Chain Urokinase-type Plasminogen Activator Research Articles

Production of recombinant proteins in Chinese hamster ovary cells using a protein-free cell culture medium.

The growth-factor prototrophic Chinese hamster ovary (CHO) SSF3 cell line was previously adapted for growth in serum-free media. Here we present a newly designed medium which allows these cells to grow in the absence of any exogenously added growth factors. To investigate the capacity of CHO SSF3 cells for the efficient production of recombinant proteins in protein-free media, expression plasmids containing either a human single chain urokinase-type plasminogen activator (uPA)-encoding cDNA or a humanized immunoglobulin G (IgG) kappa light chain cDNA were introduced by transfection. The tryptophan synthase (trpB) gene of Escherichia coli was used as a dominantly acting selection marker allowing the cells to survive in a medium containing indole in place of tryptophan. Some of the clones obtained exhibited a stable uPA expression over a period of several months under selective conditions and the yields were up to 74 mg of uPA/l in a bioreactor and the productivity was around 40 mg/day per 10(9) cells. The yields of IgG light chains were up to 118 mg/l and the productivity was in the order of 56 mg/day per 10(9) cells in a bioreactor. These results demonstrate the potential of CHO SSF3 cells for the efficient production of recombinant proteins under protein-free conditions.

Heparin and heparan sulfate enhancement of the inhibitory activity of plasminogen activator inhibitor type 1 toward urokinase type plasminogen activator

To study effects of glycosaminoglycan on the interaction between two chain urokinase type plasminogen activator (tcu-PA) (EC 3.4.21.31) and plasminogen activator inhibitor type (PAI-1) the second order rate constant ( k 1) between high molecular weight tcu-PA and active recombinant prokaryotic PAI-1 (rpPAI-1) was determined employing a continuous method using chromogenic substrate S-2444 either in the presence or absence of various kinds of glycosaminoglycans. k 1 was (5.9 ± 1.6) · 10 6/mol per s in the absence of effector molecule, and following addition of heparin (1.0 U/ml) k 1 was enhanced to 3.22 ± 0.73) · 10 7. A significant enhancement of k 1 was also obtained by heparan sulfate (1.87 ± 0.25) · 10 7. Dermatan sulfate or chondroitin sulfate did not show a significant effect of k 1 although a slight decrease was obtained by mono-dextran sulfate (4.2 ± 1.2) · 10 6. The intrinsic fluorescence of rpPAI-1 was shown to be slightly increased following addition of heparin (1.49 ± 0.22%, n = 6), suggesting that heparin may enhance the inhibitory activity of PAI-1 toward tcy-PA both by a template mechanism and by a modification of PAI-1 structure.

Regions involved in binding of urokinase-type-1 inhibitor complex and pro-urokinase to the endocytic alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Evidence that the urokinase receptor protects pro-urokinase against binding to the endocytic receptor.

The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) binds several ligands, including complex between the two chain urokinase-type plasminogen activator (uPA) and type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of uPA and PAI-1 as well as Fab fragments of monoclonal antibodies with known epitopes to identify regions in the uPA.PAI-1 complex and in pro-uPA involved in binding to alpha 2MR/LRP.uPA.PAI-1 complex bound with high affinity (EC50 about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uPA bound with lower affinity (EC50 about 10 nM), and efficient binding to alpha 2MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex formation with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA.PAI-1 complex is internalized following interaction with alpha 2 MR/LRP (Nykjaer, A., Petersen, C. M., Møller, B., Jensen, P.H., Moestrup, S.K., Holtet, T.L., Etzerodt M., Thøgersen, H.C., Munch, M., Andreasen, P.A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombinant urokinase receptor blocked the binding of pro-uPA to alpha 2MR/LRP but caused only a slight reduction in the affinity for binding of uPA.PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against degradation via alpha 2MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uPA.PAI-1 complex for binding to alpha 2MR/LRP remains sufficient to allow rapid internalization and degradation.

Single chain urokinase type plasminogen activator (SCU-PA) complexes with and is inhibited by plasminogen activator inhibitor type 1 (PAI-1)

Single chain urokinase type plasminogen activator (SCU-PA) complexes with and is inhibited by plasminogen activator inhibitor type 1 (PAI-1)

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.

