Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Abstract Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.

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Demonstration of single chain urokinase-type plasminogen activator on human platelet membrane

Blood ◽

10.1182/blood.v73.6.1421.bloodjournal7361421 ◽

1989 ◽

Vol 73 (6) ◽

pp. 1421-1425 ◽

Cited By ~ 1

Author(s):

S Park ◽

LA Harker ◽

UM Marzec ◽

EG Levin

Keyword(s):

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Neutralization Assay ◽

Platelet Membrane ◽

Immunofluorescent Staining ◽

Single Chain ◽

Outer Leaflet ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Triton X

Fibrinolytic activity was found to be associated with sonicated platelet membranes after separation from cytosol by differential centrifugation. This fibrinolytic activity was attributed to the presence of a plasminogen activator, which was immunochemically identified as urinary-type plasminogen activator (uPA) by antibody neutralization assay, immunoblotting, and immunofluorescence. The molecular weight (mol wt) of this uPA was 54,000 and was present as the single chain form, although a small amount was detected in a higher mol wt complex indicative of a uPA-inhibitor complex. Treatment of membrane preparations with Triton X-100, 3 mol/L KCl, and 0.1 mol/L glycine, (pH 2.3), but not 10 mmol/L ethylenediamine tetraacetic acid (EDTA), removed the uPA from the membrane. This suggests that uPA is a peripheral membrane protein and that metal ions do not mediate protein- membrane association. Immunofluorescent staining revealed the presence of uPA on the outer surface of the platelet in preparations of intact unstimulated platelets. Thus, uPA is associated with the outer leaflet of the platelet membrane and may be involved with the acceleration of thrombus degradation observed with platelet-rich thrombi.

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Comparison of the In Vitro Fibrinolytic Activities of Low and High Molecular Weight Single-Chain Urokinase-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649609 ◽

1993 ◽

Vol 70 (03) ◽

pp. 481-485 ◽

Cited By ~ 11

Author(s):

Gerard A W de Munk ◽

Eleonore Groeneveld ◽

Dingeman C Rijken

Keyword(s):

Molecular Weight ◽

High Molecular Weight ◽

Plasminogen Activator ◽

Fibrinolytic Activity ◽

Time Course ◽

Clot Lysis ◽

Single Chain ◽

Epidermal Growth Factor Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

SummaryThe fibrinolytic activity of low molecular weight (LMW) single-chain urokinase-type plasminogen activator (scu-PA) lacking the epidermal growth factor domain and the kringle domain was compared with the activity of high molecular weight (HMW) scu-PA. LMW scu-PA was 1-5 times less active than HMW scu-PA in a fibrin plate method, in a purified fibrin clot lysis assay and in a plasma clot lysis assay. Time course experiments in a chromogenic plasminogen activator assay suggested that LMW scu-PA was less sensitive to activation by plasmin than HMW scu-PA. This was confirmed in a scu-PA activation test, which showed that at a concentration of 40 IU/ml LMW scu-PA required a three-fold higher plasmin concentration for 50% activation in 20 min than did HMW scu-PA. Kinetic experiments in the presence of 0.1 M NaCl showed non-standard Michaelis-Menten kinetics for the activation by plasmin of both HMW and LMW scu-PA. In contrast, standard kinetics was observed at 0.15 M NaCl, showing a 2.6-fold lower catalytic efficiency for LMW scu-PA than for HMW scu-PA. It is concluded that the plasmin activation of LMW scu-PA is about three times slower than the activation of HMW scu-PA. This explains, at least partially, the lower fibrinolytic activity of LMW scu-PA in comparison with HMW scu-PA.

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In Vitro Study of the Fibrinolytic Activity via Single Chain Urokinase-Type Plasminogen Activator and Molecular Docking of FGFC1

Molecules ◽

10.3390/molecules26071816 ◽

2021 ◽

Vol 26 (7) ◽

pp. 1816

Author(s):

Chunli Gao ◽

Quan Shen ◽

Pengjie Tang ◽

Yuling Cao ◽

Houwen Lin ◽

...

