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

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.

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

1993 ◽  
Vol 70 (03) ◽  
pp. 481-485 ◽  
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.

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

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1421-1425 ◽  
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

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.

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

Blood ◽  
1989 ◽  
Vol 73 (6) ◽  
pp. 1421-1425 ◽  
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.

Purification and Characterization of Single-Chain Urokinase-Type Plasminogen Activator (Pro-Urokinase) from Human A431 Cells

1986 ◽  
Vol 56 (02) ◽  
pp. 219-224 ◽  
Author(s):  
Angelo Corti ◽  
Maria Luisa Nolli ◽  
Adolfo Soffientini ◽  
Giovanni Cassani
Keyword(s):  
Plasminogen Activator ◽  
Specific Activity ◽  
Active Form ◽  
Human Kidney ◽  
Single Chain ◽  
A431 Cells ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Sds Page ◽  
Aminoacid Sequence

SummaryA single-chain urokinase-type plasminogen activator (A431sc-uPA) was purified ˜18,000-fold from A431 human epidermoid carcinoma cell supernatants by monoclonal antibody immunoaffinity chromatography on 5B4-agarose and ion-exchange FPLC (overall yield 63%). More than 100 jig of A431sc-uPA can be recovered per liter of supernatant. The product is homogeneous by SDS-PAGE and reverse phase FPLC analysis while two main isoelectric forms of pi 9.05 and pi 9.20 were observed by IEF. SDS-PAGE in reducing and non-reducing conditions, Western blot analysis and zymography showed that A431sc-uPA is a single-chain protein of about 50,000 Mr immunologically related to urokinase (uPA) and distinct from tissue plasminogen activator (tPA). The N-terminal aminoacid sequence of A431sc-uPA (27 residues) is identical to that of human kidney single-chain uPA. A431sc-uPA does not incorporate 3H-diisopropylfluorophosphate and is virtually inactive on the synthetic substrate S-2444. Plasmin treatment converts A431sc-uPA into a two-chain active form with a fibrinolytic specific activity of 123,000 I.U./mg.

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

1994 ◽  
Vol 71 (01) ◽  
pp. 134-140 ◽  
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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