Recombinant human microplasmin: production and potential therapeutic properties.

The effect of recombinant human microplasmin was studied in ischemic stroke models in mice and in an extracorporeal loop thrombosis model in rabbits. Human microplasminogen ( micro Plg), which lacks the five 'kringle' domains of plasminogen was expressed with high yield in Pichia pastoris. It was purified, converted to microplasmin ( micro Pli) and equilibrated with 5 mmol L(-1) citrate, pH 3.1, yielding a stable preparation. In mice with middle cerebral artery (MCA) ligation, an intravenous (i.v.) bolus of 5.0 mg kg(-1) micro Pli reduced infarct size at 24 h from 27 (26-30) to 25 (21-28) mm3 (median and range, n= 16 each, P= 0.0001), whereas 4.0 mg kg(-1) rt-PA and 40 mg kg(-1) micro Plg had no effect. Infarct reduction was observed with administration at 4 h after occlusion. In mice with MCA, infarct size at 24 h was reduced from 20 (14-30) to 9.1 (3.1-25) mm3 with 5.0 mg kg(-1) micro Pli (n = 15 each, P < 0.002) and to 11 (5.2-27) mm3 with 4.0 mg kg(-1) rt-PA (n = 6; P= 0.02). Infarct reduction was still observed at 10 h after occlusion with micro Pli but not with t-PA. In rabbits with radiolabeled clots in an extracorporeal arteriovenous loop, local infusion of 2.5 mg kg(-1) micro Pli over 2 h, induced 51 +/- 15% lysis (mean +/- SD, n= 11) vs. a control value of 23 +/- 5.5%. micro Pli did not prolong template bleeding times, whereas equipotent doses of rt-PA were associated with extensive rebleeding. The potency of micro Pli in both models was similar to that of intact plasmin. These findings indicate that recombinant micro Pli may be useful for treatment of ischemic stroke and arterial thrombosis.

Polyethylene glycol-derivatized cysteine-substitution variants of recombinant staphylokinase for single-bolus treatment of acute myocardial infarction.

BACKGROUND: Thrombolytic therapy of acute myocardial infarction (AMI) is evolving toward bolus administration. Derivatization of proteins with polyethylene glycol (PEG) may reduce their clearance. METHODS AND RESULTS: A staphylokinase (SakSTAR) variant with 12 amino acid substitutions to reduce its antigenicity, SakSTAR (K35A, E65Q, K74R, E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T, K135R), and with Ser in position 3 mutated into Cys (code SY161), was derivatized with maleimide-PEG with M:(r) of 5,000 (P5), 10,000 (P10), or 20,000 (P20). The PEGylated variants recognized only one third of the antibodies elicited with wild-type SakSTAR in AMI patients. In experimental animals, plasma clearances were reduced 2. 5- to 5-fold with P5, 5- to 20-fold with P10, and 20-fold with P20, and bolus injection induced pulmonary plasma clot lysis at doses inversely related to their clearance. Intravenous bolus injection of 5 mg of the P5, P10, or P20 variants in AMI patients was associated with plasma half-lives (t(1/2alpha)) of 13, 30, and 120 minutes and clearances of 75, 43, and 8 mL/min, respectively, compared with 3 minutes and 360 mL/min for SakSTAR. Injection of 5 mg P5 variant restored TIMI-3 flow within 60 minutes in 14 of 18 AMI patients (78%, 95% CI 55% to 91%) and of 2.5 mg in 7 of 11 patients (63%, 95% CI 35% to 85%), both in the absence of fibrinogen degradation. The immunogenicity of the variants was significantly (P:<0.002) reduced. CONCLUSIONS: The staphylokinase variant SY161-P5, derivatized with one linear polyethylene glycol molecule of M:(r) 5000, is a promising fibrin-selective agent for single-bolus coronary thrombolysis.

Plasminogen binding properties of macrophage inflammatory protein (MIP)-2alpha.