The role of urokinase generation during clot lysis by pro-urokinase in a plasma milieu

The conversion of single chain urokinase-type plasminogen activator (scu-PA) to two chain urokinase-type plasminogen activator (tcu-PA) during lysis of normal and tissue-type plasminogen activator (t-PA) pre-treated clots by scu-PA was examined using a combined ELISA assay system which selectively measures either scu-PA or tcu-PA in plasma. At fibrin-specific doses, conversion did not exceed 20% against normal clots. Conversion was found to be dose-dependent and correlated with fibrinolysic efficiency. Against t-PA pre-treated clots the efficacy of lysis by scu-PA, as measured by the length of the lag-phase and maximum rate of lysis, was increased and was accompanied by 30% conversion of scu-PA to tcu-PA. An infusion of tcu-PA equivalent to that generated from scu-PA in the above experiments induced 70% less fibrinolysis and 25% more fibrinogenolysis. These findings indicate that local activation of scu-PA on the fibrin surface induces far more lysis and less non-specific effects than systemic conversion to tcu-PA, and that local activation is well correlated with scu-PA lytic efficiency.

A study of the activation of fibrin-bound plasminogen by tissue-type plasminogen activator, single chain urokinase and sequential combinations of the activators

The efficacy of tissue-type plasminogen activator (t-PA) and single chain urokinase-type plasminogen activator (scu-PA) to activate fibrin-bound plasminogen was determined. With the use of a solid-phase model of intact and plasmin-degraded fibrin, the initial rate, Vi (fmol of plasmin/min), of plasmin generation by t-PA, scu-PA and two chain u-PA (tcu-PA) and the ratio, R (mol of plasmin generated/mol of activator) were determined as indicators of the efficiency of plasminogen activation. [Glu)t-plasminogen bound to intact fibrin was most efficiently activated by t-PA (Vi=0.843 and R=4.08), while scu-PA and tcu-PA produced markedly lower activation coefficients (Vi=0.011 and 0.023, and R=0.13 and 0.50 respectively). In contrast, when [Glu] 1-plasminogen was bound to the degraded surface, it became activatable with equivalent efficiencies by both t-PA and scu-PA (R=4.95 and 4.72, respectively). No evidence of Lys-plasminogen formation was observed. Rather, the selective activation of plasminogen bound to degraded fibrin by scu-PA is consistent with a particular interaction of Glu-plasminogen with carboxy-terminal lysine residues on the degraded surface. The consequences of this interaction of scu-PA with plasminogen bound to degraded fibrin on sequential activations of plasminogen by both t-PA and scu-PA were further explored. While the activation by scu-PA and then t-PA produced only an additive effect on plasmin generation, the opposite combination induced a greater than additive affect; the concentration of activators needed to produce 50% of plasmin generation was 70pM for the scu-PA/t-PA combination; in contrast, the amount required to produce a similar degree of activation with the opposite combination (t-PA/scu-PA) was only 17 pM. These results indicate that the potentiation of the scu-PA activity requires both the previous generation of plasmin by t-PA, and plasminogen bound to carboxy-terminal lysine residues of the degraded fibrin surface as a substrate.

Remaining perspectives of mutant and chimeric plasminogen activators.

Potential approaches to improve thrombolytic agents comprise the construction of mutants and variants of tissue-type plasminogen activator (tPA) or of single chain urokinase-type plasminogen activator (scuPA, pro-urokinase), of chimeric plasminogen activators and of conjugates of plasminogen activators with monoclonal antibodies. tPA mutants have been constructed with altered pharmacokinetic properties or altered functional properties, including binding to and stimulation by fibrin, resistance to plasmin and to protease inhibitors. Mutants of tPA described to date, obtained by deletion/substitution of functional domains or of single amino acids, have markedly reduced clearances, but usually also reduced specific thrombolytic potencies. Mutants of scuPA with improved thrombolytic potencies have thus far not been reported. Chimeric molecules containing functional domains of both tPA and scuPA have intact enzymatic properties of uPA and some fibrin affinity of tPA. Surprisingly, chimeras endowed with fibrin affinity usually have unaltered or reduced thrombolytic potencies. However, a chimera consisting of amino acids 87-274 of tPA and amino acids 138-411 of scuPA, with negligible fibrin affinity, has a 10-fold higher thrombolytic potency than scuPA in animal models of venous thrombosis, as a result of a delayed in vivo clearance and a relatively maintained specific thrombolytic activity. Plasminogen activators conjugated with antifibrin or antiplatelet monoclonal antibodies, either chemically or by recombinant DNA technology, are targeted to blood clots, resulting in a 5- to 10-fold increased thrombolytic potency. Thus, it is possible to develop plasminogen activators with improved thrombolytic potency. Whether such agents will be clinically useful remains to be established.

