Effect of changes in liver blood flow on the pharmacokinetics of saruplase in patients with acute myocardial infarction.

BACKGROUND: 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. METHODS AND RESULTS: 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). CONCLUSIONS: 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.

Amyloid beta peptides stimulate tissue-type plasminogen activator but not recombinant prourokinase.

Tissue-type plasminogen activator (rt-PA) and prourokinase (rscu-PA) have been tested with respect to the influence of amyloid beta peptides on plasminogen activation which was monitored by cleavage of the chromogenic plasmin substrate S-2251. It was shown that rt-PA is stimulated by amyloid beta peptides at concentrations of 10 micrograms/ml in contrast to prourokinase, which does not alter its catalytic properties in presence of amyloid beta peptides. The stimulation of rt-PA can be inhibited by tranexamic acid indicating a molecular mode of stimulation similar to the fibrin mediated stimulation of rt-PA.

Effects of changing liver blood flow by exercise and food on kinetics and dynamics of saruplase.

OBJECTIVE: To investigate the influence of changes in liver blood flow on the pharmacokinetics and pharmacodynamics of single-chain unglycosylated urokinase-type plasminogen activator. METHODS: This open, randomized, crossover trial was carried out in the clinical research unit. Infusions of 37.5 mg saruplase and 90 mg indocyanine green were administered over 150 minutes to 10 healthy male volunteers. After 60 minutes the subjects consumed a standardized meal to increase liver blood flow or performed an exercise test (20 minutes) to decrease liver blood flow. Indocyanine green concentrations, total urokinase-type plasminogen activator (u-PA) antigen, two-chain u-PA activity, fibrinogen, total degradation products, alpha 2-antiplasmin, and factor XII-dependent fibrinolytic activity were measured. Blood flow was measured after food intake in a portal vein branch with Doppler echography. RESULTS: The weighted average indocyanine green concentration after exercise was increased by 29% compared with baseline (steady-state concentration) values (95% confidence intervals [CI]: +6%, +56%). After food, the concentration was 27% lower compared with baseline values (95% CI: -35%, -19%), and portal vein flow was increased by a maximum of 103% (95% CI: +71%, +136%). Average maximal concentrations of u-PA antigen after exercise were increased by 130 ng/ml compared with baseline concentrations (95% CI: +65, +195 ng/ml) and, unexpectedly, 156 ng/ml higher after food (95% CI: +59, +253 ng/ml). Although not significant, an increase in average u-PA antigen concentration compared with baseline values was detected after both exercise (7%) and food (13%). This tendency toward a larger effect after food compared with the effect after exercise was reflected by minor changes in the pharmacodynamics. CONCLUSIONS: u-PA plasma concentrations were increased by reduced liver blood flow induced by exercise. Food intake produced an unexpected increase in u-PA concentrations despite increases in liver blood flow.

Pharmacokinetics and pharmacodynamics of saruplase, an unglycosylated single-chain urokinase-type plasminogen activator, in patients with acute myocardial infarction.

We examined in patients with acute myocardial infarction (AMI) the pharmacokinetics of saruplase, an unglycosylated, single chain, urokinase-type plasminogen activator (rscu-PA) by measuring urokinase-type plasminogen activator (u-PA) antigen and total u-PA activity, its conversion to active two-chain urokinase-type plasminogen activator (tcu-PA) and evaluated its effect on haemostatic parameters. Twelve patients were studied during and after administration of 20 mg bolus plus 60 mg continuous 1 h i.v. infusion of saruplase. For u-PA antigen and total u-PA activity (expressed as protein equivalents), where 234 U corresponds to 1 microgram, respectively, steady state plasma concentrations were 2.75 +/- 8.3 and 2.50 +/- 7.0 micrograms/ml (mean +/- standard deviation) and were reached within 20 min, t1/2 lambda 1 was 9.1 +/- 1.8 and 7.8 +/- 1.3 min, t1/2 lambda 2 1.2 +/- 0.2 and 1.9 +/- 0.5 h, and the total clearance was 393 +/- 110 and 427 +/- 113 ml/min. Inactivation of saruplase in plasma was negligible. After 15 min, tcu-PA was detected in plasma. From the ratio of the areas under the curve of tcu-PA and total u-PA activities it was calculated that 28 +/- 9.3% of the saruplase dose is converted into active tcu-PA. Systemic plasminaemia occurs as shown by a decrease in alpha 2-antiplasmin and fibrinogen and an increase in fibrinogen degradation products. Thrombin-antithrombin complex formation indicated activation of the clotting system. Saruplase is eliminated rapidly from plasma in AMI patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of saruplase, a recombinant unglycosylated human single-chain urokinase-type plasminogen activator and its effects on fibrinolytic and haemostatic parameters in healthy male subjects.