The chemokine macrophage inflammatory protein (MIP)-2alpha was identified as a plasminogen binding protein by phage display analysis. MIP-2alpha and a truncated form lacking 5 lysine residues in the COOH-terminal region (mut-MIP-2alpha) were expressed in E. coli and purified to apparent homogeneity. Purified MIP-2alpha but not mut-MIP-2alpha bound specifically to plasminogen, with K(A) of 3.7 X 10(5) M(-1) for the interaction of plasminogen with surface-bound MIP-2alpha. Binding and competition experiments indicated that the interaction involves the region comprising the first 3 kringles of plasminogen and the COOH-terminal lysine-rich domain of MIP-2alpha. Activation of plasminogen bound to surface-associated MIP-2alpha by two-chain urokinase-type plasminogen activator (tcu-PA) was about 2.5-fold more efficient than in solution (catalytic efficiency k(cat)K(M) of 0.1 microM(-1)s(-1), as compared to 0.04 microM(-1)s(-1). In contrast, binding of plasminogen to MIP-2alpha in solution was very weak, as evidenced by the absence of competition of MIP-2alpha with lysine-Sepharose or with human THP-1 cells for binding of plasminogen. In agreement with this finding, addition of excess MIP-2alpha did not affect the main functional properties of plasmin(ogen) in solution, as indicated by unaltered activation rates of plasminogen by tcu-PA or tissue-type plasminogen activator (t-PA), t-PA-mediated fibrinolysis, and inhibition rate of plasmin by alpha2-antiplasmin. Thus, association of MIP-2alpha with surfaces exposes its COOH-terminal plasminogen-binding site, and may result in enhanced local plasmin generation.

Recombinant staphylokinase variants with reduced antigenicity due to elimination of B-lymphocyte epitopes.

Site directed mutagenesis (350 variants) of recombinant staphylokinase (SakSTAR), a potent fibrin-selective thrombolytic agent, was undertaken in order to reduce its antigenicity while maintaining its potency. Variants with K35A, (ie, Lys[K] in position 35 substituted with Ala[A]), E65D or E65Q, K74R or K74Q, E80A+D82A, K130T, and K135R displayed increased enzymatic activity or reduced binding of human staphylokinase-specific antibodies. Additive mutagenesis identified 8 variants with intact thrombolytic potencies, which absorbed down to less than a third of SakSTAR-specific antibodies. Intra-arterial administration in 61 patients with peripheral arterial occlusion caused no significant allergic reactions. Median neutralizing antibody titers (with 15 to 85 percentiles), expressed as microgram (microg) compound neutralized per milliliter plasma, were 4.4 (0.3 to 49) for the variants, compared with 12 (4 to 100) in 70 patients given wild-type SakSTAR (P =.002 by Mann-Whitney rank sum test). Overt neutralizing antibody induction (more than 5 microg compound neutralized per milliliter plasma) was observed in 57 of 70 patients (81%) given wild-type SakSTAR, but only in 28 of 60 patients (47%) treated with variants (P <.0001 by Fisher exact test). On the basis of this study, the variant SakSTAR (K35A, E65Q, K74R, D82A, S84A, T90A, E99D, T101S, E108A, K109A, K130T, K135R) (code SY155) has been selected for further clinical development. (Blood. 2000;96:1425-1432)

Outcome and one year follow-up of intra-arterial staphylokinase in 191 patients with peripheral arterial occlusion.

