Isolation, Identification and Pharmacokinetic Properties of Human Tissue-Type Plasminogen Activator Species: Possible Localisation of a Clearance Recognition Site

1988 ◽  
Vol 59 (03) ◽  
pp. 523-528 ◽  
Author(s):  
Ian Dodd ◽  
Barbara Nunn ◽  
Jeff H Robinson
Keyword(s):  
Plasminogen Activator ◽  
Active Species ◽  
Recognition Site ◽  
Tissue Type ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Pharmacokinetic Properties ◽  
Rapid Clearance ◽  
Multiple Species

SummaryPurified preparations of recombinant tissue-type plasminogen activator (t-PA) from the recombinant Bowes melanoma cell line TRBM6 were shown to contain multiple species of plasminogen activator. Using a combination of chromatography on Sephadex G25, Sephadex G75 and Heparin Sepharose CL6B we have isolated two fibrinolytically active species, which, under nonreduced SDS PAGE, have apparent Mr = 38,000 and 56,000. Double immunodiffusion studies indicated that both species were closely related to both the t-PA B chain and t-PA itself. N-terminal sequencing identified the Mr = 38,000 species as ala160-t-PA (essentially ΔFGKI t-PA) and the Mr = 56,000 species as ser1-tyr2-gln3-glyx-cys51 t-PA (ΔF t-PA), the latter probably produced by alternative splicing of the t-PA gene. The pharmacokinetic properties of N,N dirnethy1-4-aminobenzoyl (DAB) derivatives of these activators and native t-PA were determined in the guinea pig. Whereas DAB → ΔF t-PA showed a similar, rapid plasma disappearance profile to that of DAB → t-PA, DAB → ΔFGKI t-PA was cleared significantly slower. These results suggest that a rapid clearance recognition site resides on either the growth factor or kringle 1, or both, domains of t-PA.

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.

scholarly journals Pharmacokinetic and distribution analysis of variant forms of tissue- type plasminogen activator with prolonged clearance in rat

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1842-1850
Author(s):  
GR Larsen ◽  
M Metzger ◽  
K Henson ◽  
Y Blue ◽  
P Horgan
Keyword(s):  
Plasminogen Activator ◽  
Alpha Phase ◽  
Tissue Type ◽  
Distribution Analysis ◽  
Wild Type ◽  
Tissue Type Plasminogen Activator ◽  
Glycosylation Sites ◽  
Type Plasminogen Activator ◽  
Primary Determinant ◽  
Rapid Clearance

Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy. Because of its rapid half-life (T1/2) of approximately five minutes, intravenous (IV) infusion of large doses (approximately 100 mg) are required in patients treated for myocardial infarction. To identify the determinant(s) on t-PA responsible for such rapid clearance, metabolically labeled forms of recombinant t-PA were analyzed in rats following IV administration. The following seven forms of t-PA were tested: (a) natural or glycosylated wild-type t-PA; (b) nonglycosylated wild-type t-PA; (c) delta F t-PA, which lacks the fibronectin fingerlike domain; (d) delta E t-PA, which lacks the epidermal growth factor (EGF) domain; (e) delta FE t-PA, which lacks both the finger and EGF domains; (f) delta FE3X t-PA, a form of delta FE t-PA in which Asn-linked glycosylation is prevented at all known glycosylation sites (Asn-117, 184, and 448; replaced by Gln); and (f) delta FE1X t-PA, a form of delta FE t-PA in which high-mannose- type glycosylation is prevented at Asn-117. Both glycosylated and nonglycosylated wild-type t-PA cleared in an exponential biphasic manner, with an initial alpha-phase T1/2 of 0.8 and 1.9 minutes, respectively. This result demonstrates that carbohydrate is not the primary mediator of the rapid clearance of t-PA. The liver was the primary organ responsible for uptake of these molecules. All other proteins tested, except for delta E t-PA, demonstrated primarily monophasic clearance patterns with T1/2 ranging between 12 and 27 minutes, and reduced uptake in the liver. delta E t-PA however, cleared in a biphasic manner with an alpha-phase T1/2 of 2.1 minutes. Results presented suggest that the clearance of t-PA is mediated by two distinct mechanisms. The primary determinant(s) responsible for modulating the rapid clearance of t-PA appears to be resident within the polypeptide sequence encoding the finger and/or EGF domains, with emphasis on the finger domain. A second and less significant contribution to clearance is defined by the presence and type of glycosylation.

ENHANCEMENT OF PLASMINOGEN ACTIVATION BY TISSUE-TYPE PLASMINOGEN ACTIVATOR IN THE PRESENCE OF ANCROD AND FIBRINOGEN

1987 ◽  
Author(s):  
K N N Reddy ◽  
B Cercek ◽  
W Ganz
Keyword(s):  
Plasminogen Activator ◽  
Degradation Products ◽  
Tissue Type ◽  
Plasminogen Activation ◽  
Amidolytic Activity ◽  
Human Fibrinogen ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Mixture Prior ◽  
Rapid Clearance

