Lucien Nelles

Thrombolytic and pharmacokinetic properties of chimeric tissue-type and urokinase-type plasminogen activators.

BackgroundChimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, partial fibrin-binding properties of t-PA, and thrombolytic properties in animal models comparable with but not superior to those of single-chain u-PA (scu-PA). Deletion of the finger and growth factor domains (t-PA-AFE/scu-PA-e) in such chimeras further reduces their affinity for fibrin. Methods and ResultsA detailed investigation of the thrombolytic potency and the pharmacokinetics of t-PA and u-PA chimeras was performed in quantitative animal models for thrombolysis. In hamsters with pulmonary embolism, in rabbits with jugular vein thrombosis, and in baboons with femoral vein thrombosis, the thrombolytic potency (percent lysis per milligram of compound administered per kilogram of body weight) of t-PA-AFE/scu-PA-e was significantly higher than that of recombinant scu-PA (rscu-PA, Saruplase) as shown by a maximal rate of 720 ± 170% versus 45 ± 5% lysis per milligram of compound per kilogram of body weight (mean ± SEM, p < 0.01) in hamsters, 210 ± 18% versus 49 ± 3% lysis per milligram of compound per kilogram of body weight (mean ± SEM, p < 0.01) in rabbits, and 310 ± 73% versus 90 ± 0.3% lysis per milligram of compound per kilogram of body weight (p < 0.01) in baboons. However, the specific thrombolytic activity (percent lysis per microgram per milliliter steadystate plasma antigen level) of t-PA-AFE/scu-PA-e was not significantly different from that of rscu-PA in hamsters (210 ± 57% versus 160 ± 27% lysis per microgram per milliliter antigen level) and was lower than that of rscu-PA in rabbits (37 ± 4% versus 130 ± 5% lysis per microgram per milliliter antigen level; p < 0.01). In dogs with a combined femoral vein blood clot and a platelet-rich femoral arterial eversion graft thrombosis, 0.25 mg/kg body wt bolus injections of t-PA-AFE/scu-PA-e produced significantly more venous clot lysis (90 + 5%, n = 10) than 0.25 mg/kg rscu-PA (26 + 3%, n = 10) (p < 0.001) and, at the arterial side, more frequent (10 of 10 dogs versus three of 10 dogs) and more persistent (six of 10 dogs versus none of 10 dogs) recanalization (p = 0.002). After bolus injection in hamsters, rabbits, or baboons, t-PA-AFE/scu-PA-e had a fourfold to sixfold longer initial half-life than rscu-PA and a slower plasma clearance of sixfold in hamsters, 10-fold in rabbits, and more than 10-fold in baboons. ConclusionsThese results indicate that t-PA-AFE/scu-PA-e has a markedly enhanced thrombolytic potency toward venous and arterial thrombi caused by a delayed in vivo clearance with relatively maintained specific thrombolytic activity. These properties suggest that the chimera may be clinically useful for thrombolytic therapy by bolus administration in patients with thromboembolic disease.

Induction of t-pa synthesis with intravenous bolus injection of vitamin-a palmitate in vitamin-a deficient rats

Abstract Retinoic acid and vitamin A palmitate induce tissue-type plasminogen activator (t-PA) synthesis in cultured human umbilical vein endothelial cells (HUVEC) in vitro without alteration of plasminogen activator inhibitor-] (PAI-1) synthesis. Therefore the effect of intravenous bolus injection of water-miscible vitamin A palmitate on the blood fibrinolytic system was investigated in normal and vitamin A deficient rats. In 5 normal rats, injection of 200000 IU/kg of vitamin A palmitate did not induce a significant increase in euglobulin fibrinolytic activity, t-PA antigen and PAI activity in plasma nor of t-PA and PAI-1 mRNA in the heart within 240 min after injection. In groups of 3–6 vitamin A deficient rats, injection of 200000IU/kg of vitamin A palmitate induced a significant increase in t-PA antigen in plasma (1.9-fold after 60 min and 2.9-fold after 120 min), but no significant increase in plasma euglobulin fibrinolytic activity. A 2-fold increase in PAI activity was observed 90 min after injection of both vitamin A palmitate as well as of its solvent, suggesting that this induction was non-specific. The increase in t-PA antigen coincided with a significant increase in t-PA mRNA in heart tissue: 2.3-fold after 60 min and 4.3-fold after 120 min. PAI-1 mRNA in heart tissue increased 3.6-fold 90 min after injection with vitamin A palmitate but, surprisingly, not after injection of solvent. It is concluded that intravenous bolus injection of vitamin A palmitate induces a specific increase of t-PA mRNA in the heart and of t-PA antigen secretion in plasma of vitamin A deficient rats.