Pharmacokinetics of two doses of the recombinant single-chain urokinase-type plasminogen activator (r-scu-PA) saruplase (40 and 20 mg) and its effect on fibrinolytic and haemostatic parameters were studied in six healthy male subjects using a randomized, double-blind, placebo-controlled, cross-over study. Special precautions were taken to prevent artefactual in vitro effects on fibrinolytic activity. The clearance of saruplase ranged from 310 to 862 ml/min and the apparent volume of distribution of the central compartment was about 8 1. Both doses of saruplase caused alpha 2-antiplasmin consumption, indicating some systemic fibrinolytic activation. However, the 20 mg dose caused no detectable fibrinogen breakdown and only a small increase in total fibrin/fibrinogen degradation products (TDP) (from 0.16 microgram/ml [range 0.14 to 0.19] to 0.78 microgram/ml [range 0.56 to 1.26]), while the 40 mg dose produce a fibrinogen breakdown to an average value of 44% (range 19 to 60%) and TDP increased from 0.12 microgram/ml (range 0.11-0.12) to 2.29 micrograms/ml (range 0.45 to 5.55). The breakdown of fibrinogen was related to the quantity of saruplase converted to active two-chain u-PA (tcu-PA) in vivo (6 to 22% conversion). There were no important effects of saruplase on overall blood coagulation (activated partial thromboplastin time) and platelet function (collagen induced platelet aggregation, urinary [2,3-dinor]-thromboxane B2 excretion and plasminogen activator inhibitor 1 [PAI-1] release from platelets). Saruplase is cleared rapidly from the plasma and a variable amount is converted to tcu-PA. This two-chain form of u-PA probably causes the dose-dependent systemic fibrinolytic activation.

Effective activation of the proenzyme form of the urokinase-type plasminogen activator (pro-uPA) by the cysteine protease cathepsin L.

Increased levels of both the cysteine protease, cathepsin L, and the serine protease, uPA (urokinase-type plasminogen activator), are present in solid tumors and are correlated with malignancy. uPA is released by tumor cells as an inactive single-chain proenzyme (pro-uPA) which has to be activated by proteolytic cleavage. We analyzed in detail the action of the cysteine protease, cathepsin L, on recombinant human pro-uPA. Enzymatic assays, SDS-PAGE and Western blot analysis revealed that cathepsin L is a potent activator of pro-uPA. As determined by N-terminal amino acid sequence analysis, activation of pro-uPA by cathepsin L is achieved by cleavage of the Lys158-Ile159 peptide bond, a common activation site of serine proteases such as plasmin and kallikrein. Similar to cathepsin B (Kobayashi et al., J. Biol. Chem. (1991) 266, 5147-5152) cleavage of pro-uPA by cathepsin L was most effective at acidic pH (molar ratio of cathepsin L to pro-uPA of 1:2,000). Nevertheless, even at pH 7.0, pro-uPA was activated by cathepsin L, although a 10-fold higher concentration of cathepsin L was required. As tumor cells may produce both pro-uPA and cathepsin L, implications for the activation of tumor cell-derived pro-uPA by cathepsin L may be considered. Different pathways of activation of pro-uPA in tumor tissues may coexist: (i) autocatalytic intrinsic activation of pro-uPA; (ii) activation by serine proteases (plasmin, kallikrein, Factor XIIa); and (iii) activation by cysteine proteases (cathepsin B and L).

