Gunnar Pohl

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA)

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 × 108 to 2 × 108 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.

Isolation of two variants of native one-chain tissue plasminogen activator

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 M r 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.

Purification and some properties of carbonic anhydrase from bovine skeletal muscle

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 Ellmans 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.

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1-3)insulin-like growth factor I.

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.

Differences between uterine and melanoma forms of tissue plasminogen activator

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.

Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator

Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of a biologically active variant form of recombinant human secretin.

A biologically active variant form of recombinant human secretin was produced using a gene fusion system designed to facilitate the purification of the protein. The fusion protein was recovered from the culture medium of Escherichia coli by IgG affinity chromatography, and recombinant secretin was released by cyanogen bromide treatment. A novel approach involving addition of a C-terminal Gly-Lys-Arg extension, was used to overcome the lack of amidation of recombinant proteins in Escherichia coli. The biological activity of the recombinant variant of secretin was at least 80% of the porcine secretin standard.

Binding of tissue plasminogen activator to endothelial cells: The effect on functional properties. Localization of a ligand in the B-chain of tPA

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 immortalized microvascular EC (HMEC). By determinations of the concentration-dependent binding it was shown that both the A-chain and the B-chain, which were isolated after partial reduction of two-chain tPA, contain ligands for binding to EC. The affinity for the B-chain was much higher than for the A-chain according to Scatchard analysis (Kd 24 and 515 nM, respectively), whereas the number of binding sites was higher for the A-chain than for the B-chain (Bmax 8 x 10(5) and 1.2 x 10(5), respectively). There were no cross interactions between the A- and B-chains and their binding sites. The binding of tPA to EC induced an almost 100-fold increase of the activation rate when compared to the same amount of enzyme in free solution, which in contrast to the fibrin-induced stimulation was not inhibited by antibodies against fibrin. The enzymatic activity of the B-chain was much less affected by the association to the cells. Both tPA and the tPA B-chain were largely protected against inhibition by an excess plasminogen activator type-1 (PAI-1) when bound to EC, whereas the same amount of free tPA was totally inactivated. The competition studies strongly indicated that an N-terminal segment in the B-chain, AKHRRSPGER, may be the ligand part of the B-chain. It is interesting to note that this polypeptide segment also participates in a binding site for PAI-1, necessary for effective inhibition. This implies a possible competition between PAI-1 and a tPA-receptor for binding of tPA. High molecular weight urokinase had no quenching effect on the binding of the B-chain to EC.

Tissue plasminogen activator mutants lacking the growth factor domain and the first kringle domain: II: Enzymatic properties in plasma and in vivo thrombolytic activity and clearance rates in rabbits

Abstract 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.

Characterization of receptors interacting specifically with the B-chain of tissue plasminogen activator on endothelial cells

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.