Ancrod, a thrombin-like enzyme from Malayan pit viper venom, when infused into experimental animals or man converts fibrinogen into fibrin micro-clots. In a recent study we have found that in dogs pretreatment with ancrod markedly enhanced thrombolysis by recombinant tissue-type plasminogen activator (rt-PA), presumably by depleting fibrinogen and preventing new fibrin uptake by the thrombus during lysis. The rapid clearance of large amounts of fibrinogen from the circulation and the appearance of fibrin degradation products during ancrod treatment is indicative of high fibrinolytic activity. In this study we found that ancrod enhances activation of plasminogen by rt-PA indirectly via plasmin mediated digestion of fibrin. To a reaction mixture containing lys-plasminogen, human fibrinogen and val-leu-lys-pNA (S-2251), ancrod was added followed by rt-PA. At time intervals plasminogen activation was followed by measuring amidolytic activity on S-2251 at 405 nm. Ancrod or fibrinogen alone had no significant effect on the activation of plasminogen by rt-PA. However, the amidolytic activity increased with time in reaction mixture containing ancrod and fibrinogen. When a small amount of alpha-2-antiplasmin was added to the reaction mixture prior to the addition of rt-PA (the inhibitor level was sufficient to inhibit only a fraction of plasmin generated during the assay period) the activation rate was very much reduced. Thus, a small amount of plasmin initiates digestion of fibrin which may result in the exposure of new sites on fibrin that enhance the rate of activation of plasminogen by rt-PA. The rapid clearance of fibrinogen during ancrod infusion may not be due to increased susceptibility of micro-clots to lysis but to increased rate of plasmin formation. These results further confirm the observations of other investigators about the role of plasmin in the activation of plasminogen by tissue plasminogen activator.

scholarly journals Pharmacokinetic and distribution analysis of variant forms of tissue- type plasminogen activator with prolonged clearance in rat

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1842-1850 ◽  
Author(s):  
GR Larsen ◽  
M Metzger ◽  
K Henson ◽  
Y Blue ◽  
P Horgan
Keyword(s):  
Plasminogen Activator ◽  
Alpha Phase ◽  
Tissue Type ◽  
Distribution Analysis ◽  
Wild Type ◽  
Tissue Type Plasminogen Activator ◽  
Glycosylation Sites ◽  
Type Plasminogen Activator ◽  
Primary Determinant ◽  
Rapid Clearance

Abstract Human tissue-type plasminogen activator (t-PA) is a glycoprotein used currently in thrombolytic therapy. Because of its rapid half-life (T1/2) of approximately five minutes, intravenous (IV) infusion of large doses (approximately 100 mg) are required in patients treated for myocardial infarction. To identify the determinant(s) on t-PA responsible for such rapid clearance, metabolically labeled forms of recombinant t-PA were analyzed in rats following IV administration. The following seven forms of t-PA were tested: (a) natural or glycosylated wild-type t-PA; (b) nonglycosylated wild-type t-PA; (c) delta F t-PA, which lacks the fibronectin fingerlike domain; (d) delta E t-PA, which lacks the epidermal growth factor (EGF) domain; (e) delta FE t-PA, which lacks both the finger and EGF domains; (f) delta FE3X t-PA, a form of delta FE t-PA in which Asn-linked glycosylation is prevented at all known glycosylation sites (Asn-117, 184, and 448; replaced by Gln); and (f) delta FE1X t-PA, a form of delta FE t-PA in which high-mannose- type glycosylation is prevented at Asn-117. Both glycosylated and nonglycosylated wild-type t-PA cleared in an exponential biphasic manner, with an initial alpha-phase T1/2 of 0.8 and 1.9 minutes, respectively. This result demonstrates that carbohydrate is not the primary mediator of the rapid clearance of t-PA. The liver was the primary organ responsible for uptake of these molecules. All other proteins tested, except for delta E t-PA, demonstrated primarily monophasic clearance patterns with T1/2 ranging between 12 and 27 minutes, and reduced uptake in the liver. delta E t-PA however, cleared in a biphasic manner with an alpha-phase T1/2 of 2.1 minutes. Results presented suggest that the clearance of t-PA is mediated by two distinct mechanisms. The primary determinant(s) responsible for modulating the rapid clearance of t-PA appears to be resident within the polypeptide sequence encoding the finger and/or EGF domains, with emphasis on the finger domain. A second and less significant contribution to clearance is defined by the presence and type of glycosylation.

Binding of 131I-Labeled Tissue-Type Plasminogen Activator on De-Endothelialized Lesions in Rabbits

1987 ◽  
Vol 26 (05) ◽  
pp. 224-228 ◽  
Author(s):  
Y. Isaka ◽  
H. Etani ◽  
K. Kimura ◽  
S. Yoneda ◽  
T. Kamada ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Abdominal Aorta ◽  
Half Life ◽  
Balloon Catheter ◽  
Biochemical Properties ◽  
Tissue Type ◽  
Fibrin Deposition ◽  
Tissue Type Plasminogen Activator ◽  
High Affinity ◽  
Type Plasminogen Activator

Tissue-type plasminogen activator (t-PA) which has a high affinity for fibrin in the clot, was labeled with 131I by the iodogen method, and its binding to de-endothelialized lesions in the rabbit was measured to assess the detectability of thrombi. The de-endothelialized lesion was induced in the abdominal aorta with a Fogarty 4F balloon catheter. Two hours after the de-endothelialization, 131I-labeled t-PA (125 ± 46 μCi) was injected intravenously. The initial half-life of the agent in blood (n = 12) was 2.9 ± 0.4 min. The degree of binding of 131I-labeled t-PA to the de-endothelialized lesion was evaluated at 15 min (n = 6) or at 30 min (n = 6) after injection of the agent. In spite of the retention of the biochemical properties of 131I-labeled t-PA and the presence of fibrin deposition at the de-endothelialized lesion, the binding of t-PA to the lesion was not sufficiently strong. Lesion-to-control ratios (cpm/g/cpm/g) were 1.65 ± 0.40 (at 15 min) and 1.39 ± 1.31 (at 30 min), and lesion-to-blood ratios were 1.39 ± 0.32 (at 15 min) and 1.36 ± 0.23 (at 30 min). These results suggest that radiolabeled t-PA may be inappropriate as a radiopharmaceutical for the scintigraphic detection of a pre-existing thrombotic lesion.

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