Fluorescent probes as tools to assess the receptor for the urokinase-type plasminogen activator on tumor cells.

Flow cytofluorometric protocols (FACScan) are described for the rapid and quantitative real-time analysis of binding of FITC-pro-u-PA to cell surface receptors (u-PAR) on living, resting, and also on PMA-stimulated human monocytic U937 cells. Binding of pro-u-PA was visualized by CLSM. This fairly new technique is superior over conventional fluorescence microscopy and is an alternative to electron microscopic approaches. Both flow cytofluorometry and confocal laser scanning microscopy allow the analysis quantitatively and with high-sensitivity binding of FITC-pro-u-PA to single suspended or adherent cells. By CLSM u-PA/u-PAR were found to be located in heterogeneously distributed discrete patches at the cell surface on U937 and not inside the cell. This is in agreement with previous studies by Hansen et al, who applied radioiodinated u-PA and electron microscopy to locate u-PAR on microvilli of fixed U937 cells. By flow cytofluorometry, it was possible to quantify the time-dependent and temperature-dependent binding of FITC-pro-u-PA to living single U937. Apparent saturation of u-PAR was achieved at 5 nM FITC-pro-u-PA for both nonstimulated and PMA-stimulated U937 cells. Half saturation of u-PAR was also determined. Nonstimulated U937 was 0.7 nM, and PMA-stimulated U937 was 1.1 nM of FITC-pro-u-PA. This increase in half-saturation concentration in PMA-stimulated cells is paralleled by a steep increase in binding sites (3.6-fold). The use of fluoresceinated reference beads is recommended to verify changes in affinity and binding sites. Using CLSM or flow cytofluorometry, it is also possible to study the structure relationship of u-PA/u-PAR in the presence of competitive binding analogues or inhibitors. Fluorescence techniques will also permit the identification of u-PAR-positive cells in blood, ascitic fluid, or biopsies obtained from cancer patients.

Cathepsin B efficiently activates the soluble and the tumor cell receptor-bound form of the proenzyme urokinase-type plasminogen activator (Pro-uPA).

Action of purified human cathepsin B on recombinant single-chain urokinase-type plasminogen activator (pro-uPA) generated enzymatically active two-chain uPA (HMW-uPA), which was indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot from plasmin-generated HMW-uPA and from elastase- or thrombin-generated inactive two-chain urokinase-type plasminogen activator. Preincubation of cathepsin B with E-64 (transepoxysuccinyl-L-leucylamino- (4-guanidino)butane, a potent inhibitor for cathepsin B) prior to the addition of pro-uPA prevented the activation of pro-uPA. The cleavage site within the cathepsin B-treated urokinase-type plasminogen activator (uPA) molecule, determined by N-terminal amino acid sequence analysis, is located between Lys158 and Ile159. Pro-uPA is cleaved by cathepsin B at the same peptide bond that is cleaved by plasmin or kallikrein. Binding of cathepsin B-activated pro-uPA to the uPA receptor on U937 cells did not differ from that of enzymatically inactive pro-uPA, indicating an intact receptor-binding region within the growth factor-like domain of the cathepsin B-treated uPA molecule. Not only soluble but also tumor cell receptor-bound pro-uPA could be efficiently cleaved by cathepsin B to generate enzymatically active two-chain uPA. Thus, cathepsin B can substitute for plasmin in the proteolytic activation of pro-uPA to enzymatically active HMW-uPA. In contrast, no significant activation of pro-uPA by cathepsin D was observed. As tumor cells may produce both pro-uPA and cathepsin B, implications for the activation of tumor cell-derived pro-uPA by cellular proteases may be considered.

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 micrograms/kg.min) and r-scu-PA (4.64 - 46.4 microns/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.

Kinetics of the inhibition by plasminogen activator inhibitor 2 of urokinase isolated from human urine and recombinant microorganisms. Further evidence for correct tertiary structure of recombinant urokinase.

