Single Chain Urokinase Yields Relatively Low Grade Fibrinolytic Activity And As More Bioavailable Than Tissue Type Plasminogen Activator In Pleural Fluids Of Rabbits With Tetracycline-Induced Pleurodesis

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648982 ◽

1994 ◽

Vol 72 (06) ◽

pp. 906-911 ◽

Cited By ~ 8

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.

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On the Fibrinolytic System in Aged Rats, and Its Reactivity to Endotoxin and Cytokines

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648525 ◽

1992 ◽

Vol 67 (06) ◽

pp. 697-701 ◽

Cited By ~ 6

Author(s):

J J Emeis ◽

A Brouwer ◽

R J Barelds ◽

M A Horan ◽

S K Durham ◽

...

Keyword(s):

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Fibrinolytic System ◽

Tissue Type ◽

High Dose ◽

Base Line ◽

Aged Rats ◽

Tissue Type Plasminogen Activator ◽

Young Rats ◽

Type Plasminogen Activator

SummaryAged rats are more susceptible to endotoxin-induced effects, including microthrombosis and platelet aggregation, than are young rats. To investigate whether changes in the fibrinolytic system might be involved, we investigated the fibrinolytic activity in plasma euglobulin fractions and tissues (lung and heart) of young (6-months old) and aged (24-months old) rats under baseline conditions and after challenge with endotoxin. Aged rats had lower plasma levels of tissue-type plasminogen activator (t-PA) and of urokinase-type PA (u-PA) activity. PA inhibitor (PAI) activity was higher in the plasma of aged rats, as was t-PA activity in lung and heart.Rats were treated with either a low dose (1 μg/kg) or a high dose (10 mg/kg) of endotoxin. Both treatments induced a transient phase of increased blood fibrinolytic activity, as evidenced by higher levels of tissue-type plasminogen activator (t-PA) activity and decreased levels of PA inhibitor (PAI) activity. Over time, the fibrinolytic activity decreased, probably due to increased levels of PA inhibitor.Both the early increase in t-PA activity, and the subsequent increase in PAI activity, were more pronounced in the aged rats, as compared with the younger rats, after the high dose of endotoxin. The aged rats also responded to an injection of interleukin-1β or tumor necrosis factor-α with a larger increase of PAI activity than did the younger rats.Together the data suggest that, compared to young rats, aged rats have a decreased base-line plasma fibrinolytic activity, while their fibrinolytic system is more responsive to challenge by endotoxin and cytokines.

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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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661598 ◽

1986 ◽

Vol 56 (01) ◽

pp. 035-039 ◽

Cited By ~ 42

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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