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.

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.

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

Inhibitor-Resistant Tissue-Type Plasminogen Activator: An Improved Thrombolytic Agent In Vitro

1994 ◽  
Vol 71 (01) ◽  
pp. 124-128 ◽  
Author(s):  
R V Shohet ◽  
S Spitzer ◽  
E L Madison ◽  
R Bassel-Duby ◽  
M-J Gething ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pai 1

SummaryPlatelet-rich clots are inefficiently lysed by current fibrinolytic agents. Platelets contain a great deal of plasminogen activator inhibitor 1 (PAI-1), the principal endogenous inhibitor of tissue-type plasminogen activator (t-PA). We have tested whether PAI-1 resistant t-PAs would be more effective thrombolytic agents in an in vitro model of platelet rich clots. Clots were formed with recalcified human plasma without or with the addition of platelets. The lysis of these clots was followed by the release of incorporated 125I-fibrinogen. Mutant and wild-type t-PA were almost equally effective against clots lacking platelets but the mutant was twice as effective at lysing platelet-rich clots. A mechanism for this effect is suggested by the demonstration that a complex between wild-type t-PA and extruded platelet contents resembles that between purified t-PA and PAI-1 and that the PAI-1 resistant t-PA does not interfere with formation of this adduct. Because of its enhanced ability to lyse platelet-rich clots in vitro, further in vivo work may find that PAI-1 resistant t-PA is a more efficacious therapeutic agent than wild-type t-PA in situations where platelets contribute to the failure of thrombolysis.

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.

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.

HSF1 Alleviates Microthrombosis and Multiple Organ Dysfunction in Mice with Sepsis by Upregulating the Transcription of Tissue-Type Plasminogen Activator

2021 ◽  
Author(s):  
Tao Li ◽  
Huan Chen ◽  
Xueyan Shi ◽  
Leijing Yin ◽  
Chuyi Tan ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Organ Dysfunction ◽  
Knockout Mice ◽  
Multiple Organ ◽  
Tissue Type ◽  
Multiple Organ Dysfunction ◽  
Wild Type ◽  
Coagulation Activity ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

AbstractSepsis is a life-threatening complication of infection closely associated with coagulation abnormalities. Heat shock factor 1 (HSF1) is an important transcription factor involved in many biological processes, but its regulatory role in blood coagulation remained unclear. We generated a sepsis model in HSF1-knockout mice to evaluate the role of HSF1 in microthrombosis and multiple organ dysfunction. Compared with septic wild-type mice, septic HSF1-knockout mice exhibited a greater degree of lung, liver, and kidney tissue damage, increased fibrin/fibrinogen deposition in the lungs and kidneys, and increased coagulation activity. RNA-seq analysis revealed that tissue-type plasminogen activator (t-PA) was upregulated in the lung tissues of septic mice, and the level of t-PA was significantly lower in HSF1-knockout mice than in wild-type mice in sepsis. The effects of HSF1 on t-PA expression were further validated in HSF1-knockout mice with sepsis and in vitro in mouse brain microvascular endothelial cells using HSF1 RNA interference or overexpression under lipopolysaccharide stimulation. Bioinformatics analysis, combined with electromobility shift and luciferase reporter assays, indicated that HSF1 directly upregulated t-PA at the transcriptional level. Our results reveal, for the first time, that HSF1 suppresses coagulation activity and microthrombosis by directly upregulating t-PA, thereby exerting protective effects against multiple organ dysfunction in sepsis.

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