Identification of novel modifiers of Aß toxicity by transcriptomic analysis in the fruitfly.

The strongest risk factor for developing Alzheimer's Disease (AD) is age. Here, we study the relationship between ageing and AD using a systems biology approach that employs a Drosophila (fruitfly) model of AD in which the flies overexpress the human Aß42 peptide. We identified 712 genes that are differentially expressed between control and Aß-expressing flies. We further divided these genes according to how they change over the animal's lifetime and discovered that the AD-related gene expression signature is age-independent. We have identified a number of differentially expressed pathways that are likely to play an important role in the disease, including oxidative stress and innate immunity. In particular, we uncovered two new modifiers of the Aß phenotype, namely Sod3 and PGRP-SC1b.

Recombinant human factor X: high yield expression and the role of furin in proteolytic maturation in vivo and in vitro.

Factor X/Xa plays a pivotal role in the coagulation cascade and exhibits a therapeutic potential for the treatment of factor X-deficient as well as FVIII and FIX inhibitor patients. This report describes the establishment of Chinese hamster ovary cell clones expressing recombinant human factor X up to 120 microg/mL x day and 78 microg/10(6) cells x day, that is to 100-fold higher levels than reported previously. Although propeptide removal and single chain precursor to light and heavy chain processing as well as vitamin K-dependent gamma-carboxylation became impaired at these expression levels, up to 25% of the recombinant human factor X produced was active. This represents the highest functional activity ever reported for a vitamin K-dependent protein at such an expression level. Expression of recombinant human factor X in Chinese hamster ovary cells lacking the endoprotease Furin revealed that propeptide removal still occurred, whereas single chain precursor to light/heavy chain processing was abolished. This suggests that a protease different from Furin mediates propeptide removal, a unique finding compared with the other vitamin K-dependent coagulation factors. In contrast, exposure of incompletely processed rFX molecules to soluble recombinant Furin in vitro mediated both of these cleavage reactions despite the absence of a typical argP4-xP3-lys/argP2-argP1 Furin cleavage site in the propeptide, indicating relaxed specificity in vitro. Concomitantly with the degree of processing, the functional activity of recombinant human factor X increased. Interestingly, Furin was shown to even perform correct N-terminal proteolytic trimming of FX molecules truncated amino-terminal to the P3 residue in vitro. Depending on the absence or presence of warfarin in the culture media, as well as on the processing state, four distinct recombinant human factor X light chain isoforms were observed and their structure characterized. One of these light chain forms correlated with the functional activity. Finally, the distribution of the individual light chain isoforms suggests that gamma-carboxylation may be a prerequisite for propeptide removal.

Recombinant von Willebrand factor: potential therapeutic use.

Human von Willebrand factor (vWF) produced by recombinant technology offers a new perspective in treatment of von Willebrand disease (vWD). Several limitations connected with plasma-derived vWF concentrates, such as proteolytic degradation during the manufacture process, variation in multimer composition, lack of high molecular weight multimers, and donor dependence, can be overcome by rec-vWF. Recombinant vWF (rec-vWF) is produced by continuous fermentation of transformed mammalian cells. Biotechnological processes have been developed to isolated rec-vWF fractions with low, medium, and high degrees of multimerization. Structural analysis of rec-vWF demonstrated that it undergoes post-translational modifications comparable with plasma-derived vWF, such as multimerization, pro-peptide processing, and glycosylation. Functional analysis showed that rec-vWF exhibited activities comparable with plasma-derived vWF, such as platelet binding, platelet aggregation, collagen binding, and coagulation factor VIII (FVIII) binding. Collagen binding and platelet aggregation activity increased with the increasing multimer size of rec-vWF. Infusion of rec-vWF in antibody-induced vWF-deficient mice resulted in a significant decrease in bleeding. Infusion of rec-vWF in vWF-deficient dogs and pigs with severe vWD caused an increase in the endodenous FVIII level. Stabilization of FVIII in vivo was mediated both by high and low molecular weight rec-vWF molecules. Apparently, rec-vWF resisted proteolytic degradation in the circulation and no satellite bands were formed. Functional analysis in vitro and in vivo demonstrated the therapeutic potentials of rec-vWF, correction of vWF level, and stabilization of FVIII in plasma.

Selectivity of von Willebrand factor triplet bands towards heparin binding supports structural model.