Wild-type or equipotent variants of recombinant staphylokinase (rSak) were given intra-arterially (as a 2 mg bolus injection followed by an infusion of 1 mg/h or 0.5 mg/h overnight, with concomitant heparin [1000 IU/h]) to 191 patients of less than 80 years (62 +/- 1 years, mean +/- SEM), with a peripheral arterial occlusion (PAO) of less than 120 days (mean 14 +/- 1 days, median 11 days, 5 to 95 percentiles 3 to 30 days). Ninety nine patients presented with acute or subacute ischemia, 57 with severe claudication, 33 with chronic rest pain and 2 with gangrene. Occlusion occurred in 122 native arteries and in 69 grafts. Revascularization was complete in 83 percent (158/191), partial in 13 percent (24/191) and absent in 4 percent (7/191) after administration of 12 +/- 0.5 mg rSak over 14 +/- 0.7 h. Complete revascularization of acute occlusions of popliteal or more distal arteries was less frequent (60 percent, 15/25) than of acute occlusions of more proximal native arteries (95 percent, 37/39, p <0.001) or grafts (89 percent, 50/56, p = 0.005). Additional endovascular procedures were performed in 47 percent and subsequent elective bypass surgery in 23 percent of patients. Major bleeding occurred in 12 percent (23/191), one month mortality was 3.1 percent (6/191) and one year mortality was 6.9 percent (12/174). However, four patients (2.1 percent) had an intracranial bleeding following therapy: a 85 year old woman with severe diabetic arteriopathy, who was included in violation of the protocol, a 79 and a 74-year-old woman and a 74-year-old man, all with severe hypertension and limb threatening ischemia; these four patients died within two months after treatment. Amputations were performed within the first year in 16 of 162 surviving patients (9.8 percent): in 7 percent (7/96) with an occluded native artery and 14 percent (9/66) with an occluded graft (p = 0.19). No significant difference in lysis rate, one month mortality or one year amputation-free survival was observed in occlusions of recent onset (< or =14 days, n = 126) as compared to occlusions of longer duration (>14 days, n = 65). Treatment was interrupted prematurely in 4 patients because of a suspected allergic reaction. Fibrinogen levels remained unaffected during treatment (3.3 +/- 0.1 g/l before vs. 3.3 +/- 0.1 g/l after infusion, n = 167). In conclusion, rSak appears to be a highly effective thrombolytic agent in patients with PAO, resulting in a low one month mortality (3.1 percent) and a high one year amputation free survival (84 percent), with an acceptable incidence of major bleedings, but with occasional fatal intracranial hemorrhages.

Recombinant staphylokinase variants with altered immunoreactivity. III: Species variability of antibody binding patterns.

BACKGROUND: The "charged cluster-to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%. Therefore, the effect of the reversal of one or more of these substituted amino acids on the ratio of activity to antigenicity was studied. METHODS AND RESULTS: Fourteen mutants with one to four "alanine-to-wild-type" reversals were expressed in Escherichia coli and highly purified (> 95%). In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodominant residue could be identified. In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epitopes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent. In pooled plasma from patients with peripheral arterial occlusion treated with wild-type SakSTAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R77 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible contribution toward antibody recognition. CONCLUSIONS: The recognition pattern by SakSTAR variants of antibodies induced with wild-type SakSTAR differs markedly among species. This implies that a systematic evaluation of reduced antigen recognition and antibody induction in humans will require the development of human or humanized systems. Surprisingly, SakSTAR(K74), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies induced in patients by treatment with wild-type SakSTAR.

Recombinant staphylokinase variants with altered immunoreactivity. I: Construction and characterization.