Biochemical properties of recombinant mutants of nonglycosylated single chain urokinase-type plasminogen activator

The role of glycosylation on the enzymatic properties of single chain urokinase-type plasminogen activator (scu-PA) was investigated by site-specific mutagenesis of the glycosylated Asn-302 residu to Gln. In addition, the role of the NH2-terminal polypeptide chain and of the Cys-148 to Cys-279 interchain disulphide bond on the activity of non-glycosylated scu-PA was investigated. Therefore, variants of recombinant scu-PA (rscu-PA) were produced by transfecting Chinese hamster ovary cells with cDNA encoding rscu-PA N302Q (rscu-PA with Asn-302 to Gln mutation), rscu-PA C279A,N302Q (rscu-PA with Cys-279 to Ala and Asn-302 to Gln mutations) or rscu-PA del(N2-F157)C279A,N302Q (rscu-PA C279A,N302Q with deletion of Asn-2 through Phe-157). These mutants were purified to homogeneity from conditioned cell culture medium and were obtained essentially as single chain molecules with specific activities on fibrin plates of (mean +/- S.E.; n = 6) 45,000 +/- 5000. IU/mg, 19,000 +/- 800 IU/mg and < or = 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64,000 +/- 2600 IU/mg for wild-type rscu-PA obtained in the same expression system. Plasmin quantitatively converts rscu-PA N302Q and rscu-PA C279A,N302Q to amidolytically active two-chain derivatives with a specific activity of 56,000 IU/mg and 32,000 IU/mg, respectively, as compared to 75,000 IU/mg for wild-type rscu-PA. Plasminogen activation as a function of time was comparable for rscu-PA N302Q and wild-type rscu-PA, and somewhat slower for rscu-PA C279A,N302Q. In a human plasma milieu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 micrograms/ml rscu-PA N302Q and 6.0 micrograms/ml rscu-PA C279A,N302Q, as compared to 3.2 micrograms/ml wild-type rscu-PA. In contrast, rscu-PA del(N2-F157)C279A,N302Q was not converted to an amidolytically active two chain derivative by plasmin, and did not induce significant plasminogen activation in purified systems or clot lysis in a human plasma milieu. Following bolus injections in hamsters, the initial half-lives (1.8-2.6 min) and the plasma clearances (0.6-1.5 ml min-1) were comparable for wild-type rscu-PA and for the three rscu-PA mutants. These results suggest that the fibrinolytic activity in a plasma milieu in vitro and the in vivo turnover of rscu-PA are not markedly affected by the absence of carbohydrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Essential validation of gene trap mouse ES cell lines: a test case with the gene Ttrap

Gene trapping in mouse embryonic stem (ES) cells enables near-saturation vector-based insertional mutagenesis across the genome of this model organism. About 135,000 trapped ES cell lines are made available to the scientific community by the International Gene Trap Consortium (IGTC; www.genetrap.org). A search of one of its databases identified an ES cell line (RRS512) with a betaGeo-based gene trap (gt) vector insertion in intron 5 of Ttrap, a gene that encodes an intracellular signalling protein, which is implicated in gastrulation movement and left-right asymmetry in zebrafish embryos. We have determined the exact gt insertion point in the mutant ES cell clone RRS512 and confirmed the production of a chimaeric transcript consisting of the upstream Ttrap exons and the gene trap vector encoded marker/selection fusion sequences. This ES cell line was used to generate heterozygous Ttrap mutant mice, which were further crossed to obtain Ttrap(gt/gt) mice. In contrast to Ttraps documented essential role during nodal and Smad3 controlled zebrafish early embryogenesis, Ttrap(gt/gt) mice were born with a normal Mendelian distribution. However, subsequent analysis of these Ttrap(gt/gt) mice has revealed a duplication of the wild-type Ttrap allele that was already present in the RRS512 cell line. Based on our detailed analysis presented here, we suggest an extensive procedure for the characterization of gene trap ES cell lines prior to generating gene trap mice with these.

Pharmacokinetic properties of mutants of recombinant single chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

Abstract Single chain urokinase-type plasminogen activator (scu-PA, prourokinase) is converted to two chain urokinase-type plasminogen activator (tcu-PA) by hydrolysis of the Lys158-Ile159 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-Arg158 (rscu-PA with Lys158 substituted with Arg), rscu-PA-Gly158 (Lys158 substituted with Gly), rscu-PA-Glu158 (Lys158 substituted with Glu), rscu-PAG1y159 (Ile159 substituted with Gly), rscu-PA-Pro159 (Ile159 substituted with Pro) and rscu-PA-Gly158,Lys160 (Lys158 substituted with Gly and Ile160 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.