Human plasma-derived von Willebrand factor (hp-vWF) and recombinant von Willebrand factor (r-vWF) have been fractionated by heparin affinity chromatography followed by multimer analysis using SDS-agarose gel electrophoresis. Because heparin binding sites are contained in each vWF subunit, high molecular weight multimers of r-vWF and hp-vWF, respectively, were eluted with higher salt concentration, in comparison to r-vWF and hp-vWF molecules with a low degree of multimerization. Heparin affinity chromatography did not affect the multimer composition of r-vWF. By contrast, faster migrating satellite bands and slower migrating satellite bands of hp-vWF exhibited reduced and increased heparin affinity, respectively, compared to the intermediate band of the same triplet. Because heparin binding sites are localised in the N-terminal domain of the hp-vWF subunit, this result confirms a structural model of hp-vWF (Fischer et al., Biochem. J. 1998;331:483-488) suggested recently, in which the slower migrating satellite bands have excess of one N-terminal fragment and the faster migrating satellite bands lack one N-terminal fragment, respectively, in comparison with the corresponding intermediate triplet band.

Strategies for recombinant Furin employment in a biotechnological process: complete target protein precursor cleavage.

Coagulation factors, amongst many other proteins, often require posttranslational endoproteolytic processing for maturation. Upon high yield expression of recombinant forms of these proteins, processing frequently becomes severely limiting, resulting in a hampered function of the protein. In this report, the human endoprotease Furin was used to achieve complete propeptide removal from recombinant von Willebrand Factor (rvWF) precursors in CHO cells. At expression beyond 200 ng rvWF/106 cells x day, processing became insufficient. Stable co- and overexpression of full length Furin resulted in complete precursor cleavage in cell clones expressing 2 mug rvWF/106 cells x day. Rather than occuring intracellularly, processing was found to be mediated by a naturally secreted form of rFurin, present in 100 fold higher concentrations than endogenous Furin and accumulating in the cell culture supernatant. Attempts to increase rFurin yield by amplification, in order to ensure complete rvWF precursor processing at expression rates beyond 2 mug rvWF/106 cells x day, failed. Truncation of the trans-membrane domain resulted in immediate secretion of rFurin and approximately 10 fold higher concentrations in the conditioned medium. In cases where these high rFurin concentrations are not sufficient to ensure complete processing, an in vitro downstream processing procedure has to be established. Secreted affinity epitope-tagged rFurin derivatives were constructed, the fate of which, at expression, was dependent on the size of the C-terminal truncation and the type of the heterologous epitope added. A suitable candidate was purified by a one step affinity procedure, and successfully used for in vitro processing. This allows complete proteolytic processing of large amounts of precursor molecules by comparably small quantities of rFurin. Complete precursor cleavage of a target protein at expression rates of up to approximately 200 ng, 2 mug, and 20 mug, as well as beyond 20 mug/106 cells x day can thus be anticipated to be accomplished by endogenous Furin, additional expression of full length rFurin, co-expression of truncated and hence secreted rFurin, and a protein-chemical in vitro procedure, respectively.

Binding of hirudin to meizothrombin.

Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.

von Willebrand factor: measuring its antigen or function? Correlation between the level of antigen, activity, and multimer size using various detection systems.

von Willebrand factor (vWF) from normal human plasma was purified and separated into three fractions containing high, medium, and low molecular weight vWF multimers. vWF fractions were tested for (1) vWF-antigen (vWF:Ag); (2) vWF-ristocetin cofactor activity (vWF:RiCof); (3) vWF-collagen binding activity (vWF:CBA); and (4) a monoclonal antibody-binding ELISA (mAB-binding ELISA), based on the vWF binding to immobilized monoclonal antibody directed to the glycoprotein Ib-binding region within the A1 domain of vWF. The three different fractions of vWF showed a correlation between multimer size and vWF:RiCof/vWG:Ag and vWF:CBA/vWF:Ag, respectively. In contrast, results obtained with the mAB-binding ELISA showed identical levels of mAB-binding/vWF:Ag, without regard for the multimer size present in the tested fraction. Our results therefore suggest that in the case of structurally normal vWF the mAB-binding ELISA reflects the concentration of vWF:Ag rather than vWF function. It is feasible that while the mAB-binding ELISA may show reduced levels for abnormal vWF protein, structurally altered within the A1 domain of vWF as in some patients with vWD type 2, this assay does not appear to be suitable for functional analysis of structurally intact vWF.

Collagen covalently immobilized onto plastic surfaces simplifies measurement of von Willebrand factor-collagen binding activity.