BACKGROUND: Recombinant staphylokinase offers promise for thrombolytic therapy in acute myocardial infarction, but it is immunogenic. Although reduced immunogenicity of heterologous proteinaceous drugs by protein engineering has not previously been reported, an attempt was made to achieve this in staphylokinase by site-specific mutagenesis. METHODS AND RESULTS: Biospecific interaction analysis of a panel of 17 murine monoclonal antibodies against recombinant staphylokinase (SakSTAR variant) identified three nonoverlapping immunodominant epitopes, two of which could be eliminated by substitution mutagenesis of clusters of two or three charged amino acids with alanine. Circulating anti-staphylokinase antibodies elceted in patients by treatment with SakSTAR were incompletely (< 90%) absorbed by these mutants. Therefore, the combination variants K35A,E38A,K74A,E75A,R77A (SakSTAR.M38) and K74A,E75A,R77A,E80A,D82A (SakSTAR.M89) were constructed, expressed in Escherichia coli, highly purified by ion-exchange and hydrophobic interaction chromatography, and characterized. These variants had specific activities that were approximately half that of SakSTAR, and they combined the reduced reactivity with the panels of monoclonal antibodies of their parent molecules. Absorption of circulating antibodies elicited in patients by treatment with SakSTAR was incomplete in 13 of 16 patients (median values, 68% and 65% with SakSTAR.M38 and SakSTAR.M89, respectively). CONCLUSIONS: SakSTAR contains three immunodominant epitopes, two of which were eliminated by site-directed mutagenesis, yielding combination mutants with relatively maintained specific activities that were not recognized by a significant fraction of the antibodies elicited in patients by treatment with wildtype SakSTAR. These mutants appear to be suitable for more detailed investigation of their thrombolytic and antigenic properties.

Anticoagulant repertoire of the hookworm Ancylostoma caninum.

Hookworms are hematophagous nematodes that infect a wide range of mammalian hosts, including humans. There has been speculation for nearly a century as to the identity of the anticoagulant substances) used by these organisms to subvert host hemostasis. Using molecular cloning, we describe a family of potent small protein (75-84 amino acids) anticoagulants from the hookworm Ancylostoma caninum termed AcAP (A. caninum anticoagulant protein). Two recombinant AcAP members (AcAP5 and AcAP6) directly inhibited the catalytic activity of blood coagulation factor Xa (fXa), while a third form (AcAPc2) predominantly inhibited the catalytic activity of a complex composed of blood coagulation factor VIIa and tissue factor (fVIIa/TF). The inhibition of fVIIa/TF was by a unique mechanism that required the initial formation of a binary complex of the inhibitor with fXa at a site on the enzyme that is distinct from the catalytic center (exo-site). The sequence of AcAPc2 as well as the utilization of an exo-site on fXa distinguishes this inhibitor from the mammalian anticoagulant TFPI (tissue factor pathway inhibitor), which is functionally equivalent with respect to fXa-dependent inhibition of fIIa/TF. The relative sequence positions of the reactive site residues determined for AcAP5 with the homologous regions in AcAP6 and AcAPc2 as well as the pattern of 10 cysteine residues present in each of the inhibitors suggest that the AcAPs are distantly related to the family of small protein serine protease inhibitors found in the nonhematophagous nematode Ascaris lumbricoides var. suum.

High-level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris.

Tick anticoagulant peptide (TAP) is a potent and specific inhibitor of the blood coagulation protease Factor Xa. We designed and assembled a synthetic TAP-encoding gene (tapo) based on codons preferentially observed in the highly expressed Pichia pastoris alcohol oxidase 1 gene (AOX1), and fused it to a novel hybrid secretory prepro leader sequence. Expression from this gene yielded biologically active rTAP, which was correctly processed at the amino-terminal fusion site, and accumulated in the medium to approximately 1.7 g/l. This corresponds to a molar concentration of 0.24 mM, and is the highest yet described for a recombinant product secreted from P. pastoris. It also represents a seven-fold improvement in productivity compared to rTAP secretion from Saccharomyces cerevisiae, making P. pastoris an attractive host for the industrial-scale production of this potential therapeutic agent. This system was also used to prepare 21 mg 15N-rTAP, 11 mg 13C-rTAP and 27 mg 15N/13C-rTAP, with isotope incorporation levels higher than 98%, and purities sufficient to allow their use in determining the solution structure of the tick anticoagulant peptide using high field NMR.

A hookworm glycoprotein that inhibits neutrophil function is a ligand of the integrin CD11b/CD18.

