Recombinant Single-chain Urokinase-type Plasminogen Research Articles

Effect of Changes in Liver Blood Flow on the Pharmacokinetics of Saruplase in Patients with Acute Myocardial Infarction

The recombinant unglycosylated single chain urokinase-type plasminogen activator saruplase is cleared for a large part by the liver. A large interindividual variation in saruplase concentration is found in acute myocardial infarction (AMI) patients. The variable cardiac performance after an infarct may induce differences in liver blood flow that could explain the concentration diversity. This study was performed to investigate the relation between hepatic blood flow and the pharmacokinetic and pharmacodynamic properties of saruplase. Thirteen AMI patients were enrolled in this open label study. Patients received a bolus injection of 20 mg saruplase followed by a one-hour infusion of 60 mg saruplase. Concurrently 36 mg intravenous indocyanine green (ICG) was given over 1 h to measure hepatic blood flow. Blood samples were taken at regular time intervals to measure plasma levels of urokinase-type plasminogen activator (u-PA) antigen and activity, the two-chain form (tcu-PA) activity, indocyanine green, fibrinogen, fibrin and fibrin degradation products, alpha2-antiplasmin and thrombin antithrombin III complex. A correlation was seen between the clearance of ICG and both those of u-PA antigen (r = 0.62; p <0.05) and u-PA activity (r = 0.57; p <0.05). A negative correlation was seen between the area under the curve of tcu-PA activity and the areas under the effect curves of both fibrinogen and alpha2-antiplasmin (r = -0.84; p <0.01 and r = -0.65; p <0.05). Liver blood flow is an important determinant of the clearance of u-PA antigen and activity and reduction of flow in patients with heart failure will lead to an increase in plasma concentrations. High plasma concentrations of tcu-PA activity lead to increased systemic fibrinogenolysis. These results may be used to optimize saruplase treatment in patients with impaired cardiac function or after co-medication with drugs that affect liver blood flow.

15 Role of the low density lipoprotein receptorrelated protein (LRP) in the clearance and liver uptake of recombinant single chain urokinase-type plasminogen activator (rscu-PA) in rats

Urokinase-type plasminogen activator (u-PA) is used as a thrombolytic agent in the treatment of acute myocardial infarction. In vitro, E.coli produced rscu-PA is recognized by LRP on parenchyma! liver cells. In this study we investigated the role of LRP in the liver uptake and plasma clearance of rscu-PA. A preinjection of the LRP inhibitor GSTRAP reduced the maximal liver uptake of IMl-rscu-PA from 50 to 30 % of the injected dose and decreased the clearance 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 40 % reduction in liver uptake of rscu-PA by preinjection of GST-RAP was caused by a 91 % and 62 % reduction in the uptake by the parenchymal and Kupffer cells, respectively. Deletion of residue 11 -135 from rscu-PA (=delta125-rscu-PA) 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 the uptake of 60.2, 32.9 and 7.0 % of 125l-delta125-rscu-PA in the liver. Preinjection of GST-RAP completely reduced the liver uptake and reduced the clearance to 0.79 ml/min. Treatment of Kupffer cells with pl-PLC reduced the binding of rscu-PA by 40 %, indicating the involvement of u-PAR in the recognition of rscu-PA by the Kupffer cells. Our results demonstrate that in vivo LRP is responsible for the parenchymal liver cell mediated uptake of rscu-PA and for 60 % of the Kupffer cell interaction. It is also shown that u-PAR is involved in the Kupffer cell recognition of rscu-PA.

Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of lipoprotein(a) on thrombolysis

Lipoprotein(a) (Lp(a)) and plasminogen share a high degree of structural homology. Therefore it has been suggested that elevated levels of Lp(a) may inhibit the profibrinolytic activity at the cell surface and increase the risk of thrombosis by competitive inhibition of plasminogen. In the present study we evaluated whether high levels of Lp(a) affect thrombolytic therapy in patients with acute myocardial infarction. Forty-one patients with acute myocardial infarction were treated with a combination of recombinant tissue-type plasminogen activator and human single-chain urokinase-type plasminogen activator. Coronary patency was assessed angiographically 90 min after initiation of treatment. Thrombolysis was successful in 30 and unsuccessful in 11 patients. Patients with high Lp(a) levels (> 25 mg/dl) ( n = 9) responded equally well to thrombolytic therapy (8 of 9, patency 89%) as did patients with normal or low levels of Lp(a) (22 of 32, patency 70%, difference P > 0.1). The results demonstrate that high levels of Lp(a) do not influence thrombolysis in patients with acute myocardial infarction when low-dose pharmacologic concentrations of recombinant tissue-type plasminogen activator and human single chain urokinase-type plasminogen activator are applied in combination.

