Thrombolytic and Pharmacokinetic Properties of a Recombinant Chimeric Plasminogen Activator Consisting of a Fibrin Fragment D-Dimer Specific Humanized Monoclonal Antibody and a Truncated Single-Chain Urokinase

1992 ◽  
Vol 68 (02) ◽  
pp. 170-179 ◽  
Author(s):  
M Dewerchin ◽  
A-M Vandamme ◽  
P Holvoet ◽  
F De Cock ◽  
G Lemmens ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Venous Thrombosis ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Humanized Monoclonal Antibody ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

SummaryA recombinant chimeric plasminogen activator consisting of a humanized monoclonal antibody specific for cross-linked human fibrin (MA-15C5Hu) and a 32 kDa single-chain urokinase-type plasminogen activator (scu-PA-32k) comprising amino acids Leu144-Leu411, MA-15C5Hu/scu-PA-32k, was previously found to have a 12-fold higher fibrinolytic potency than recombinant scu-PA-32k towards a human plasma clot in a human plasma milieu in vitro (Vandamme et al., Eur J Biochem 1992; 205: 139–46). Therefore, the thrombolytic and pharmacokinetic properties of MA-15C5Hu/scu-PA-32k were compared with those of recombinant single-chain urokinase-type plasminogen activator (scu-PA) in 3 different venous thrombosis models in vivo. In hamsters with a pulmonary embolus consisting of a human plasma clot, the thrombolytic potency (% lysis per dose in mg/kg administered) of MA-15C5Hu/scu-PA-32k was 23-fold higher than that of scu-PA (p <0.0005). In rabbits with a jugular vein clot prepared from human plasma, the thrombolytic potency of MA-15C5Hu/scu-PA-32k was 11-fold higher than that of scu-PA (p = 0.012). In baboons with an autologous whole blood clot in the femoral vein, the chimera had a 5-fold higher thrombolytic potency than scu-PA. In all three animal species, the clearance of the chimera was 10- to 27-fold reduced as compared to scu-PA. The specific thrombolytic activity (% lysis per µg/ml steady-state plasma u-PA antigen) was increased up to 7-fold with MA-15C5Hu/scu-PA-32k as compared with scu-PA, which is indicative of targeting of the chimera to the clot. No fibrinogen breakdown or α2-antiplasmin depletion was observed during thrombolysis with the chimera.Thus, MA-15C5Hu/scu-PA-32k constitutes a recombinant chimeric plasminogen activator with a significantly enhanced thrombolytic potency in 3 different animal models of venous thrombosis.

scholarly journals A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1794-1800 ◽  
Author(s):  
PJ Declerck ◽  
HR Lijnen ◽  
M Verstreken ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

Role of α2-Antiplasmin in Fibrin-Specific Clot Lysis with Single-Chain Urokinase-Type Plasminogen Activator in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 394-398 ◽  
Author(s):  
P J Declerck ◽  
H R Lijnen ◽  
M Verstreken ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Clot Lysis ◽  
Minor Role ◽  
Single Chain ◽  
Plasma Clot ◽  
Normal Plasma ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryThe role of plasma α2-antiplasmin (α2-AP) in the fibrinspecificity of clot lysis by recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and in the conversion of rscu-PA to its two-chain derivative (rtcu-PA, urokinase) was investigated in an in vitro human plasma clot lysis system. Fifty % lysis in 2 h of a 0.1 ml 125l-fibrin labeled human plasma clot immersed in 0.5 ml normal human plasma was obtained with 1.4 ± 0.15 µg/ml rscu-PA (mean ± SD, n = 8). This was associated with degradation of 23 ± 7% of fibrinogen and generation of 0.20 ± 0.09 µg/ml rtcu-PA. In α2-AP-depleted plasrna 50% clot lysis in 2 h required 2-fold less rscu-PA which was associated with 3-fold more extensive fibrinogen degradation and 2-fold more rtcu-PA generation. Fifty % lysis in? h, of a 0.1 ml α2-AP-depleted plasma clot, subriersed in 0.5 ml normal plasma, was obtained with 0.80 ± 0.05 µg/ml rscu-PA (n = 3, p <0.001 vs normal clot) and was associated with 17 ± 6% fibrinogen breakdown (p : 0.22 vs normal clot) and 0.08 ± 0.02 µg/ml rtcu-PA generation (p < 0.05 vs normal clot). In α2-AP-depleted plasma the equipotent rscu-PA concentration was 4-fold lower than in normal plasma and was associated with 3-fold more fibrinogen degradation and a similar extent of rtcu-PA generationThus, α2-AP in plasma contributes significantly to the fibrinspecificity of rscu-PA, primarily via prevention of conversion in plasma of rscu-PA to rtcu-PA. Clot associated α2-AP increases the resistance of the clot to lysis with rscu-PA, but plays an only minor role in the fibrin-specificity of clot lysis in normal plasma.