The chronic survival of many endoparasites is dependent on the ability of these organisms to escape the host immune response. Identification of the molecular mechanisms by which these organisms evade this response may yield novel approaches in the development of anti-inflammatory agents. We describe here the discovery and characterization of a novel 41-kilodalton glycoprotein from the canine hookwork (Ancylostoma caninum) that potently inhibits CD11/CD18-dependent neutrophil function in vitro. Neutrophil inhibitory factor (NIF) blocks the adhesion of activated human neutrophils to vascular endothelial cells as well as the release of H2O2 from activated neutrophils, over a similar concentration range (IC50 10-20 nM). Studies aimed at determining the nature of the NIF binding site on neutrophils revealed selective, high affinity binding of this protein to the integrin CD11b/CD18. A cDNA encoding NIF was isolated from a canine hookworm cDNA library. NIF comprises a mature polypeptide of 257 amino acids, preceded by a 17-amino acid leader. The mature protein has 10 cysteines and has seven potential N-linked glycosylation sites. NIF has no significant sequence homologies to any previously reported protein. As such, NIF represents a prototype of a novel class of leukocyte function inhibitors.

Pharmacokinetic and thrombolytic properties of chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody fused to single-chain urokinase.

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single-chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady-state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody-targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.

Biochemical characterization of single-chain chimeric plasminogen activators consisting of a single-chain Fv fragment of a fibrin-specific antibody and single-chain urokinase.

K12G0S32 is a 57-kDa recombinant single-chain chimeric plasminogen activator consisting of scFv-K12Go, a single-chain variable-region antigen-binding fragment (Fv) of the monoclonal antibody MA-15C5, which is specific for fragment D-dimer of human cross-linked fibrin, and a low-molecular-mass (33 kDa) urokinase-type plasminogen activator (u-PA-33k) containing amino acids Ala132-Leu411 (Holvoet, P., Laroche, Y., Lijnen, H. R., Van Cauwenberghe, R., Demarsin, E., Brouwers, E., Matthyssens, G. & Collen D. (1991) J. Biol. Chem. 266, 19717-19724). In addition, the Arg156-Phe157 thrombin-cleavage site in the u-PA moiety of K12G0S32 is removed by substitution of Phe157 with Asp. In the present study, the fibrinolytic potency of K12G0S32, determined in a system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated plasma, was found to be only twofold higher than that of intact single-chain u-Pa (rscu-PA), but 17-fold higher than that of rscu-PA(M), a variant of rscu-PA in which the thrombin-cleavage site was removed by substitution of Phe157 with Asp. The fibrinolytic potency of K12G0S32T, with an intact thrombin-cleavage site, was 6-15-fold higher than that of rscu-PA. Conversion of 1 microM single-chain K12G0S32 or rscu-PA(M) into their two-chain derivatives with plasmin occurred at a rate of 1.0 +/- 0.15 nmol.min-1.nmol plasmin-1 and 0.85 +/- 0.074 nmol.min-1.nmol plasmin-1, compared to 14 +/- 2.3 nmol.min-1.nmol plasmin-1 and 18 +/- 2.6 nM.min-1.nmol plasmin-1 for K12G0S32T and rscu-PA, respectively. Purified fragment D-dimer of human cross-linked fibrin inhibited the fibrinolytic potency of single-chain K12G0S32T, but not of two-chain K12G0S32T, in a dose-dependent manner. Furthermore, the fibrinolytic potencies of two-chain K12G0S32 and K12G0S32T were not significantly higher than those of recombinant two-chain u-PA (rtcu-PA) or of rtcu-PA(M). These findings suggest that the 59-fold increase in fibrinolytic potency of K12G0S32T, relative to that of rscu-PA(M), is due both to targeting of the activator to the clot via the single-chain Fv fragment (sixfold increase) and to a more efficient conversion of single-chain K12G0S32T to its two-chain derivative (eightfold increase). Thus, targeting to clots by means of fibrin-specific antibodies results in a significant increase of the fibrinolytic potency of single-chain but not of two-chain u-PA.(ABSTRACT TRUNCATED AT 400 WORDS)

Arginine residues as stabilizing elements in proteins.