Nerve growth factor-gamma activates soluble and receptor-bound single chain urokinase-type plasminogen activator

Nerve growth factor-gamma (NGF-gamma) is a serine proteinase which reversibly associates with the well characterized neurotrophin NGF-beta. In this study, we demonstrated that NGF-gamma cleaves recombinant single chain urokinase-type plasminogen activator (scu-PA), converting the zymogen into a two-chain form (tcu-PA). The apparent masses of the two u-PA chains were 33 and 22 kDa, as determined by SDS-polyacrylamide gel electrophoresis (PAGE). There was no evidence for secondary cleavage sites or further digestion of tcu-PA by NGF-gamma, even when conversion of scu-PA was complete. The NH2-terminal sequence of the 33-kDa band was Ile-Ile-Gly-Gly-Glu, indicating that NGF-gamma cleaved scu-PA at Lys158-Ile159, the plasmin cleavage site. Cleavage of scu-PA by NGF-gamma resulted in scu-PA activation. The kcat and Km for this reaction, as determined in a continuous assay with the tcu-PA-specific substrate L-pyroglutamyl-glycyl-arginine-p-nitroanilide hydrochloride (S-2444), were (4.1 +/- 0.6) x 10(-2) s-1 and 2.3 +/- 0.4 microM, respectively. The catalytic efficiency (kcat/Km) for scu-PA activation by NGF-gamma was 1.3 x 10(4) M-1 s-1, compared with 6.2 x 10(5) M-1 s-1 for the activation of scu-PA by plasmin. NGF-gamma-cleaved scu-PA which was bound to receptors on U937 monocytoid cells. The apparent masses of the resulting u-PA cleavage products were identical to those generated in solution as determined by SDS-PAGE. Cell-associated scu-PA was activated by NGF-gamma, as determined by the generation of activity against the tcu-PA-specific fluorogenic substrate, glutamyl-glycyl-arginine-7-amino-4-methyl coumarin. By activating scu-PA, NGF-gamma may initiate the u-PA-dependent cell-surface proteinase cascade and support NGF-beta activities which involve cellular migration and/or extracellular matrix remodeling.

Time course of left ventricular function and coronary patency after saruplase vs streptokinase in acute myocardial infarction

As part of a prospective randomized double-blind trial (PRIMI) to study the early patency rate of the infarct-related artery after saruplase (INN for recombinant unglycosylated full-length human single-chain urokinase-type plasminogen activator) vs streptokinase in a subgroup of patients, left ventricular function was compared between both treatment groups at 90 min and 24 h after thrombolysis and at discharge, and ventricular function was related to the coronary perfusion grade. Despite a higher patency rate in the saruplase group 60 min after initiating thrombolysis, neither global ejection fraction nor hypokinesia at the infarct site were significantly different between the treatment groups at any of the three time points when function was measured. Hypokinesia at the infarct site remained almost equally severe throughout the study in patients with perfusion grade O, I, and II, and was consistently significantly milder in patients with perfusion grade III. In contrast, in patients with perfusion grade II there was a significant drop in hyperkinesia at the opposite wall at 24 h after thrombolysis and before discharge despite unchanged wall motion at the infarct site. Although patients treated with saruplase had a higher patency rate in the infarct related vessel shortly after the start of thrombolysis, no difference was found in left ventricular function compared to patients treated with streptokinase. Complete reperfusion (TIMI grade III) seems to be a prerequisite for left ventricular function recovery after thrombolysis, whereas only an occluded vessel (TIMI grade O and I) seems to be related to a longer lasting hyperkinesia at the opposite wall.