The specific plasminogen activator inhibitor 2 (PAI-2) from human placenta was used to compare the kinetics of inactivation of recombinant urokinase and urokinase from human urine. The inactivation of the enzymes proceeded with identical second order rate constants. The result is considered a further indication of corresponding 3-dimensional structures of urokinases from human urine and of recombinant origin.

Selective fibrinolytic activity of recombinant non-glycosylated human pro-urokinase (single-chain urokinase-type plasminogen activator) from bacteria.

To assess their therapeutic value recombinant non-glycosylated human pro-urokinase (cPUK) and recombinant non-glycosylated human low molecular mass urokinase (cLUK) both obtained from genetically transformed bacteria were compared with respect to their thrombolytic efficacy and their potential to induce systemic plasminogen activation which impairs the coagulation system. The investigations were performed in in vitro and in vivo test systems. In vitro, both substances significantly and concentration-dependently lysed radiolabelled human thrombi in rotating loops of tubes (Chandler-loops) with equivalent efficacy. Fibrinolytic activity of cLUK was accompanied by a decrease in alpha 2-antiplasmin, plasminogen and fibrinogen. In contrast, cPUK did not change the plasminogen and fibrinogen levels and induced a substantially smaller decline in alpha 2-antiplasmin than cLUK. In the lysis of pulmonary embolized radiolabelled blood clots in anesthetized rabbits cPUK and cLUK dose-dependently exerted significant effects of similar extent. Whereas cLUK significantly decreased plasma levels of alpha 2-antiplasmin, plasminogen and fibrinogen, cPUK caused only a marginal decrease in alpha 2-antiplasmin and left the plasminogen and fibrinogen levels unchanged. The results indicate that cPUK exerts fibrinolytic efficacy without systemic plasminogen activation and breakdown of fibrinogen. Therefore, cPUK might be expected to be a more specific and safer thrombolytic agent than urokinase and other traditional fibrinolytics.

Chemical, enzymological and pharmacological equivalence of urokinases isolated from genetically transformed bacteria and human urine.

Low molecular weight urokinase (LUK), which was prepared from E. coli containing a plasmid coding for human pro-urokinase, has an amino acid sequence identical to that of LUK isolated from human urine (uLUK) but lacks the carbohydrate side chain at Asn 144 of the B chain. This chemical difference results in an altered mobility in SDS polyacrylamide gel electrophoresis and an apparently increased specific activity of the E. coli-derived product (cLUK) in diffusion-limited test systems (fibrin agar plate tests). Comparative enzymological investigations in homogeneous phases reveal that the active centers and the substrate recognition sites of cLUK and uLUK are congruent. No significant difference between the enzymes was detectable in the following parameters: Michaelis constants and maximum velocities with the synthetic substrate S-2444; activation rates of human and porcine plasminogen; specificity for ten chromogenic substrates; inhibition constants for the competitive inhibitor benzamidine; inhibition by placental urokinase inhibitor and polyclonal antibodies. Further, cLUK and uLUK dissolved fibrin clots prepared from human plasma in vitro with essentially identical velocities. Both, cLUK and uLUK efficiently lysed injected emboli in rabbits and prevented renal fibrin deposition and death due to endotoxin infusion in rats. It is concluded that cLUK, despite the lack of the carbohydrate side chain, is functionally identical and pharmacologically equivalent to uLUK.

The amino-acid sequence of bovine glutathione peroxidase.

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.

Influence of supidimide on brain neurotransmitter systems of rats and mice.