Site-specific substitutions of arginine for lysine in the thermostable D-xylose isomerase (XI) from Actinoplanes missouriensis are shown to impart significant heat stability enhancement in the presence of sugar substrates most probably by interfering with nonenzymatic glycation. The same substitutions are also found to increase heat stability in the absence of any sugar derivatives, where a mechanism based on prevention of glycation can no longer be invoked. This rather conservative substitution is moreover shown to improve thermostability in two other structurally unrelated proteins, human copper, zinc-superoxide dismutase (CuZnSOD) and D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus subtilis. The stabilizing effect of Lys----Arg substitutions is rationalized on the basis of a detailed analysis of the crystal structures of wild-type XI and of engineered variants with Lys----Arg substitution at four distinct locations, residues 253, 309, 319, and 323. Molecular model building analysis of the structures of wild-type and mutant CuZnSOD (K9R) and GAPDH (G281K and G281R) is used to explain the observed stability enhancement in these proteins. In addition to demonstrating that even thermostable proteins can lend themselves to further stability improvement, our findings provide direct evidence that arginine residues are important stabilizing elements in proteins. Moreover, the stabilizing role of electrostatic interactions, particularly between subunits in oligomeric proteins, is documented.

Characterization of a chimeric plasminogen activator consisting of a single-chain Fv fragment derived from a fibrin fragment D-dimer-specific antibody and a truncated single-chain urokinase.

An Mr 57,000 single-chain chimeric plasminogen activator, K12G0S32, consisting of a variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 and of a 33-kDa (amino acids Ala132 to Leu411) recombinant single-chain urokinase-type plasminogen activator (rscu-PA-33k) was studied. K12G0S32, secreted by infected Spodoptera frugiperda insect cells at a rate of 1.5 micrograms/10(6) cells/48 h, was purified to homogeneity by ion-exchange chromatography and gel filtration. It was obtained essentially as a single-chain molecule with a Ka = 5.5 x 10(9) M-1 for immobilized fragment D-dimer, similar to that of MA-15C5. The specific activity of both its single-chain and two-chain forms on fibrin plates was 100,000 IU/mg of urokinase-type plasminogen activator (u-PA) equivalent. Activation of plasminogen by two-chain K12G0S32 obeyed Michaelis-Menten kinetics with Km = 2.9 +/- 0.6 microM and a k2 = 3.7 +/- 0.6 s-1 (mean +/- S.D.; n = 3), as compared to Km = 12 microM and k2 = 4.8 s-1 for rtcu-PA-32k (recombinant low Mr two-chain u-PA consisting of amino acids Leu144 to Leu411). Single-chain K12G0S32 induced a dose- and time-dependent lysis of a 125I-fibrin-labeled human plasma clot immersed in citrated human plasma; 50% lysis in 2 h was obtained with 0.70 +/- 0.07 micrograms/ml (mean +/- S.D.; n = 5), as compared with 8.8 +/- 0.1 micrograms/ml for rscu-PA-32k (recombinant low Mr single-chain u-PA consisting of amino acids Leu144 to Leu411) (mean +/- S.D.; n = 3). With two-chain K12G0S32, 50% clot lysis in 2 h required 0.25 +/- 0.03 micrograms/ml (mean +/- S.D.; n = 3), as compared with only 0.62 +/- 0.04 micrograms/ml (mean +/- S.D.; n = 2) for rtcu-PA-32k. These results indicate that low Mr single-chain u-PA can be targeted to a fibrin clot with a single-chain Fv fragment of a fibrin-specific antibody, resulting in a 13-fold increase of the fibrinolytic potency of the single-chain form and a 2.5-fold increase of the potency of the two-chain form.

Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal antibody specific for human fibrin fragment D-dimer.

