 1 minute sanple. One of these peaks (about 30% of labelled t-PA) was compatible with a t-PA/ PAI-1 complex, having a molecular weight of about 120,000, while the other (comprising about 60% of labelled t-PA) had a molecular weight of about 350,000 and was undetectable by ELISA, bioimmunoassay or fibrin plate assay, while showing low activity by a promoter-type t-PA assay. The two major activity peaks in these HPLC profiles of rat plasma were associated with low levels of radiolabelled t-PA and were compatible with free t-PA and a complex of t-PA having a molecular weight of about 200,000. It was observed that free t-PA was retarded during the HPLC column separations and eluted as a broad trailing peak despite the presence of 0.1% - 0.5% Tween-80 in the column. Thus molecular weights of the various complexes formed are subject to further examination. All three t-PA peaks had the same initial half-life of 2-3 minutes. Since about 60% of the t-PA is contained in the high molecular weight inhibitor complex, we propose that the formation of this complex may be a major mechanism by which t-PA is cleared from the rat circulation, despite inactive catabolic breakdown products reappearing in the circulation following clearance of these complexes.

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.

Characteristics of Tissue Plasminogen Activator from Paranasal Mucous Membrane in Chronic Sinusitis

1982 ◽  
Vol 91 (1) ◽  
pp. 101-105 ◽  
Author(s):  
T. Kosugi ◽  
O. Matsuo ◽  
H. Mihara ◽  
K. Hirano ◽  
M. Hamaya
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Mucous Membrane ◽  
Chronic Sinusitis ◽  
Gel Filtration ◽  
Tissue Extract ◽  
Molecular Weights ◽  
Tissue Plasminogen ◽  
Filtration Technique

Tissue extract of paranasal mucous membrane from patients with chronic sinusitis was found to show a large lysis area on plasminogen-rich fibrin plates, but not on plasminogen-free fibrin plates. This indicates the existence of tissue plasminogen activator in the tissue extract. Further studies by the gel filtration technique showed that two plasminogen activators of different molecular weights were present in the tissue extract. The existence of tissue plasminogen activator with a low molecular weight has not previously been reported. This activator is labile at neutral pH at 37°C, but stable on fibrin under the above conditions. The molecular weight of this compound is lower than that of cytochrome c. It may be a compound which is proteolytically modified by proteases, ie, trypsin-like enzymes, existing in the paranasal mucous membrane tissue of patients with chronic sinusitis.

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.

Bioavailability in Rats of Human Recombinant Tissue Plasminogen Activator After Subcutaneous and Intramuscular Injection

1986 ◽  
Vol 56 (03) ◽  
pp. 299-301 ◽  
Author(s):  
L J Garcia Frade ◽  
S Poole ◽  
S Hanley ◽  
L J Creighton ◽  
A D Curtis ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Inferior Vena ◽  
Peak Plasma ◽  
Plasma Concentrations ◽  
Vena Cava ◽  
Recombinant Tissue Plasminogen Activator ◽  
Fibrin Plate ◽  
Tissue Plasminogen

SummaryThe bioavailability of human recombinant tissue plasminogen activator (rt-PA) in rats was measured after subcutaneous (s.c.) and intramuscular (i.m.) injection. Rt-PA was absorbed after both i.m. and s.c. injection, giving peak plasma concentrations within 30 min and 1 h, respectively, with detectable concentrations up to 6 h. These peak values of bioavailable t-PA were obtained in a functional fibrin plate assay of euglobulin precipitates and expressed as +88% and +243% (for s.c. and i.m. routes respectively) above basal rat fibrinolytic activity. Prior injection of rt-PA, s.c. or i.m., significantly reduced the weights of thrombi induced in the inferior vena cava after injection.

Sign in / Sign up

Export Citation Format

Share Document