scholarly journals Comparative thrombolytic properties of single-chain forms of urokinase- type plasminogen activator

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 592-596
Author(s):  
DC Stump ◽  
JM Stassen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Molecular Forms ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Thrombosis Model ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu- PA-32k). All four molecular forms induced significant lysis of a 125I- labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2- antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

scholarly journals Comparative thrombolytic properties of single-chain forms of urokinase- type plasminogen activator

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 592-596 ◽  
Author(s):  
DC Stump ◽  
JM Stassen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Molecular Forms ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Thrombosis Model ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu- PA-32k). All four molecular forms induced significant lysis of a 125I- labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2- antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

scholarly journals Effect of chemical conjugation of recombinant single-chain urokinase- type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1005-1018 ◽  
Author(s):  
M Dewerchin ◽  
HR Lijnen ◽  
JM Stassen ◽  
F De Cock ◽  
T Quertermous ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Human Platelet ◽  
Single Chain ◽  
Plasma Clot ◽  
Antiplatelet Antibodies ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Induced Aggregation

Abstract The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

scholarly journals Effect of chemical conjugation of recombinant single-chain urokinase- type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1005-1018 ◽  
Author(s):  
M Dewerchin ◽  
HR Lijnen ◽  
JM Stassen ◽  
F De Cock ◽  
T Quertermous ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Human Platelet ◽  
Single Chain ◽  
Plasma Clot ◽  
Antiplatelet Antibodies ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Induced Aggregation

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

scholarly journals A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1794-1800 ◽  
Author(s):  
PJ Declerck ◽  
HR Lijnen ◽  
M Verstreken ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

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.

scholarly journals Thrombolytic and pharmacokinetic properties of an immunoconjugate of single-chain urokinase-type plasminogen activator (u-PA) and a bispecific monoclonal antibody against fibrin and against u-PA in baboons

Blood ◽  
1992 ◽  
Vol 79 (9) ◽  
pp. 2322-2329 ◽  
Author(s):  
Y Imura ◽  
JM Stassen ◽  
T Kurokawa ◽  
S Iwasa ◽  
HR Lijnen ◽  
...  
Keyword(s):  
Body Weight ◽  
Plasminogen Activator ◽  
Autologous Blood ◽  
Clot Lysis ◽  
Maximal Rate ◽  
Antigen Level ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Pharmacokinetic Properties

Abstract Targeting of plasminogen activators to the fibrin component of a thrombus with the use of monoclonal antibodies (MA) directed against human fibrin may enhance their thrombolytic potency and fibrin- specificity. The thrombolytic and pharmacokinetic properties of rscu- PA/MA-FU1–74, an immunoconjugate of recombinant single-chain urokinase- type plasminogen activator (rscu-PA) and a bispecific MA directed against u-PA and against the beta-chain of human fibrin (MA-FU1–74), were investigated in baboons with a [125I]fibrin-labeled autologous blood clot in the femoral vein. Continuous intravenous infusion of rscu- PA/MA-FU1–74 (1:1.2 molar ratio) over 2 hours showed a fivefold increased thrombolytic potency (lysis per unit dose) over that of unconjugated rscu-PA, as evidenced both by a higher maximal rate of lysis (380% +/- 68% v 78% +/- 25% lysis per mg u-PA equivalent of compound administered per kg body weight, P less than .001), and by a lower dose at which the maximal rate of lysis occurs (0.19 +/- 0.03 v 0.82 +/- 0.10 mg compound per kg body weight, P less than .001). The specific thrombolytic activity (percent lysis per unit steady-state plasma u-PA antigen level) was lower for rscu-PA/MA-FU1–74 than for rscu-PA, as shown by both a lower maximal rate of lysis (60% +/- 13% v 220% +/- 22% lysis per microgram/mL u-PA antigen level in plasma, P less than .001) and a higher plasma antigen level at which maximal lysis is achieved (1.2 +/- 0.17 v 0.20 +/- 0.01 microgram/mL, P less than .001). The thrombolytic potency of rscu-PA/MA-UK1–3, an immunoconjugate of rscu-PA with the parental anti-u-PA antibody was similar to that of unconjugated rscu-PA. Clot lysis was achieved without systemic fibrinogen or alpha 2-antiplasmin consumption, and with a minor transient prolongation of the bleeding time. After the end of the infusions, u-PA-related antigen disappeared from plasma in a biphasic manner, with an initial half-life of 3.3 +/- 0.4 minutes for rscu-PA, 13 +/- 1 minutes for rscu-PA/MA-FU1–74, and 13 +/- 1 minutes for rscu-PA/MA-UK1–3, with corresponding plasma clearances of 340 +/- 28, 10 +/- 1, and 37 +/- 4 mL/min, respectively (mean +/- SEM). rscu- PA/MA-FU1–74 has a fivefold higher thrombolytic potency than unconjugated rscu-PA, as a result both of fibrin targeting by the specific idiotype of the antibody and of a slower plasma clearance.

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