The influence of 2-(2-oxo-3-piperidyl)-1,2-benzisothiazoline-3-one-1, 1-dioxide (supidimide), a representative of a new class of sedative drugs, on the noradrenergic, dopaminergic, serotoninergic and gamma-aminobutyric acid (GABA)ergic neuronal systems of rodent brains was investigated. In each case the brain transmitter levels after administration of supidimide were determined. Utilisation of noradrenaline (norepinephrine, NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) was also investigated ex vivo. The study was complemented with in vitro investigations of biosynthesis, synaptosomal uptake, degradation, and receptor binding of the transmitters. Based on a preliminary study of the distribution of [35S]-supidimide in rat brain, in vitro effects observed at greater than 10(-4) mol/l were considered irrelevant. Similarly, in vivo effects requiring dosages higher than 300 mg/kg i.p. were not regarded adequate to explain the sedative and antiaggressive efficacy of supidimide. With the above restrictions, the following parameters can be rated as not influenced by supidimide: levels of tryptophan in rat brain and serum (free and total); 5-HT biosynthesis in vivo (rat brain; 5-HT accumulation after monoamine oxidase (MAO) blockade); activity of MAO-A and MAO-B (rat brain mitochondria); uptake of 5-HT, NE and DA (rat synaptosomes); 5-HT receptor binding ( [3H]-LSD binding assay in rat cortical membranes); tyrosine hydroxylase activity (rat adrenal glands); catechol-O-methyl transferase (COMT) (rat liver); NE binding to central alpha 1- and alpha 2-receptors (rat brain; radioligand assay with [3H]-dihydroergocryptine, [3H]-prazosin and [3H]-WB 4101 (2',6'-dimethoxy-(G-3H]-phenoxy]-ethylaminomethylbenzo-1,4-dioxane ); DA levels (whole rat brain and striata); dihydroxyphenylacetic acid (DOPAC) levels (whole rat brain without cerebellum and striata); elevated DOPAC levels after pretreatment with haloperidol; DA-dependent adenylate cyclase in vitro (rat striatum); D2 receptor binding ( [3H]-spiperone binding assay, rat striatum); GABA levels (mouse brain); GABA transaminase activity (mouse brain stem); sodium-independent [3H]-GABA receptor binding (rat brain) and benzodiazepine binding (rat cortical membranes, [3H]-diazepam binding assay). Two effects on the GABAergic system were induced by supidimide. Starting at 300 mg/kg i.p., supidimide slowed down the GABA accumulation in brains of aminooxyacetate-treated mice. At 10(-4) mol/l supidimide caused a significant inhibition of GABA uptake (rat synaptosomes).(ABSTRACT TRUNCATED AT 400 WORDS)

Adaptation of plasminogen activator sequences to known protease structures.

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

The primary structure of Cu-Zn superoxide dismutase from Photobacterium leiognathi: evidence for a separate evolution of Cu-Zn superoxide dismutase in bacteria.

The complete amino-acid sequence of the copper-zinc superoxide dismutase of the Photobacterium leiognathi was determined. The fragmentation strategy employed included cyanogen bromide cleavage at its methionine residues and the only tryptophan residue. The S-carboxymethylated chain was further cleaved by means of trypsin, in order to obtain overlapping fragments. For sequence determination automated solid or liquid-phase techniques of Edman degradation were used. C-Terminal amino acids of the entire chain were determined after treatment with carboxypeptidase A. Comparison of the primary structure of this bacterial Cu-Zn superoxide dismutase with the established amino-acid sequences of the other eukaryotic Cu-Zn superoxide dismutases revealed clear homologies. Correspondingly, the Cu-Zn-binding amino-acid residues of the active centre were localized: His45, His47, His70, His79, His125 and Asp91. The two cysteine residues in position 52 and 147 were homologous to the cysteine residues, modelling the essential intrachain disulfide bridge of the corresponding bovine enzyme. As only 25-30% of aligned sequence positions were found to be identical, the enzyme of P. leiognathi shows only a remote phylogenetic relationship towards eukaryotic Cu-Zn superoxide dismutases. When compared to the established phylogenetic tree of the cytochrome c family, this indicates a separate evolution of Cu-Zn superoxide dismutase in Photobacterium. Therefore, a natural gene transfer from the eukaryotic host (ponyfish) to the prokaryotic photobacterium, which Martin and Fridovich postulated 1981 (J. Biol. Chem. 256, 6080-6089) on the basis of amino-acid compositions, can be excluded.

The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain.

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.

The complete amino acid sequence of low molecular mass urokinase from human urine.

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.