Differential detection of recombinant single chain urokinase-type plasminogen activator (rec.scu-PA) from its two chain derivative (rec.tcu-PA) in plasma

Differential detection of recombinant single chain urokinase-type plasminogen activator (rec.scu-PA) from its two chain derivative (rec.tcu-PA) in plasma

Comparison of intravenous bolus injection or continuous infusion of recombinant single chain urokinase-type plasminogen activator (saruplase) for thrombolysis. A canine model of combined coronary arterial and femoral venous thrombosis

The thrombolytic efficacy of recombinant unglycosylated full length single chain urokinase-type plasminogen activator (rscu-PA, saruplase), applied either as single intravenous bolus or as a continuous infusion over 60 minutes, was studied in 5 randomized blinded groups of 5 dogs with combined copper coil induced coronary artery thrombosis and 125I-fibrin labeled femoral vein clots. Infusion of 1 mg/kg recu-PA (group I) induced coronary recanalization in 4 of 5 dogs and 98 +/- 1% (mean +/- SEM) venous clot lysis. Bolus injection of 1 mg/kg recu-PA (group II) caused reflow in 3 of 5 dogs and 88 +/- 5 percent venous clot lysis. Infusion of 0.5 mg/kg rescu-PA (group III) achieved reflow in 3 of 5 dogs and 52 +/- 6% venous clot lysis. Bolus injection of 0.5 mg/kg rscu-PA (group IV) induced reflow in 4 of 5 dogs and 48 +/- 12% venous clot lysis. Placebo infusion (group V) was associated with late recanalization in 1 of 5 dogs and 18 +/- 8% venous clot lysis. Coronary artery reocclusion after reflow was not observed in groups I and II, but occurred in 2 of 3 animals in group III and in 3 of 4 animals in group IV (P = .02). The time to reflow in responsive animals was 22 +/- 5 minutes with infusion of 0.5 or 1 mg/kg rscu-PA and 14 +/- 1 minute with bolus injection of 0.5 or 1 mg/kg (P = .14). Depletion of fibrinogen and alpha 2-antiplasmin to less than 25% of baseline levels was observed in the 5 dogs given 1 mg/kg rscu-PA by bolus and in 3 of the 5 dogs given 1 mg/kg rscu-PA via infusion, but in none of the dogs that received 0.5 mg/kg rscu-PA (P less than .001). Plasma clearance rates were 170 +/- 44 and 230 +/- 30 mL/minute after bolus injection and 190 +/- 47 and 310 +/- 56 mL/minute during infusion of rscu-PA for the 1 mg/kg and 0.5 mg/kg doses respectively. Thus, intravenous bolus injection of rscu-PA (saruplase) appears to be equipotent to an infusion over 60 minutes for both coronary and venous thrombolysis. This animal model of combined arterial and venous thrombolysis may be useful for the evaluation of new thrombolytic strategies.

Comparison of Intravenous Bolus Injection or Continuous Infusion of Recombinant Single Chain Urokinase-Type Plasminogen Activator (Saruplase) for Thrombolysis. A Canine Model of Combined Coronary Arterial and Femoral Venous Thrombosis

Abstract The thrombolytic efficacy of recombinant unglycosylated full length single chain urokinase-type plasminogen activator (rscu-PA, saruplase), applied either as single intravenous bolus or as a continuous infusion over 60 minutes, was studied in 5 randomized blinded groups of 5 dogs with combined copper coil induced coronary artery thrombosis and 125I- fibrin labeled femoral vein clots. Infusion of 1 mg/kg recu-PA (group I) induced coronary recanalization in 4 of 5 dogs and 98 +/- 1% (mean +/- SEM) venous clot lysis. Bolus injection of 1 mg/kg recu-PA (group II) caused reflow in 3 of 5 dogs and 88 +/- 5 percent venous clot lysis. Infusion of 0.5 mg/kg rescu-PA (group III) achieved reflow in 3 of 5 dogs and 52 +/- 6% venous clot lysis. Bolus injection of 0.5 mg/kg rscu-PA (group IV) induced reflow in 4 of 5 dogs and 48 +/- 12% venous clot lysis. Placebo infusion (group V) was associated with late recanalization in 1 of 5 dogs and 18 +/- 8% venous clot lysis. Coronary artery reocclusion after reflow was not observed in groups I and II, but occurred in 2 of 3 animals in group III and in 3 of 4 animals in group IV (P = .02). The time to reflow in responsive animals was 22 +/- 5 minutes with infusion of 0.5 or 1 mg/kg rscu-PA and 14 +/- 1 minute with bolus injection of 0.5 or 1 mg/kg (P = .14). Depletion of fibrinogen and alpha 2-antiplasmin to less than 25% of baseline levels was observed in the 5 dogs given 1 mg/kg rscu-PA by bolus and in 3 of the 5 dogs given 1 mg/kg rscu-PA via infusion, but in none of the dogs that received 0.5 mg/kg rscu-PA (P less than .001). Plasma clearance rates were 170 +/- 44 and 230 +/- 30 mL/minute after bolus injection and 190 +/- 47 and 310 +/- 56 mL/minute during infusion of rscu-PA for the 1 mg/kg and 0.5 mg/kg doses respectively. Thus, intravenous bolus injection of rscu-PA (saruplase) appears to be equipotent to an infusion over 60 minutes for both coronary and venous thrombolysis. This animal model of combined arterial and venous thrombolysis may be useful for the evaluation of new thrombolytic strategies.