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 NH 2-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) 45000 ± 5000 IU/mg, 19000 ± 800 IU/mg and <- 100 IU/mg for rscu-PA N302Q, rscu-PA C279A,N302Q and rscu-PA del(N2-F157)C279A,N302Q, respectively, as compared to 64000 ± 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 56000 IU/mg and 32000 IU/mg, respectively, as compared to 75000 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 milicu in vitro, consisting of a 125I-fibrin labeled plasma clot submerged in plasma, 50 percent clot lysis in 2 h required 2.2 μg/ml rscu-PA N302Q and 6.0 μg/ml rscu-PA C279A,N302Q, as compared to 3.2 μg/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 t-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. Deletion of the interchain disulphide bond in the non-glycosylated molecule reduces its fibrinolytic activity somewhat, whereas additional deletion of the NH 2-terminal polypeptide chain results in abolishment of activity.

Biochemical and biological properties of a recombinant chimera consisting of amino acids Asn-1 to Lys-12 of α2-antiplasmin (fibrin cross-linking site) and amino aids Leu-4 to Leu-411 of single chain urokinase-type plasminogen activator

A recombinant chimeric plasminogen activator (APUK) consisting of amino acids Asn 1 to Lys 12 of α 2-antiplasmin (fibrin cross-linking site) and amino acids Leu 4 to Leu 411 of single chain urokinase-type plasminogen activator (scu-PA) was expressed in E. coli and purified to homogeneity. APUK was obtained as a single chain molecule which was quantitatively converted to a two chain moiety by treatment with plasmin; this was associated with an increase in specific amidolytic activity from ⩽300IU/mg to 95 000IU/mg. Its specific activity on fibrin plates was approximately 120 000IU/mg. Activation of plasminogen by two chain APUK obeyed Michaelis-Menten kinetics with K m=3.7 μM and k 2=3.4s −1. APUK induced dose-dependent lysis of a 0.12ml 125I-fibrin labelled human plasma clot submersed in 0.5 ml citrated human plasma; 50% clot lysis in 2 h was obtained with 0.80 or 1.0 μg/ml single chain or two chain APUK and was associated with respectively 0 or 52% fibrinogen degradation. None of these properties of APUK was significantly different from those of recombinant scu-PA (rscu-PA) obtained in the same expression system. In the presence of Ca 2+, however, significant binding of APUK, but not of rscu-PA, to forming fibrin clots was observed. The thrombolytic properties of APUK were compared with those of rscu-PA following a 1 h intravenous infusion in a combined femoral arterial whole blood clot and femoral venous blood clot model in the dog. At the arterial side, similar recanalisation rates were obtained with APUK at doses of 0.30–1.20 mg/kg (8 of 11 dogs) and with rscu-PA at doses of 0.25–1.0 mg/kg (5 of 9 dogs). Persistence of recanalisation was also similar, with patency at 2h occurring in 6 of 11 dogs with APUK and in 4 of 9 dogs with rscu-PA. At the venous side, the thrombolytic potency of APUK was 84 ± 33% lysis per mg compound administered per kg body weight, versus 140 ± 51% lysis per mg/kg for rscu-PA (p=0.3). APUK disappeared from plasma after the end of the infusion with an initial half-life of 1.8–2.7 min, corresponding to a plasma clearance of 210–240 ml/min; corresponding values for rscu-PA were 2.5–3.0 min and 200–270 ml/min. These findings indicate that APUK, a chimera comprising the fibrin cross-linking site of α 2-antiplasmin in addition to scu-PA, has the potential to bind to forming fibrin. However, this does not result in an increased thrombolytic potency in a combined arterial and venous thrombosis model in the dog.

K 1K 2P u, a recombinant t-PA/u-PA Chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man

K 1K 2P u, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K 1K 2P u cDNA, high producer cell lines were selected and scaled up in 800 cm 2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 ± 4 liter (mean ± SD, n= 28) containing 1.8 ± 0.6 mg/l of K 1K 2P u related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-IC8). Yields were 8.6 ± 3.4 mg K 1K 2P u per batch with a specific activity of 83,000 ± 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HC1 buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 ± 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 μm Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 ± 16,000 IU/mg (mean ± SD, n= 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K 1K 2P u in patients with thromboembolic disease.

