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Summary We have previously shown that the catalytic domain of tissue plasminogen activator (t-PA), the B-chain, binds specifically to hydrophobic components on the endothelial cell (EC) surface. The binding is mediated by amino acids within the sequence, AKHRRSPGER (B 20–29 ). The complex imparts a strong fibrinolytic potential to the EC surface, which is not inhibited by plasminogen activator inhibitor 1 (PAI-1). Here we characterize membrane proteins from EC that interact with the binding peptide and have hydrophobic properties. Specific labelling of cell surface components with affinity for the t-PA B-chain was performed using B-chain derivatized with the heterobifunctional and cleavable cross-linked SASD labelled with 125 I. Analysis of the cell extract by SDS/PAGE before and after reduction demonstrated the presence of a complex between the B-chain and a membrane protein of about 56 kDa. Radiolabelled proteins from the surface of EC were affinity purified on B 19–30 -Sepharose. The adsorbed proteins were eluted with lysine and the peptide B 20–29 . The hydrophobic fraction from the peptide eluate contained one main component with a molecular mass 56 kDa, which was absent in the hydrophilic fraction. In addition, four minor components were observed. Only traces of radiolabelled proteins were found in the hydrophobic fraction of the lysine eluate. Complexes between 125 I-t-PA and hydrophobic proteins from EC obtained by affinity chromatography on B 19–30 -Sepharose were crosslinked with disuccinylsuberate (DSS). Two radioactive complexes were identified after electroblotting to a PVDF membrane, one major component with a molecular mass of about 120 kDa and one minor 200 kDa complex. The 120 kDa compound had a significant activator activity before and after treatment with an excess of PAI-1. This is in contrast to free t-PA, which completely loses its activity in the presence of PAI-1. This indicates that t-PA (about 65 kDa) can form complexes with membrane components from EC with an approximate molecular mass of 55 kDa.

The binding of 125I-labelled tissue plasminogen activator (tPA), the tPA A- or B-chain to endothelial cells (EC) were studied in suspensions of cultured human umbilical vein EC (HUVEC) or immortali ...

The way in which three thrombolytic agents, tissue-type plasminogen activator (t-PA), streptokinase (SK) and a genetically engineered variant of t-PA composed of the second kringle and the protease domain (K2P), cause the dissolution of haemostatic plugs of differing ages was investigated in a novel rabbit model. Standardized incisions were made on the rabbit ear and the wounds were left to heal for 0.5 h or 24 h, before the thrombolytic agents were infused. In the absence of heparin, t-PA showed little discrimination between clots of different ages (36% and 28% lysis of the 0.5 h and 24 h wounds, respectively). In contrast, K2P and SK showed a pronounced fresh clot selectivity since they were significantly more effective in lysing fresh clots than old ones (68% and 4% lysis for K2P and 72% and 36% for SK, respectively). In the presence of heparin the potency of t-PA on fresh clots was considerably increased whilst the effect on old clots was not affected, a fresh clot selectivity for t-PA (64% lysis of fresh clots, 24% lysis of old clots) was thus effected. Heparin did not significantly affect the fresh clot selectivity of K2P or SK, although lysis of old clots was increased (from 4% to 36%) when it was given together with K2P. Furthermore, heparin did not affect the time to onset of bleeding nor was the bleeding time prolonged by its addition. The bleeding time observed for t-PA (20-25 min) was markedly shorter than that found for K2P or SK (40-50 min).(ABSTRACT TRUNCATED AT 250 WORDS)

The aim of the present study was to investigate the binding of tissue plasminogen activator (tPA) to cultured endothelial cells and to characterize binding structures present in the cultures. Studies on the binding of 125I-tPA to cultured endothelial cells from human umbilical-cord veins (HUVEC) indicated that the number of sites for specific binding of tPA is 8 x 10(5) per cell. Treatment with an excess of antibodies against plasminogen-activator inhibitor type 1 (PAI-1) caused an 80% decrease in the binding, leaving about 1.6 x 10(5) unoccupied binding sites per cell, which appeared to be different from PAI-1. About 1.9 x 10(5) binding sites/cell for tPA were found on the surface of HUVEC that had been detached from the matrix. This indicates that only minor amounts of PAI-1 occur on the surface of the cells. In addition, immunocytochemical analysis showed that PAI-1 antigen is present almost exclusively in the cytoplasm but was not observed on the surface of the cells, whereas tPA antigen is abundant on the plasma membrane of tPA-treated cells as well as intracellularly. Competition studies using unlabelled compounds showed that native tPA and tPA B-chain (the proteinase domain), as well as the inactive derivatives, B-chain inactivated with D-Phe-Pro-Arg-chloromethane and tPA-PAI-1 complex, caused a considerable quenching of the binding of 125I-tPA to HUVEC, whereas the isolated A-chain had no demonstrable effect. Two components (apparent molecular masses 38 kDa and 56 kDa) reacting with tPA but lacking PAI-1 antigen determinants were identified. Thus the data suggest that tPA binds to HUVEC by two principally different mechanisms. One is mediated by PAI-1, which binds and inactivates tPA with a functional active site. The other binding is achieved by components which react with sites on the activator molecule other than structures of the A-chain or the active site.