A recombinant single-chain molecule, scFv-K12G0, containing the variable domains of the monoclonal antibody MA-15C5, specific for fragment D-dimer of human cross-linked fibrin, was constructed and expressed in Spodoptera frugiperda, Sf9, insect cells. The Arg108 carboxyl-terminal amino acid of the variable domain of the light-chain of the antibody was connected through a synthetic Ala-Gly-Gln-Gly-Ser-Ser-Val peptide linker with the Gln1 amino-terminal amino acid of the variable domain of its heavy chain. scFv-K12G0 was secreted by the infected Sf9 cells at a rate of 10 micrograms/10(6) cells within 48 h, resulting in conditioned medium with a maximal concentration of 15 mg of scFv-K12G0/liter. The molecule, purified to homogeneity by ion exchange chromatography and gel filtration, migrated as a single Mr band on reduced sodium dodecyl sulfate-gel electrophoresis. It bound to immobilized fragment D-dimer with an affinity constant of 4.0 x 10(9) M-1 (2.0 x 10(10) M-1 for intact MA-15C5). Clearing of scFv-K12G0 from the circulation in rabbits occurred with an initial half-life (t1/2 alpha) of 10 min and a clearance of 5.1 ml min-1, as compared to 90 min and 210 ml min-1 for intact MA-15C5. Nephrectomy resulted in a prolongation of t1/2 alpha to 110 min, suggesting that the rapid clearance of scFv-K12G0 occurs primarily via the kidney, presumably by glomerular filtration. The results indicate that the single-chain recombinant molecule scFv-K12G0 is secreted in functionally intact form and suggest that it may be useful for targeting of radioisotopes or plasminogen activators to blood clots in vivo.

Yersinia enterocolitica, a primary model for bacterial invasiveness.

Yersinia enterocolitica is now the species of Yersinia most frequently isolated from human and animal infections. The species includes pathogens and ubiquitous strains. Among the human pathogens, those isolated in America are more virulent than those isolated elsewhere, especially in Europe and Japan, and these isolates differ biochemically and serologically. The relation between Y. enterocolitica and Y. pestis only became obvious in 1980 with the discovery that at 37 degrees C Y. enterocolitica requires Ca++, a phenotype described in the 1960s for Y. pestis. This requirement as well as virulence is dependent on a 70-kilobase plasmid found later in Y. pseudotuberculosis and Y. pestis. Thus, many bacteriologists elected Y. enterocolitica as a model for bacterial invasiveness. However, studies with non-American strains were impeded by the lack of an inexpensive, simple animal test, a difficulty now circumvented by supplying an appropriate siderophore to the bacteria. Ca++ dependence can be viewed as a transition between free growth and protection against the immune system. In the latter phase, Y. enterocolitica synthesizes and releases large amounts of six plasmid-encoded outer membrane proteins. Most of these are under the control of the plasmid region governing Ca++ dependence. Mutants in this region either lose the Ca++ requirement at 37 degrees C or become unable to grow at 37 degrees C irrespective of the Ca++ concentration. The complex events leading to Ca++ dependence is still not understood. Virulence in Y. enterocolitica also depends on chromosomal genes: the endocytosis in intestinal epithelial cells seems not to be encoded by the pYV plasmid. Studies of Y. pseudotuberculosis suggest that this property depends on a single chromosomal locus, the study of which might be particularly important in the understanding of the first step in infection.

Comparison of serogrouping and polyacrylamide gel electrophoresis for typing Clostridium difficile.