Pharmacokinetic properties of mutants of recombinant single chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys 158, Ile 159 and Ile 160

Single chain urokinase-type plasminogen activator (scu-PA, prourokinase) is converted to two chain urokinase-type plasminogen activator (tcu-PA) by hydrolysis of the Lys 158-Ile 159 peptide bond with plasmin. In this study we have evaluated the pharmacokinetic properties in rabbits of rscu-PAs with specific amino acid substitutions at the cleavage site, including rscu-PA-Arg 158 (rscu-PA with Lys 158 substituted with Arg), rscu-PA-Gly 158 (Lys 158 substituted with Gly), rscu-PA-Glu 158 (Lys 158 substituted with Glu), rscu-PAG1y 159 (Ile 159 substituted with Gly), rscu-PA-Pro 159 (Ile 159 substituted with Pro) and rscu-PA-Gly 158,Lys 160 (Lys 158 substituted with Gly and Ile 160 with Lys). The mutant or wild type rscu-PAs (at doses between 1.6 and 4.0 mg/kg) were infused intravenously over 4 h in groups of 2 to 5 rabbits, and the initial disposition rate of rscu-PA related antigen from plasma was monitored over a 30 min period after the end of the infusion. The disappearance rate of antigen from plasma was biphasic for rscu-PA and for the mutants and could be described by a sum of two exponential terms with an a half-life of 2.0 to 3.0 min (2.6 min for rscu-PA) and a β half-life of 11 to 14 min (11 min for rscu-PA). The plasma clearance rate ranged between 21 and 31 ml per min (30 ml per min for rscu-PA). These results indicate that the amino acid sequence around the plasmin cleavage site in scu-PA is not a significant determinant for its in vivo pharmacokinetic properties and that scu-PA is not cleared via activation to tcu-PA and neutralisation by protease inhibitors.

Characterization of Thrombin- and Plasmin-Resistant Mutants of Recombinant Human Single Chain Urokinase–Type Plasminogen Activator1

Recombinant human single-chain urokinase-type plasminogen activator (suc-PA) (SM0: wild type) and its variants resistant to plasmin and/or thrombin (SM1: Lys135 to Gln; SM3: Phe157 to Asp; and SM4: Lys135 to Gln and Phe157 to Asp) have been constructed by site-directed mutagenesis with the aim of producing more efficient thrombolytic agents [Miyake, T. et al. (1988) J. Biochem. 104, 643-647]. In the present study, we characterized the recombinant variant scu-PAs expressed in Escherichia coli. They appeared to have structural integrity because their heat-stabilities, immunological reactivities, and circular dichroism spectra were essentially identical to those of each other and of native scu-PA (nscu-PA). In the presence of thrombin, SM3 and SM4 showed efficient clot lysis by all of the assays used, compared with SM0, SM1, and nscu-PA. While in the absence of thrombin, when measured by a fibrin plate method in a purified system, SM3 and SM4 had lower specific activities than SM0, SM1, and nscu-PA, because of their catalytic constants for conversion to the two-chain form (tcu-PA) by plasmin are lower. However, SM4 lysed clots as efficiently as SM0 in plasma by retaining the single-chain form, whereas SM0 was partly converted to the two-chain form.