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1-3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1-3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1-3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.

SummaryA modified variant of human tissue-type plasminogen activator (t-PA) lacking the finger domain (F), the growth factor domain (G) and the first kringle domain (K1), has an extended plasma half-life in vivo, compared to that of t-PA. When the variant (denoted K2P) was tested in vitro for its ability to lyse human plasma clots we found that the activity was characterized by a time lag phase and a sigmoidal dose-response curve. However, an attenuation of the lag phase in vitro was observed both when K2P was mixed with t-PA in a w/w ratio of 4 : 1 and when K2P was allowed to lyse a clot that had been pre-exposed to t-PA i.e. submitted to a limited plasmic digestion. Dosis that in vitro caused 50% lysis within 6 h were calculated from individual dose-response curves and were for K2P, t-PA and K2P/t-PA (4 : 1 w/w) 540 ng/ml, 360 ng/ml and 310 ng/ml, respectively. These results indicated a synergistic effect between K2P and t-PA. However, the data from individual dose-response curves showed that the effect of the K2P/t-PA mixture never was better than that of t-PA alone, and the synergistic effect in vitro is therefore considered to be of limited use. The thrombolytic activity in vivo was evaluated in a rabbit jugular vein thrombus model. Despite the lag phase observed in vitro, K2P was approximately 3 times as effective as t-PA in vivo (bolus injection). The thrombolytic effect of K2P was further potentiated when it was administred together with a small amount of t-PA (4 : 1 w/w). This potentiation in vivo was, in contrast to the effect in vitro, a useful synergistic effect as the dose-response curve for the K2P/t-PA mixture was steeper than that of t-PA and K2P alone. Doses that caused 50% lysis within 3 h were for t-PA, K2P and K2P/t-PA 1.28 mg/kg, 0.56 mg/kg and 0.35 mg/kg, respectively.

Six variants of tissue-type plasminogen activator (t-PA) were produced in mouse C127 cells using a bovine papilloma virus expression vector. All variants lacked the growth factor (G) domain and the first kringle domain (K1) and three of the variants also lacked the finger domain (F). Furthermore, the specific changes, Lys 277 → Val and Asn 448 → Gln were introduced into some of the molecules. The variants were denoted K2P, K2P(Val277), K2P(Gln448), FK2P, FK2P(Va1277), and FK2P(Gln448). Amino acid sequence analyses revealed that the variants were proteolytically processed in the amino terminus and at Arg 275 -Ile 276 in the same way as the full sized molecule. A proteolytically sensitive site was identified in the F domain at Arg 27 -Ser 28 . The two variants that lacked glycosylation at Asn 448 , K2P(GIn448) and FK2P(GIn448), were cleaved at Arg 449 -Thr 450 , indicating that the oligosaccharide normally present at Asn 448 protects this site against proteolysis. The fibrin affinity for all variants was markedly reduced compared with normal t-PA. The plasminogen activator activity of all variants was stimulated by cyanogen bromide fragments of fibrinogen. In an indirect chromogenic assay K2P and K2P(Val277) showed specific activities that were 23% and 36%, respectively, of that of wild type t-PA, while the corresponding non-glycosylated variant K2P(Gln448) was as active as t-PA. The activity of the three F domain-containing variants were between 88 and 98% of the value determined for t-PA. When the specific activity was determined with the fibrin plate assay all variants were found to have higher specific activities than t-PA (1.8–4.7 fold). The lack of correlation between the activity of t-PA and the variants in these two assays indicate that the reaction mechanism may differ between the variants and wild type t-PA. The kinetic constants K m and k cat were determined for two-chain forms of t-PA and the variants with the chromogenic peptide substrate DIleProArgpNA. The results show that the t-PA heavy chain is not affecting the reaction with small peptide substrates as the K m and k cat values were essentially identical for t-PA, K2P, and FK2P (K m , 0.18–0.21 mM and k cat , 8.8–11.1s −1 ). The values for the two non-glycosylated variants K2P(Gln448) and FK2P(Gln448) were 0.28mM, 9.4s −1 and 0.24mM, 5.3s −1 , respectively. Interestingly, the Val277 variants showed significantly reduced K m values, suggesting that Lys 277 is important for the substrate interaction. For the variant K2P(Val277) K m and k cat were 0.06mM and 6.5s −1 , respectively, and for FK2P(Val277) the corresponding values were 0.06mM and 6.0s −1 .