Keyword(s):

Molecular Docking ◽

Plasminogen Activator ◽

Binding Sites ◽

Fibrinolytic Activity ◽

Hydrophobic Interactions ◽

Single Chain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

Fungi fibrinolytic compound 1 (FGFC1) is a rare marine-derived compound that can enhance fibrinolysis both in vitro and in vivo. The fibrinolytic activity characterization of FGFC1 mediated by plasminogen (Glu-/Lys-) and a single-chain urokinase-type plasminogen activator (pro-uPA) was further evaluated. The binding sites and mode of binding between FGFC1 and plasminogen were investigated by means of a combination of in vitro experiments and molecular docking. A 2.2-fold enhancement of fibrinolytic activity was achieved at 0.096 mM FGFC1, whereas the inhibition of fibrinolytic activity occurred when the FGFC1 concentration was above 0.24 mM. The inhibition of fibrinolytic activity of FGFC1 by 6-aminohexanoic acid (EACA) and tranexamic acid (TXA) together with the docking results revealed that the lysine-binding sites (LBSs) play a crucial role in the process of FGFC1 binding to plasminogen. The action mechanism of FGFC1 binding to plasminogen was inferred, and FGFC1 was able to induce plasminogen to exhibit an open conformation by binding through the LBSs. The molecular docking results showed that docking of ligands (EACA, FGFC1) with receptors (KR1–KR5) mainly occurred through hydrophilic and hydrophobic interactions. In addition, the binding affinity values of EACA to KR1–KR5 were −5.2, −4.3, −3.7, −4.5, and −4.3 kcal/moL, respectively, and those of FGFC1 to KR1–KR5 were −7.4, −9.0, −6.3, −8.3, and −6.7 kcal/moL, respectively. The findings demonstrate that both EACA and FGFC1 bound to KR1–KR5 with moderately high affinity. This study could provide a theoretical basis for the clinical pharmacology of FGFC1 and establish a foundation for practical applications of FGFC1.

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DEMONSTRATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA) ON HUMAN PLATELET MEMBRANES

10.1055/s-0038-1642906 ◽

1987 ◽

Author(s):

S Park ◽

L A Harker ◽

E G Levin

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Human Platelets ◽

Platelet Membrane ◽

Single Chain ◽

Plate Method ◽

Platelet Membranes ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Sds Page

A role for platelets in enhancing fibrinolysis has been suggested. To elucidate the nature of fibrinolysis-accelerating effect of human platelets, different fractions of the platelets were tested for fibrinolytic activity by the fibr:i.n plate method in the presence of plasminogen and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography. The platelets were washed in Tyrode’s buffer containing 0.5 ug/ml prostaglandin Ef, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM phenylmethylsulfonyl fluoride by albumin density gradient separation and gel filtration techniques. Platelet membranes were isolated by differential centrifugation after disruption of the platelets by ultrasonication. The sedimented platelet membranes demonstrated plasminogen activation with subsequent fibrinolysis which was completely abolished in the presence of affinity-purified antibody to high molecular weight urokinase-type plasminogen activator (u-PA). Minimal u-PA activity was observed associated with the supernatant from sonicated platelets indicating that most of the detectable u-PA was membrane-bound. Fibrin autography following SDS-PAGE of the platelet membrane fraction revealed a lysis zone at the molecular weight level of 54,000. Single chain nature of the u-PA was demonstrated by immunoblot analysis of the platelet membrane after SDS-PAGE under reducing conditions. Treatment of the platelets with 1.2 u/ml alpha-thrombin eliminated membrane-associated u-PA activity and probably resulted from the cleavage and inactivation of scu-PA. u-PA activity was quantitated by the 125I_fibrin plate method: values in the range of 0.15-0.25 mU/109 platelets were observed. Most of the activity was liberated from the platelet membrane by exposure to 3 M KC1 or 0.1 M glycine, pH 2.3, but not to 10 mM EDTA. We conclude that human platelets have scu-PA on their membranes that may play a role as a carrier of scu-PA in blood circulation. Binding studies are in progress.

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Expression and Characterization of Clustered Charge-to-Alanine Mutants of Low Mr Single-Chain Urokinase-Type Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642397 ◽

1994 ◽

Vol 71 (01) ◽

pp. 134-140 ◽

Cited By ~ 6

Author(s):

S Ueshima ◽

P Holvoet ◽

H R Lijnen ◽

L Nelles ◽

V Seghers ◽

...

Keyword(s):

Plasminogen Activator ◽

Solvent Accessibility ◽

Site Directed Mutagenesis ◽

Clot Lysis ◽

Wild Type ◽

Single Chain ◽

Charged Amino Acids ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Pharmacokinetic Properties

SummaryIn an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M r single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala).The rscu-PA 32k moictic3 were expressed in High Five Ttichoplasiani cells, and purified to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/1. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion In lwo chain moieties by plasmin were comparable for mutant and wild-type rscu PA 32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant lysis with 16 pg/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min). In hamsters with pulmonary embolism, continuous intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type rscu-PA-32k.These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed to eliminate charged regions with the highest solvent accessibility, do not have significantly improved functional, fibrinolytic or pharmacokinetic properties.