A hamster pulmonary embolism model for the evaluation of the thrombolytic and pharmacokinetic properties of thrombolytic agents

A pulmonary embolus model in adult hamsters (body weight less than 100 g) was developed for the quantitative evaluation of the thrombolytic and pharmacokinetic properties of plasminogen activators. A clot was made from a mixture of 125I-labeled fibrinogen, citrated human plasma, thrombin and CaCl 2, and was injected into the jugular vein of heparinized hamsters. The time course of in vivo clot lysis was continuously measured with an external gamma counter, placed over the thorax. The extent of thrombolysis within 90 min was determined both as the difference between the initial and final external gamma count and as the difference between the radioactivity injected in the jugular vein and that recovered in the heart and lungs. In control animals infused with solvent for 60 min, spontaneous progressive clot lysis occurred resulting in 25 ± 1 % lysis (mean ± SEM; n = 87) by ex vivo isotope recovery and in 15 ± 2% lysis (n = 31) by external gamma counting. Recombinant tissue-type plasminogen activator (Activase®), recombinant single chain urokinase-type plasminogen activator (rscu-PA) and urokinase (Winkinase®), infused intravenously over 60 min, caused dose-dependent progressive thrombolysis. With 1.0 mgAkg Activase®, lysis was 91 ± 3% (n = 4) by ex vivo isotope recovery and 61 ± 6% (n = 4) by external gamma counting. With streptokinase (Streptase®) infusion or with bolus injection of anisoylated plasminogen streptokinase activator complex (Eminase®), dose-response curves were not linear. Fibrinogen and α 2-antiplasmin levels at the end of the experiments were unchanged with Activase® and rscu-PA, and moderately decreased with Winkinase®, Streptase® and Eminase®. Linear regression analysis of all results of the percent lysis at 90 min, determined by ex vivo isotope recovery (IR) and by external gamma counting (EC) (n = 101) yielded the equation (IR) = 1.08 (EC) + 21 with r = 0.87. The failure rate of the thrombolysis experiments, due to technical reasons or to death of the animals, was less than 5 %. After i.v. bolus injection of plasminogen activators in hamsters, the pharmacokinetic properties were determined from the disappearance rate of antigen from plasma, which were for Activase®, i.e., an initial half-life of 1.5 min and a plasma clearance of 2.8 ml/min. The hamster pulmonary embolism model is simple, reproducible, quantitative, and allows for one investigator to perform up to 10 experiments per day. This model may be useful for the evaluation of the potency, fibrin-specificity and pharmacokinetics of thrombolytic agents.

Characterisation of a chemical conjugate between a low molecular weight form of recombinant single chain urokinase-type plasminogen activator (comprising Leu 144 through Leu 411) and F(ab′) 2-fragments of a fibrin D-dimer-specific monoclonal antibody

Summary A recombinant low M r derivative of single chain urokinase-type plasminogen activator consisting of amino acids Leu 144 through Leu 411 (rscu-PA-32k) was conjugated to F(ab′) 2 -fragments of a murine monoclonal antibody (MA-15C5), directed against fragment D-dimer of crosslinked human fibrin, by means of the crosslinking reagent N-succinimidyl 3-(2-pyridyldithio) propionate (rscu-PA/MA-15C5-F(ab′) 2 ). The conjugate was purified by immunoadsorption on a monoclonal antibody directed against urokinase followd by affinity chromatography on insolubilised fibrin fragment D-dimer. Recoveries were 26±3.1% for rscu-PA-32k and 27±2.7% for total protein (mean±SD, n=3). The purified conjugate contained 1.0±0.03 mol of F(ab′) 2 -fragment per mol of rscu-PA-32k. The specific amidolytic activity on a chromogenic substrate for urokinase was 2 and rscu-PA-32k, whereas the specific fibrinolytic activity on fibrin plates was 29000 iu/mg u-PA antigen in the conjugate as compared to 53000 iu/mg u-PA antigen in rscu-PA-32k. Activation of plasminogen by rscu-PA-32k/MA-15C5-F(ab′) 2 obeyed Michaelis-Menten kinetics with a K m value of 0.71±0.26 μM and a k 2 value of 0.00023±0.00010s −1 (n=3), as compared to K m =1.6 μM and k 2 =0.00028s −1 for rscu-PA-32k. Both rscu-PA-32k/MA-15C5-F(ab′) 2 and rscu-PA-32k induced dose- and time-dependent lysis of a 125 I-fibrin labelled human plasma clot immersed in citrated human plasma. Fifty per cent lysis in 2h was obtained with a concentration of 6.4 μg/ml (n=4) for rscu-PA-32k, whereas only 1.2μg u-PA/ml (n=4) was required of rscu-PA-32k/MA-15C5-F(ab′) 2 . We conclude that low M r scu-PA can be targetted to a fibrin clot with the F(ab′) 2 -fragment of a fibrin-specific antibody, resulting in a 5-fold increased thrombolytic potency, which is comparable to that of intact scu-PA coupled to intact antibody. These fragments may serve as preferred moieties for the construction of recombinant chimaeric scu-PA/antibody proteins for fibrin-targetted thrombolytic therapy.