The in vitro fibrinolytic activities and fibrin specificity in human plasma as well as in vivo fibrinolytic and pharmacokinetic properties in rabbits were studied for six genetically engineered variants of human tissue-type plasminogen activator (t-PA). All variants lacked the growth factor (G) domain and the first kringle (K1) domain and varied in the presence or absence of the finger (F) domain. Additional specific site modifications were Lys 277 →Val and Asn 448 →Gln. The variants were denoted K2P, K2P(Val277), K2P(Gln448), FK2P, FK2P(Val277) and FK2P(G1n448). The highest specific activity in vitro in a plasma clot lysis assay was observed for the wild type t-PA and decreased in the order FK2P(Val277)≈K2P(Val277)>FK2P≈K2P>K2P(Gln448)>FK2P(Gln448). All variants displayed a time lag phase of 30–60 min before fibrinolysis was detected. This lag phase was far less pronounced for the wild type molecule. The fibrin specificity of the variants in vitro was studied by analyses of clottable fibrinogen and consumption of α 2 -antiplasmin after incubating human plasma with fibrinolytically equipotent doses of the different activators. Variants K2P(Val277) and FK2P(Val277) were as fibrin specific as t-PA, whereas K2P, FK2P and the non-glycosylated K2P(Gln448) consumed more fibrinogen and α 2 -antiplasmin at fibrinolytically equipotent doses. However, all t-PA variants were more fibrin specific than streptokinase. The thrombolytic and pharmacokinetic properties of t-PA and five of the variants were examined in a rabbit jugular vein thrombosis model. The thrombolytic activity in vivo following a bolus injection of 0.3mg/kg was, in order of descent, K2P>FK2P>K2P(Val277)≈FK2P(Val277)>K2P(Gln448)≈rt-PA. Two of the variants and rt-PA were investigated at two additional doses (0.6 and 1.2mg/kg). Their ED 50 (dose required to lyse 50% of the thrombus within 3h), as calculated from the dose-response curve, were 1.4, 1.25 and 0.4 mg/kg for rt-PA, K2P(Val277) and K2P, respectively. When rt-PA and the variants were compared at equivalent doses (weight) the variants were found to cause a significantly higher consumption of fibrinogen and α 2 -antiplasmin. However, when the compounds were compared at a thrombolytically equivalent dose the two most potent variants, K2P and FK2P, caused the same haemostatic effect as t-PA, whereas the less potent variants were found to have a reduced fibrin specificity in vivo. All of the variants had a significantly slower clearance and a longer plasma half-life compared to rt-PA. Differences in pharmacokinetic profile were also seen among the five variants, as those containing the finger domain had a faster clearance and a shorter half-life than those without the finger.

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 x 10(8) to 2 x 10(8) M-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.

A gene encoding a variant (lacking amino acids 6–173) of human tissue-type plasminogen activator (t-PA), consisting only of the second kringle domain (K2) and the serine protease domain (P), was fused to a DNA segment coding for the signal peptide of staphylococcal protein A and a synthetic gene coding for a protein with ability to bind immunoglobulin G (IgG). The fusion protein which was synthesized in Escherichia coli and secreted into the growth medium, was found to be fibrinolytically active. Purification of the fusion protein was performed in a single step by affinity chromatography with immobilized IgG. Enzymatically active K2P was liberated from the fusion protein by cleavage at a unique Asn-Gly dipeptide sequence using hydroxylamine. These results demonstrate that a variant of human t-PA can be synthesized and secreted by E. coli as a fibrinolytically active fusion protein, which upon specific cleavage yields an active variant t-PA of the expected size.