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Inhibition in Purified Systems and in Human Plasma of Chimaeric Plasminogen Activators Consisting of the NH2-Terminal Region of Tissue-Type Plasminogen Activator and the COOH-Terminal Region of UrokinaseType Plasminogen Activator

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647039 ◽

1988 ◽

Vol 60 (02) ◽

pp. 247-250 ◽

Cited By ~ 1

Author(s):

H R Lijnen ◽

L Nelles ◽

B Van Hoef ◽

F De Cock ◽

D Collen

Keyword(s):

Plasminogen Activator ◽

Rate Constants ◽

Plasminogen Activators ◽

Second Order ◽

Tissue Type ◽

Single Chain ◽

Chain Molecules ◽

Order Rate ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator

SummaryRecombinant chimaeric molecules between tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) or two chain urokinase-type plasminogen activator (tcu-PA) have intact enzymatic properties of scu-PA or tcu-PA towards natural and synthetic substrates (Nelles et al., J Biol Chem 1987; 262: 10855-10862). In the present study, we have compared the reactivity with inhibitors of both the single chain and two chain variants of recombinant u-PA and two recombinant chimaeric molecules between t-PA and scu-PA (t-PA/u-PA-s: amino acids 1-263 of t-PA and 144-411 of u-PA; t-PA/u-PA-e: amino acids 1-274 of t-PA and 138-411 of u-PA). Incubation with human plasma in the absence of a fibrin clot for 3 h at 37° C at equipotent concentrations (50% clot lysis in 2 h), resulted in significant fibrinogen breakdown (to about 40% of the normal value) for all two chain molecules, but not for their single chain counterparts. Preincubation of the plasminogen activators with plasma for 3 h at 37° C, resulted in complete inhibition of the fibrinolytic potency of the two chain molecules but did not alter the potency of the single chain molecules. Inhibition of the two chain molecules occurred with a t½ of approximately 45 min. The two chain variants were inhibited by the synthetic urokinase inhibitor Glu-Gly-Arg-CH2CCl with apparent second-order rate constants of 8,000-10,000 M−1s−1, by purified α2-antiplasmin with second-order rate constants of about 300 M−1s−1, and by plasminogen activator inhibitor-1 (PAI-1) with second-order rate constants of approximately 2 × 107 M−1s−1.It is concluded that the reactivity of single chain and two chain forms of t-PA/u-PA chimaers with inhibitors is very similar to that of the single and two chain forms of intact u-PA.

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An Enzyme-Linked Immunosorbent Assay for Urokinase-Type Plasminogen Activator (u-PA) and Mutants and Chimeras Containing the Serine Protease Domain of u-PA

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648387 ◽

1992 ◽

Vol 67 (01) ◽

pp. 095-100 ◽

Cited By ~ 18

Author(s):

Paul J Declerck ◽

Leen Van Keer ◽

Maria Verstreken ◽

Désiré Collen

Keyword(s):

Serine Protease ◽

Plasminogen Activator ◽

Active Site ◽

Enzyme Linked Immunosorbent Assay ◽

Single Chain ◽

Protease Domain ◽

Serine Protease Domain ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Normal Human

SummaryAn enzyme-linked immunosorbent assay (ELISA) for quantitation of natural and recombinant plasminogen activators containing the serine protease domain (B-chain) of urokinase-type plasminogen activator (u-PA) was developed, based on two murine monoclonal antibodies, MA-4D1E8 and MA-2L3, raised against u-PA and reacting with non-overlapping epitopes in the B-chain. MA-4D1E8 was coated on microtiter plates and bound antigen was quantitated with MA-2L3 conjugated with horseradish peroxidase. The intra-assay, inter-assay and inter-dilution coefficients of variation of the assay were 6%, 15% and 9%, respectively. Using recombinant single-chain u-PA (rscu-PA) as a standard, the u-PA-related antigen level in normal human plasma was 1.4 ± 0.6 ng/ml (mean ± SD, n = 27).The ELISA recognized the following compounds with comparable sensitivity: intact scu-PA (amino acids, AA, 1 to 411), scu-PA-32k (AA 144 to 411), a truncated (thrombin-derived) scu-PA comprising A A 157 to 411, and chimeric t-PA/u-PA molecules including t-PA(AA1-263)/scu-PA(AA144-411), t-PA(AA1-274)/scu-PA(AA138-411) and t-PA(AA87-274)/scu-PA(AA138-411). Conversion of single-chain to two-chain forms of u-PA or inhibition of active two-chain forms with plasminogen activator inhibitor-1 or with the active site serine inhibitor phenyl-methyl-sulfonyl fluoride, did not alter the reactivity in the assay. In contrast, inactivation with α2-antiplasmin or with the active site histidine inhibitor Glu-Gly-Arg-CH2Cl resulted in a 3- to 5-fold reduction of the reactivity. When purified scu-PA-32k was added to pooled normal human plasma at final concentrations ranging from 20 to 1,000 ng/ml, recoveries in the ELISA were between 84 and 110%.The assay was successfully applied for the quantitation of pharmacological levels of scu-PA and t-PA(AA87_274)/scu-PA(AA138-411) in plasma during experimental thrombolysis in baboons.Thus the present ELISA, which is specifically dependent on the presence of the serine protease part of u-PA, is useful for measurement of a wide variety of variants and chimeras of u-PA which are presently being developed for improved thrombolytic therapy.