Transfection of human endothelial cells with plasminogen activator inhibitor type-1 promoter/reporter gene fragments reveals phorbol ester induction of gene expression

Transfection of human endothelial cells with human plasminogen activator inhibitor type-1 promoter-reporter gene fragmentes reveals phorbol ester induciton of gene expression

Hormonal regulation of the expression of fibrinolytic components in HT1080 fibrosarcoma and endothelial cells

Summary The effects of the synthetic glucocorticoid dexamethasone (DEX) and of all-trans-retinoic acid (RA) on the expression of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and the plasminogen activator inhibitor (PAI)-1 were investigated in the HT1080 fibrosarcoma cell line and in human umbilical vein endothelial (HUVE) cells. DEX significantly reduced the u-PA steady state mRNA level in HT1080 cells to 0.41±0.08-fold control. It induced the t-PA transcript 2.2±0.36-fold and the 2.8 and 3.4 kb PAI-1 transcripts 2.5±0.13-fold and 2.8±0.48-fold, respectively. RA increased both PAI-1 transcripts to a minor but significant extent (1.3±0.03-fold and 1.2±0.03-fold), and the t-PA and u-PA transcripts more strongly (11±0.4-fold and 2.1±0.02-fold). When added together, DEX enhanced the induction of t-PA mRNA by RA to 34±1.5-fold, inhibited the RA-mediated induction of u-PA mRNA to 0.85±0.02-fold control, and further increased the levels of the 2.8 and 3.4 kb PAI-1 transcripts to 3.6±0.13-fold and 5.0±0.05-fold, respectively. The secretion of the corresponding antigens in the conditioned medium paralleled the changes in mRNA. The effects of DEX and RA were abolished by simultaneous treatment with cycloheximide, suggesting that they required protein synthesis. In HUVE cell culture only a minor effect on t-PA-related antigen secretion was observed when DEX was added alone (1.2±0.02-fold), but DEX increased the induction by RA from 6.2±0.1-fold to 9.6±0.15-fold. In contrast, DEX and RA, alone or in combination, had very little or no effect on the generation of PAI-1-related antigen (maximally 1.2±0.4-fold). It is concluded that the glucocorticoid and the RA signal transduction pathways can interact in a complex manner resulting in a differential regulation of the fibrinolytic parameters.

Characterisation of rt-PA I276G, a recombinant human tissue-type plasminogen activator mutant with altered plasmin cleavage site

Abstract rt-PA I276G, a mutant of recombinant human tissue-type plasminogen activator (rt-PA) with altered plasmin cleavage site, was obtained by site-directed mutagenesis of Ile 276 to Gly. rt-PA I276G, purified to homogeneity from the conditioned medium of transfected Chinese hamster ovary cells, was obtained as a single chain molecule, which was quantitatively converted to a two chain moiety by cleavage of the Lys 277 -Gly 278 peptide bond with plasmin. The specific activities on fibrin films of the single chain and two chain forms of rt-PA I276G were 8900 and 15000 IU/mg respectively, as compared to 210000 and 130000 IU/mg respectively for the single chain and two chain forms of wild-type rt-PA obtained in the same expression system. The amidolytic activity of the rt-PA I276G moieties was 3- to 5-fold lower and their catalytic efficiency for plasminogen activation 20- to 50-fold lower than those of the wild-type rt-PA moieties. Both single chain and two chain rt-PA I276G induced concentration-dependent lysis of a 125 I-fibrin labelled plasma clot submersed in human plasma; equi-effective concentrations (causing 50% clot lysis in 2 h) were 0.55 and 1.40 μg/ml respectively, as compared to 0.36 and 0.60 μg/ml for single chain or two chain wild-type rt-PA respectively. Continuous infusion over 60 min of single chain rt-PA 1276G or wild-type rt-PA in hamsters with a pulmonary embolus, revealed an approximately 2-fold lower thrombolytic potency (clot lysis versus dose) for the mutant, but a comparable specific thrombolytic activity (clot lysis versus steady state plasma antigen level). It is concluded that replacement of Ile 276 by Gly in single chain rt-PA significantly reduces the intrinsic enzymatic activity in purified systems. In a plasma milieu in the presence of fibrin the fibrinolytic potential of the mutant is, however, only 2-fold lower than that of wild-type rt-PA obtained in the same expression system.