Procedures for the purification of bovine muscle carbonic anhydrase (isoenzyme III) are described. The purified enzyme has a molecular weight near 29,000 and contains one Zn2+ ion per molecule. The sedimentation coefficient, s(0)20,w, is 2.8 X 10(-13) s, the isoelectric pH is 8.5, and A280(0.1%) = 2.07 cm-1. The CO2 hydration activity, expressed as kcat/Km, is about 1.5% of that of human isoenzyme I (or B) and about 0.3% of that of human isoenzyme II (or C) at pH 8 and 25 degrees C. The activity is nearly independent of pH between pH 6.0 and 8.6. The muscle enzyme is weakly inhibited by the sulfonamide inhibitor, acetazolamide, whereas some anions, particularly sulfide and cyanate, are efficient inhibitors. Bovine carbonic anhydrase III contains five thiol groups, two of which react readily with Ellman's reagent without effect on the catalytic activity. A reinvestigation of the amino acid sequences of cysteine-containing tryptic peptides has shown that cysteine residues occur at sequence positions 66, 183, 188, 203, and 206.

Several fusions between the gene for human insulin-like growth factor I (IGF-I) and the genes for different IgG-binding fragments of staphylococcal protein A were assembled and compared regarding expression, secretion, and purification of the peptide hormone. After IgG affinity purification of the fusion proteins from the growth medium of Staphylococcus aureus or Escherichia coli, native IGF-I was released by cleavage of an Asn-Gly peptide bond with hydroxylamine. An optimized expression system based on a modified synthetic IgG-binding domain (z), resistant to hydroxylamine, gave the highest yield of fusion protein. After cleavage, the hormone could be separated from the IgG-binding moiety and from noncleaved fusion protein by a second passage through the IgG affinity column. The biological activity and the purity of the IGF-I obtained were confirmed by a radioreceptor assay, N-terminal sequence analysis, polyacrylamide gel electrophoresis, isoelectric focusing, and high-performance liquid chromatography.

Tissue plasminogen activator, separated into variants I and II (differing in Mr by 2000-3000), was reduced and [14C]carboxymethylated. Fragments from cleavages with enzymes and cyanogen bromide (CNBr) were separated by reverse-phase high-performance liquid chromatography and subjected to sequence degradations. All seven CNBr fragments were purified and found to be compatible with the cDNA-derived amino acid sequence [Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N., Vehar, G. A., Ward, C. A., Bennett, W. F., Ylverton, E., Seeburg, P. H., Heynecker, H. L., Goeddel, D. V., & Collen, D. (1983) Nature (London) 301, 214-221]. Chemical characterization of 93% of the 527 residues recovered in 50 peptides confirmed the indirectly deduced primary structure of the protein. The tryptic peptide patterns from the two variants were found to differ for one peptide (T15). Since carbohydrate was present in this peptide for variant I and since a marked difference in chromatographic behavior for T15 was observed in variant II, we conclude that carbohydrate differences in this peptide (i.e., Asn-184 in the numbering system of the cDNA-derived amino acid sequence) are the explanation for the size differences between variants I and II. Carbohydrate was also found at two other positions in the protein, corresponding to Asn-117 and Asn-448. However, a fourth potential glycosylation site, Asn-218, is apparently not utilized for carbohydrate attachment. The enzyme is inactivated by diisopropyl phosphorofluoridate, which covalently modifies the serine residue corresponding to position 478, identifying this as the active site serine residue.(ABSTRACT TRUNCATED AT 250 WORDS)

The plasminogen activator from a human melanoma cell line was purified with immunoadsorption as a major step. The cells were cultured in the presence of aprotinin in order to avoid proteolysis. A three-step purification involved adsorption on antibodies to porcine tissue plasminogen activator before chromatographies on arginine-Sepharose and Sephadex G-150. All solvents contained Tween-80 (0.01%) and, except for the last step, aprotinin. The final product had a specific activity of about 220000 IU/mg measured against the WHO urokinase standard. The activator obtained has an apparent Mr of 72000 and consists of single-chain molecules. Evidence was obtained that four different types of activator variants occur. First and known previously, the one-chain form can be proteolytically cleaved into a two-chain form. Secondly, both the one-chain and two-chain molecules exhibit two forms with molecular weight differences of about 3000 (possibly due to carbohydrate differences). Thirdly, the one-chain preparations contain two variants, each constituting about 50% of the material and differing in length by three N-terminal amino acids. Finally, a possible positional microheterogeneity was detected. Digestion with plasmin yields the two-chain form with disulfide-bonded polypeptide chains, 'A' and 'B' (from the N-terminal and C-terminal parts, respectively). At the same time, the variability of the original N terminus is removed. The A chain keeps the two Mr variants (now about 40000 and 37000, respectively). The B chain (Mr about 33000) contains the active site of the molecule, as demonstrated by labelling with [3H]diisopropyl phosphofluoridate, and is homologous to the enzymatically active chains of thrombin, plasmin and other serine proteases. In contrast to these enzymes, the plasminogen activator is enzymatically active in the one-chain form. A speculative explanation for this activity may possibly be the presence of an epsilon-amino group of a lysine residue at a position close to the bond cleaved in the two-chain form.