Differential detection of single chain urokinase-type plasminogen activator and two chain urokinase-type plasminogen activator in human and monkey plasma

An ELISA-based assay system is described for the selective measurement of recombinant single chain (rscu-PA) and two chain urokinase-type plasminogen activator (rtcu-PA) when present together in human or in monkey plasma. The assay system consists of two separate sandwich ELISAs, one for rscu-PA which utilises two monoclonal antibodies, and another one for total u-PA, which utilises a polyclonal antibody both as capture and as tagging antibody. Since the latter was found to be less sensitive to rtcu—PA-inhibitor complexes than to uncomplexed rscu-PA in plasma, plasmin activation of rscu-PA was introduced in order to eliminate this variable. Complete inhibitor complex formation was shown to occur after incubation (⩾2h). Excess inhibitors were provided by making dilutions with normal plasma. The assay detection limit, in plasma, for total u-PA antigen and rscu-PA was 0.8 and 3 ng/ml, respectively. The rtcu-PA concentration was calculated by subtracting the rscu-PA from the total u-PA concentration. The assay system was used to analyse blood samples from Cynomolgus monkeys infused with 90000 IU (0.6 mg)/kg of rscu-PA infused over 1h. By the end of the infusion, the rtcu-PA concentration was 0.5 μ/ml, whereas that of rscu-PA was 1 μg/ml. No fibrinogenolysis occurred. However, when the rscu-PA infusion was preceded by a bolus of 400000 IU (0.8 mg)/kg of rt-PA, rtcu-PA generated reached 1.35 μg/ml with 30% fibrinogenolysis. The ELISA system described can be used to provide a better understanding of the relationship between systemic scu-PA activation and thrombolytic activity, fibrin specificity and the co-use of other agents such as t-PA or tcu-PA.

Thrombolytic Activity of Two Chimeric Recombinant Plasminogen Activators (FK2tu-PA and K2tu-PA) in Rabbits

The aim of this study was to evaluate the thrombolytic activity of two hybrid plasminogen activators (HPAs) in a rabbit jugular vein thrombosis model. In the two HPAs the kringle-2 domain (K2tu-PA) or the finger and the kringle-2 domains (FK2tu-PA) of tissue-type plasminogen activator (t-PA) are linked to the catalytic protease domain of single chain urokinase type plasminogen activator (scu-PA). The two HPAs were compared with rt-PA and scu-PA on a weight/weight basis. K2tu-PA, FK2tu-PA, rt-PA and scu-PA were infused at doses of 0.4, 0.8 and 1.2 mg/kg over 3 h. Saline served as control. Saline produced 11 +/- 2% thrombolysis. The three doses of K2tu-PA led to 38 +/- 4%, 66 +/- 5% and 89 +/- 7% thrombolysis, respectively; the three doses of FK2tu-PA: 18 +/- 3%, 29 +/- 5% and 33 +/- 6%, respectively; the three doses of rt-PA 32 +/- 2%, 49 +/- 3% and 68 +/- 6%, respectively; the three doses of scu-PA 16 +/- 2%, 24 +/- 3% and 32 +/- 4%, respectively. K2tu-PA and rt-PA showed a statistically significant higher thrombolytic activity than FK2tu-PA and scu-PA at the three tested doses (p less than 0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p less than 0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, alpha 2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.

The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.

Influence of Intrinsic and Extrinsic Plasminogen upon the Lysis of Thrombi In Vitro

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

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

Serine protease and metallo protease cascade systems involved in pericellular proteolysis

Cultures of transformed fibroblasts actively involved in extracellular matrix degradation have been examined for initial activation of serine and metallo protease cascade systems. Rous sarcoma virus transformed chick embryo fibroblasts (RSVCEF), in contrast to transformed mammalian cells, produce active, two chain urokinase-type plasminogen activator (tcu-PA). Active tcu-PA is found in serum-free, plasmin-free conditioned medium from RSVCEF cultures as determined by two independent methods, immunoprecipitation and differential DFP sensitivity. RSVCEF cultures synthesize and secrete inactive, single chain uPA (scu-PA) which is converted to tcu-PA in a time dependent manner by a catalytic mechanism that appears to involve a functioning uPA receptor on the surface of intact cells. The enzyme activity responsible for this conversion may represent the initiating catalytic event in the PA/plasminogen serine protease cascade system. A 70 kDa prometalloprotease capable of degrading denatured collagen following its activation also is significantly elevated in RSVCEF cultures over that of normal CEF. Trace amounts of the active 62 kDa form of the metalloprotease (gelatinase) is found in the transformed RSVCEF cultures indicating that these cultures produce a natural activator of the prometalloprotease. Plasmin and/or PA do not appear to be the activator of this enzyme as determined by indirect inhibition assays and direct assays employing purified enzymes. The possible central position of pro PA and the 70 kDa prometalloprotease in an interacting, complex protease cascade system involved in extracellular matrix degradation is discussed.

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.