A typing scheme for Clostridium difficile based on slide agglutination with rabbit antisera was previously described. It allows the differentiation of 10 serogroups designated A, B, C, D, F, G, H, I, K, and X. We studied the correlation between serogrouping and polyacrylamide gel electrophoresis (PAGE) of whole-cell proteins. A total of 202 isolates from different sources were analyzed by PAGE after ultrasonic disintegration of cells from an 18-h liquid culture and treatment with sodium dodecyl sulfate and 2-mercaptoethanol. A total of 21 different patterns were observed. The reference strains from the 10 serogroups showed different profiles. For each serogroup except A, the patterns obtained with the clinical isolates were identical to the patterns obtained with the reference strains. For the 48 strains belonging to serogroup A, 12 different profiles were observed. Five of these involved strains isolated from patients with antibiotic-associated diarrhea. Typing by sodium dodecyl sulfate-PAGE thus correlates with serogrouping. In addition, it allows discrimination within the heterogeneous serogroup A.

Genetic analysis of the plasmid region controlling virulence in Yersinia enterocolitica 0:9 by Mini-Mu insertions and lac gene fusions.

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid (pYVe plasmid) of 70 kilobases (kb) which specifies, at 37 degrees C, a calcium requirement for growth, autoagglutinability, resistance to the bactericidal activity of human serum, and the expression of outer membrane proteins (OMP). Some mutations suppress the calcium requirement for growth while others make the bacteria unable to grow at 37 degrees C, even in the presence of calcium. To analyse the genes involved in these latter phenotypes, the plasmid of a serogroup 0:9 strain was subjected to transposon mutagenesis with a Mini-Mu (Kan, lac) element. The mapping of 15 insertions and the analysis of transcription showed that at least four transcription units, spanning 22 kb, are involved in the phenomenon of calcium dependence. Mutations in two divergent units (virA and virB) suppressed the requirement for calcium at 37 degrees C. When insertions occurred in the other units (virC and virD), the Y. enterocolitica host became thermosensitive for growth. VirA, B and C mutants did no longer express the pYVe dependent OMPs. VirD mutants expressed and released these proteins save two of mol. wt 37,400 and 40,800 daltons. Transcription of the lac genes in the four groups of mutants was dependent on temperature.

Genetic analysis of virulence plasmid from a serogroup 9 Yersinia enterocolitica strain: role of outer membrane protein P1 in resistance to human serum and autoagglutination.

Enteropathogenic strains of Yersinia enterocolitica harbor a virulence plasmid (70 kilobases) which specifies, at 37 degrees C, a calcium requirement for growth, autoagglutinability, resistance to the bactericidal activity of human serum, and the expression of some outer membrane proteins (OMPs). To map the genes encoding these properties, the virulence plasmid of a serogroup 9 strain (W22708) was subjected to transposon mutagenesis. A set of 68 independent mutations was obtained in Escherichia coli by transposon Tn813 (a tnpR mutant of Tn21)-mediated cointegration with the self-transmissible R388 plasmid. The resulting cointegrates were introduced and studied in Y. enterocolitica W22708. One mutant lost the calcium dependence property. Two other mutants presented a peculiar phenotype: they grew poorly at 37 degrees C, especially in the presence of calcium. Lastly, two mutants were affected in the properties of autoagglutination and resistance to human serum. Analysis of the OMP pattern of these two mutants revealed the absence of the largest OMP, called P1 (I. Bölin, and H. Wolf-Watz, Infect. Immun. 43:72-78, 1984). Complementation of one of these mutations with the cloned structural gene of OMP P1 restored the wild-type phenotype. However, OMP P1 was not sufficient by itself to specify the serum resistance property and a rapid autoagglutination of the host.

A simple adult-mouse test for tissue invasiveness in Yersinia enterocolitica strains of low experimental virulence.

The virulence of Yersinia enterocolitica depends on the presence of a 70-kilobase plasmid, called the Vwa plasmid. This situation is particularly favourable for studies of the mechanism of pathogenicity, but these are hindered by the lack of a suitable animal test to monitor the virulence of the human-pathogenic strains isolated outside the USA which belong to serogroups O:3, O:9 and O:5,27. We observed that, after oral administration to the mouse, the Vwa-positive strains of these serogroups produce a discrete systemic infection while the Vwa-negative strains do not. We present here a simple mouse-virulence test based on this observation.