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The Role of the Low-Density Lipoprotein Receptor-Related Protein (LRP) in the Plasma Clearance and Liver Uptake of Recombinant Single-chain Urokinase-type Plasminogen Activator in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656039 ◽

1997 ◽

Vol 77 (04) ◽

pp. 710-717 ◽

Cited By ~ 5

Author(s):

Marieke E van der Kaaden ◽

Dingeman C Rijken ◽

J Kar Kruijt ◽

Theo J C van Berkel ◽

Johan Kuiper

Keyword(s):

Plasminogen Activator ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Single Chain ◽

Liver Uptake ◽

Type Plasminogen Activator ◽

Parenchymal Liver ◽

Urokinase Type Plasminogen Activator ◽

Density Lipoprotein Receptor

SummaryUrokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, recombinant single-chain u-PA (rscu-PA) expressed in E.coli is recognized by the Low-Density Lipoprotein Receptor-related Protein (LRP) on rat parenchymal liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA in rats. A preinjection of the LRP inhibitor GST-RAP reduced the maximal liver uptake of 125I-rscu-PA at 5 min after injection from 50 to 30% of the injected dose and decreased the clearance of rscu-PA from 2.37 ml/min to 1.58 ml/min. Parenchymal, Kupffer and endothelial cells were responsible for 40, 50 and 10% of the liver uptake, respectively. The reduction in liver uptake of rscu-PA by the preinjection of GST-RAP was caused by a 91 % and 62% reduction in the uptake by parenchymal and Kupffer cells, respectively. In order to investigate the part of rscu-PA that accounted for the interaction with LRP, experiments were performed with a mutant of rscu-PA lacking residues 11-135 (= deltal25- rscu-PA). Deletion of residues 11-135 resulted in a 80% reduction in liver uptake and a 2.4 times slower clearance (0.97 ml/min). The parenchymal, Kupffer and endothelial cells were responsible for respectively 60, 33 and 7% of the liver uptake of 125I-deltal25-rscu-PA. Preinjection of GST-RAP completely reduced the liver uptake of delta 125-rscu-PA and reduced its clearance to 0.79 ml/min. Treatment of isolated Kupffer cells with PI-PLC reduced the binding of rscu-PA by 40%, suggesting the involvement of the urokinase-type Plasminogen Activator Receptor (u-PAR) in the recognition of rscu-PA. Our results demonstrate that in vivo LRP is responsible for more than 90% of the parenchymal liver cell mediated uptake of rscu-PA and for 60% of the Kupffer cell interaction. It is also suggested that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

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Differential Detection of Single-Chain and Two-Chain Urokinase-Type Plasminogen Activator by a New Immunoadsorbent-Amidolytic Assay (IAA)

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661692 ◽

1986 ◽

Vol 56 (03) ◽

pp. 407-410 ◽

Cited By ~ 10

Author(s):

Angelo Corti ◽

Maria Luisa Nolli ◽

Giovanni Cassani

Keyword(s):

Plasminogen Activator ◽

Carcinoma Cell ◽

Differential Detection ◽

Single Chain ◽

Response Curves ◽

Type Plasminogen Activator ◽

Urokinase Type Plasminogen Activator ◽

Before And After ◽

Culture Supernatants ◽

Human Epidermoid Carcinoma

SummaryA new immunoadsorbent-amidolytic assay (IAA) for the specific differential detection of two-chain urokinase-type plasminogen activator (tcu-PA) and its single-chain precursor (scu-PA) in cell culture supernatants has been developed. The assay combines the selectivity of immunoassays with the specificity of enzyme activity assays exploiting both the antigenic and enzymatic properties of the two proteins. tcu-PA and scu-PA are selectively immunoadsorbed on the wells of a microtiterplate coated with the monoclonal antibody 5B4 and tested for enzymatic activity before and after activation by plasmin treatment. Both proteins are determined with similar efficiency since overlapping dose-response curves were obtained in the range between 12.5-200 ng/ml. The assay has been used to determine tcu-PA and scu-PA in A431 human epidermoid carcinoma cell supernatants. The analytical recoveries for tcu-PA and scu-PA added to A431 cell supernatants were 95.2% and 96.9% respectively. The intra- and inter-assay variations (CV) were 5.5% and 9.0% for tcu-PA and 9.7% and 9.8% for scu-PA respectively.

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