Characterisation of conjugates of thrombin-treated single chain urokinase-type plasminogen activator with a monoclonal antibody specific for crosslinked fibrin

Summary rscu-PA/MA-15C5, a chemical conjugate of recombinant single chain urokinase-type plasminogen activator (rscu-PA) with a murine monoclonal antibody (MA-15C5) directed against fragment D-dimer of human crosslinked fibrin, has a 6.4-fold higher fibrinolytic potency than unconjugated rscu-PA towards a fibrin clot in a plasma milieu in vitro (Dewerchin et al). In the present study we have evaluated the thrombin-derived two chain form of this conjugate. Treatment of rscu-PA/MA-15C5 or of rscu-PA with thrombin caused a conversion of the single chain molecules to two chain derivatives (rtcu-PA/MA-15C5/T or rtcu-PA/T respectively) with a very low specific amidolytic activity ( In an in vitro sytem composed of a 125I-fibrin labelled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained with rtcu-PA/MA-15C5/T; 50% lysis in 2 h was reached at a concentration of 1.1 μg u-PA/ml without associated plasma fibrinogen breakdown. In contrast, at concentrations up to 50 μg/ml, no fibrinolytic activity was observed with rtcu-PA/T or with a thrombin-treated conjugate made with rscu-PA and an irrelevant murine monoclonal antibody. We conclude that the fibrinolytic potency of rtcu-PA/MA-15C5/T is at least 50-fold higher than that of its unconjugated counterpart. This may be explained by targetting of the conjugate to the fibrin clot and reactivation by plasmin at the fibrin surface.

Spin Filter Perfusion System for High Density Cell Culture: Production of Recombinant Urinary Type Plasminogen Activator in CHO Cells

We have used a 20 liter stirred tank fermentor, equipped with a 127 mesh ethylene-tetrafluoroethylene rotating screen for cell recycle, for the continuous production of recombinant single chain urokinase-type plasminogen activator (rscu-PA) from Chinese hamster ovary (CHO) cells. Viable cell densities between 60 and 74 million per ml were maintained at medium perfusion rates of 3.0 to 4.0 fermentor volumes per day. Cells were retained by the 120 micron nominal opening filter through the formation of "clumped" cell aggregates of 200 to 600 microns in size, which did not foul the filter. In 31 days of culture, a total of 51 grams of rscu-PA were produced in 1,000 liters of medium. The rscu-PA produced over the course of this continuous culture was purified and characterized both in vitro and in vivo and shown to be comparable to natural scu-PA produced from the transformed human kidney cell line, TCL-598.

Biochemical properties of conjugates of urokinase‐type plasminogen activator with a monoclonal antibody specific for cross‐linked fibrin

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.

Thrombolytic and pharmacokinetic properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for crosslinked fibrin

A primarily 1:1 stoichiometric conjugate between a murine monoclonal antibody directed against fragment D-dimer of crosslinked human fibrin (MA-15C5) and recombinant single chain urokinase-type plasminogen activator (rscu-PA) was produced by chemical crosslinking with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (Dewerchin et al, in press). The thrombolytic and pharmacokinetic properties of this conjugate (rscu-PA/MA-15C5) and of its two chain derivatives obtained by digestion with thrombin (rtcu-PA/MA-15C5/T) or plasmin (rtcu-PA/MA-15C5/P) were compared with those of their unconjugated counterparts. Continuous intravenous infusion of these compounds over 4 h in rabbits with a 0.2 ml t 2 s1-fibrin labelled human plasma clot in the jugular vein resulted in linear dose-response curves of clot lysis. The thrombolytic potency of rscu-PA/MA-15C5 was 8 to 9-fold higher (50% lysis with 0.3 mg/kg) than that of rscu-PA (50% lysis with 2.6 mg/kg), while that of rtcu-PA/MA-15C5/P (25% lysis with 0.3 mg/kg) was 4-fold higher than that of rtcu-PA/P 25% lysis with 1.2 mg/kg). Both rtcu-PA/MA-15C5/T and rtcu-PA/T, which are inactive towards purified plasminogen, had significant thrombolytic potency in vivo, causing 15–26% lysis at doses of 1.0–2.0 mg/kg. Systemic fibrinogen degradation during infusion was only observed with rtcu-PA/P but not with rtcu-PA/MA-15C5/P, which is suggestive of an increased fibrin specificity of the conjugate. The enhanced thrombolytic potency of the conjugates as compared to their unconjugated counterparts was not observed when jugular vein clots were produced with rabbit plasma or when u-PA was conjugated with an irrelevant antibody. This suggests that the increased potency is due to specific antibody targetting of u-PA to human fibrin. After intravenous bolus injection in rabbits, u-PA related antigen disappeared from plasma in a biphasic manner with t 1 2 of 2.0 and 7.0 min for rscu-PA and 12 and 90 min for rscu-PA/MA-15C5. Plasma clearance rates were 14 ± 1.1 ml/min for rscu-PA and 3.4 ± 0.56 ml/min for rscu-PA/MA-15C5. In conclusion, conjugation of rscu-PA with MA-15C5 increases its in vivo specific thrombolytic potency about 8-fold and decreases its plasma clearance rate about 4-fold.