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.

Tissue plasminogen activator purified from human uterine tissue exhibits differences in N-terminal starting positions in relation to the melanoma cell plasminogen activator usually studied. A new starting position is compatible with an additional N-terminal processing apart from those already known. Like the melanoma activator, the uterine activator was found to yield protein chains starting at either of two positions. One of these was identical between uterine and melanoma activators, whereas the other was unique in each case. The most abundant starting position for the uterine preparation was at a valine residue, apparently from cleavage of a Gln-Val bond, and corresponding to Val-7 of the longest form of the melanoma activator chain.

The native one-chain tissue plasminogen activator from the human melanoma cell line (Bowes) occurs in two variants as demonstrated by sodium dodecylsulphate—polyacrylamide gel electrophoresis of reduced and carboxymethylated samples. The two variants were isolated by affinity chromatography on arginine—Sepharose using a guanidinium—HCl gradient. in the order of elution, the variants were designated tissue plasminogen activator I and II. Variant I was found to be 3000 Mr larger than variant II and the difference was found to reside in the N-terminal half of the molecule. No substantial difference in carbohydrate content nor in enzymatic activity was demonstrated.

A cDNA fragment encoding the human tissue-type plasminogen activator was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus downstream from the polyhedrin promoter. The induction kinetics of t-PA was followed, after infection of Spodoptera frugiperda cells, at both mRNA and protein levels. Fibrinolytically active plasminogen activator accumulated in the culture medium and reached 2.5 μg/ml after 120 h. The protein was compared with recombinant plasminogen activator produced in mouse cells and was found to be slightly smaller. This difference in size was found to be caused by N -linked oligosaccharides which are shorter in the recombinant activator obtained from insect cells. The molecules produced in such cells contain at least two different types of N -linked glycans, since only one out of three oligosaccharides is sensitive to endoglycosidase H. However, all glycan structures bind strongly to concanavalin A-Sepharose.

Tissue plasminogen activator was treated with Sepharose-bound trypsin or chymotrypsin. Trypsin rapidly converted the one-chain activator to the two-chain form. This caused a marked increase in the amidolytic activity, while plasminogen activation initially increased but then decreased again. SDS/polyacrylamide gel electrophoresis in combination with [3H]diisopropylfluorophosphate active-site labeling revealed that after the conversion to the two-chain activator a minor cleavage occurred in the B chain, while the A chain was substantially degraded. Chymotrypsin caused a marked decrease in both amidolytic activity and plasminogen activation. SDS/polyacrylamide gel electrophoresis under reducing conditions revealed that two pairs of new bands had appeared, with Mr or about 50000/52000 and 17000/20000 respectively. N-terminal sequence analysis identified cleavage sites at peptide bonds 420–421 and 423–424. These bonds are located in a region of the activator which is homologues to the segments of trypsin and chymotrypsin, where autocatalytic cleavages occur during their activations. However, treatment of two-chain activator with chymotrypsin had markedly less effect on plasminogen activation and amidolytic activity. By treatment of samples of chymotrypsin-digested one-chain activator with plasmin, amidolytic activity could be largely restored. Thus, chymotrypsin may, by cleaving bonds 420–421 and 423–424, convert the active one-chain activator into an ‘inactive’ zymogen, which is again ‘activated’ by plasmin cleavage.

Tissue plasminogen activator was purified in high yield from pig heart by immunoaffinity chromatography and characterized by analysis of the glycosylation pattern and the N-terminal amino acid sequence. Comparisons with the human enzyme reveals residue exchanges in the A-chain at positions 3 (porcine Arg/human Gin) and 5 (Thr/Ile), and in the B-chain at positions 6 (Tyr/Phe), 10 (Thr/Ala) and 20 (Val/Ala). The glycosylation pattern for the porcine activator was determined by endoglycosidase treatment followed by gel electrophoresis. The A-chain contains a single high-mannose type of JV-linked glycan structure and the B-chain contains a complex type of oligosaccharide. A similar but not identical pattern has been observed for the human activator, purified from melanoma cells.