Additive fibrinolysis by recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase type plasminogen activator (r-scu-PA) in rabbit pulmonary thrombosis

The mode of interaction between recombinant tissue-type plasminogen activator (r-t-PA) and recombinant single-chain urokinase-type plasminogen activator (r-scu-PA) has been investigated in in vivo experiments. 125J-fibrin-labeled clots were embolized via a jugular vein into the lungs of anesthetized rabbits. In saline-treated control rabbits the spontaneous lysis, shown as decrease in radioactivity of the retrieved clots, amounted to 10.4 ± 1.4 % at 255 min after the pulmonary embolization. r-t-PA (0.464 - 4.64 μg/kg min) and r-scu-PA (4.64 - 46.4 μm/kg - min), infused for 60 min, produced dose-dependent lytic effects to a similar extent (maximum lysis rate 53.9 ± 5.8 and 55.4 ± 7.2 %, resp.). When various ratios of submaximal doses of r-t-PA and r-scu-PA were combined the lytic effects of these combinations were not higher than the calculated summation of the lysis rates by the single components. The fractional dose-response curves of r-t-PA and r-scu-PA and the combination of them, fitted by linear regression analysis, are overlaying each other. The results indicate that r-t-PA and r-scu-PA produce in vivo lysis in rabbits with pulmonary embolized clots in a purely additive manner.

Production and characterisation of human recombinant single chain urokinase-type plasminogen activator from mouse cells

Human chromosomal DNA coding for single chain urokinase-type plasminogen activator (scu-PA) has been inserted into a vector and expressed in LB6 cells. The recombinant clone termed 1C9 is stable and produces scu-PA at about 0.8 mg/I/day in a bioreactor. The scu-PA produced is human by amino- and carboxy-terminal analysis and immunoblotting. The recombinant scu-PA is glycosylated and fully activatable by plasmin.

Effect of Retraction on the Lysis of Human Clots with Fibrin Specific and Non-Fibrin Specific Plasminogen Activators

The effect of the serum content of human clots on their sensitivity to lysis with plasminogen activators was studied in a system composed of 125I-fibrin labeled clots immersed in buffer or in citrated plasma. The effect was studied with plasma clots before or after mechanical compression and with whole blood clots before or after retraction, using either the fibrin specific plasminogen activators recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA), and the non-fibrin specific activators recombinant two chain urokinase-type plasminogen activator (rtcu-PA), or streptokinase (SK). In a buffer milieu, all plasminogen activators had a similar fibrinolytic potency towards serum-rich clots (non-compressed plasma clots or non-retracted blood clots): 50% clot lysis in 4 h required 50 to 85 ng plasminogen activator per ml. Serum-poor clots (compressed plasma clots or retracted blood clots) were resistant to lysis in a buffer milieu but became sensitive to lysis following preincubation in plasma for 48 h. These findings indicate that plasma proteins entrapped in clots contribute significantly to their sensitivity to lysis and suggest that the amount of bound or entrapped plasminogen may be a limiting factor. In a plasma milieu, all plasminogen activators lysed serum-rich plasma or blood clots, albeit at higher concentrations (3 to 40 times higher than in the buffer milieu) and with different efficiencies: 50% clot lysis in 4 h required approximately 600 ng/ml of rtcu-PA but 1,500 to 2,000 ng/ml of rscu-PA.(ABSTRACT TRUNCATED AT 250 WORDS)