Human collagen type III was immobilized covalently via activated carbohydrate moieties onto hydrazine-treated microtiter plates which could be used to measure von Willebrand factor (vWF) collagen binding activity (vWF:CBA) in an ELISA. Such plates were simple to prepare and remained stable at 4 degrees C and -20 degrees C for at least 2 months. Samples analyzed by this system included (a) normal human vWF fractionated according to the degree of multimerization, (b) normal citrated and EDTA plasma and corresponding serum, and (c) plasma from patients with von Willebrand disease (vWD) types 1 and 2. When related to the concentration of vWF antigen (vWF:Ag), proportionally low levels of vWF:CBA were found for samples lacking the high-molecular-weight multimers, while higher values were obtained for samples containing these multimers. The ratio of vWF:CBA/vWF:Ag sensitively reflected the functional and structural intactness of the vWF molecules for all analyzed samples. Monoclonal antibody directed to the region within the A1 domain of vWF which interacts with the glycoprotein Ib completely inhibited the vWF ristocetin cofactor (vWF:RistCof), while vWF:CBA was not affected. Thus vWF:CBA and vWF:RistCof clearly represent separate, noninterchangeable functional parameters of vWF. In conclusion, our results indicate that the newly described method for the immobilization of collagen onto microtiter plates is suitable for the determination of vWF:CBA. In conjunction with vWF:Ag and the calculated ratio of vWF:CBA/vWF:Ag, this method simplifies the detection and classification of patients with vWD and assists in quality control during the purification of normal vWF.

Triplet structure of human von Willebrand factor.

Human von Willebrand factor (hp-vWF) is a high-molecular- mass protein found in plasma as a series of multimers. It consists of subunits comprising 2050 amino acids linked by disulphide bonds into multimers of various size ranging in molecular mass up to greater than 10000kDa. Partial proteolysis at position Tyr842-Mer843 of the subunit [Dent et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6306-6310] by a vWF-specific protease [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] results in the generation of an N-terminal and a C-terminal fragment and the appearance of hp-vWF triplet bands. It has been suggested [Furlan et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 7503-7507] that (i) the intermediate triplet band of the primary dimer represents a dimer of two C-terminal fragments, (ii) the slower migrating satellite band of the primary dimer represents an asymmetric structure composed of a mature subunit to which one N-terminal and one C-terminal fragment are linked by disulphide bonds, and (iii) the faster migrating satellite band of the primary dimer contains two N-terminal fragments. Here we used recombinant vWF (r-vWF) for structural analysis of hp-vWF multimers. r-vWF exhibited no proteolytic degradation and all multimers contained mature subunits. High-resolution agarose-gel electrophoresis and two-dimensional electrophoresis demonstrated that (i) r-vWF multimers and hp-vWF intermediate triplet bands exhibited identical molecular mass and electrophoretic mobilities, (ii) the faster and slower migrating satellite bands of hp-vWF differ by less than the molecular mass of one subunit from the corresponding intermediate triplet band, and (iii) the triplet bands of hp-vWF are composed of mature and degraded subunits. The results support a structural model of hp-vWF triplet bands according to which the intermediate triplet bands represent multiple numbers of symmetric and/or asymmetric dimers, the slower migrating satellite bands have one extra N-terminal fragment, and the faster migrating satellite band lacks one N-terminal fragment respectively in comparison with the corresponding intermediate triplet band.

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale.

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.

Production of highly homogeneous and structurally intact recombinant von Willebrand factor multimers by furin-mediated propeptide removal in vitro.

Recombinant human von Willebrand Factor (rvWF), a multimeric glycoprotein essential to haemostasis, has been developed as a potential therapeutic agent for treatment of von Willebrand disease (vWD). Permanent Chinese-hamster ovary (CHO)-rvWF cell clones co-expressing recombinant furin (rfurin) were established in order to ensure complete rvWF propeptide removal [Fischer, Schlokat, Mitterer, Reiter, Mundt, Turecek, Schwarz and Dorner (1995) FEBS Lett. 375, 259-262]. Large quantities of material are required for in vivo tests and clinical studies. This demand is commonly met by achieving high-yield expression of the desired protein via amplification. Co-amplification of rfurin, necessary to completely process increasing amounts of rvWF precursor, could not be accomplished, presumably due to lethal effects of overexpressed rfurin for the host cells [Creemers (1994) Ph.D. Thesis, University of Leuven]. Recent reports have inferred that rfurin can only mediate rvWF processing intracellularly [Rehemtulla and Kaufman (1992) Blood 79, 2349-2355; Rehemtulla, Dorner and Kaufman (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 8235-8239]. We report here that rvWF-precursor processing, however, occurs predominantly extracellularly upon rfurin co-expression. Mixing experiments employing rfurin- as well as rvWF-precursor-containing conditioned media demonstrate that rvWF precursors are accessible and cleavable by rfurin in vitro. Exposure to rfurin in vitro converts the heterogeneous multimer pattern typical of incompletely processed rvWF multimers into highly homogeneous and structurally intact multimers superior to the ones exhibited by plasma-derived vWF. These findings thus demonstrate the feasibility of large-scale production of a completely processed, intact and homogeneous rvWF preparation, based on individual rvWF-precursor high-yield expression and subsequent propeptide removal by rfurin in vitro.

Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus.

Recombinant human prothrombin (r-prothrombin) and recombinant mutant prothrombin with active site Asp419 substituted by Asn (D419N-prothrombin) were expressed in recombinant CHO cells, isolated and purified from the fermentation supernatant. The r-Prothrombin and D419N-prothrombin were digested by both Echis carinatus venom and Oxyuranus scutellatus venom. Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the prothrombin polypeptide chain were analysed. Owing to the recombinant preparation and inactivity of D419N-prothrombin and its activation products, the proteolytic action of E.carinatus and O.scutellatus venoms could be studied without addition of thrombin inhibitor, without interference from autocatalytic digestion of prothrombin and in the absence of any other blood coagulation protease. The comparison between the activation of r-prothrombin and D419N-prothrombin by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of prothrombin. Incubation of D419N-prothrombin with E.carinatus venom resulted in the generation of stable D419N-meizothrombin by hydrolysis of the peptide bond Arg320-Ile321. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg320-Ile321 and Arg271-Thr272 and lower activity towards peptide bond Arg155-Ser156, thus converting D419-prothrombin into D419N-thrombin and also liberating Fragment-1, Fragment-2 and Fragment-1/2 activation peptide. Activation of r-prothrombin by E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of prothrombin-derived molecules and indicated that meizothrombin hydrolysed the cleavage between Fragment-2 and thrombin A-chain in the meizothrombin molecule, but not in prothrombin, preferentially at position Arg284-Thr285. By contrast, both meizothrombin and thrombin exhibited no detectable activity towards peptide bond Arg320-Ile321 between thrombin A- and B-chain, although this site exhibits the optimum sequence for thrombin cleavage.

Effect of multimerization of human and recombinant von Willebrand factor on platelet aggregation, binding to collagen and binding of coagulation factor VIII.

The smallest circulating von Willebrand factor (vWF) molecule is a dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction resulted in a binding rate constant of about 3 x 10(6) M-1 s-1 and an equilibrium dissociation constant of about 0.4 x 10(-9) M.

Rational design, recombinant preparation, and in vitro and in vivo characterization of human prothrombin-derived hirudin antagonists.

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

Immobilized hirudin and hirudin-based peptides used for the purification of recombinant human thrombin prepared from recombinant human prothrombin.

A simple and efficient activation-affinity purification system was developed to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification strategy for the recombinant thrombin generated. At a constant flow rate and with a contact time limited to few seconds, recombinant prothrombin was filtered through immobilized trypsin. In a closed flow system, the recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin inhibitor, and hirudin-based peptides were covalently immobilized to Sepharose, thus creating thrombin-specific affinity gels that immediately absorb the thrombin generated from the activation mixture. Prothrombin and incompletely activated molecules did not bind to the affinity gel and were recirculated for a further activation cycle. Due to the specificity of the affinity gels for thrombin and the elimination of thrombin from the activation mixture, proteolytic degradation and autocatalytic inactivation of the recombinant thrombin was prevented. Recombinant thrombin was isolated from the hirudin-based affinity gels by chaotrope salt elution, resulting in high yields of highly pure, active thrombin. Affinity purification of thrombin was not deleteriously affected by contamination of the starting material with other proteins. Activation and affinity purification were equally effective for recombinant and human plasma-derived prothrombin as well as for human and recombinant thrombin.

Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.

Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345-348]. In contrast to pd-vWF, r-vWF preparations consisted of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers. In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r-vWF obtained from these cells was fully processed. Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands. Two-dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r-vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.

Renaturation of recombinant proteins produced as inclusion bodies.

Expression of recombinant proteins in Escherichia coli often results in the formation of insoluble inclusion bodies. Within the last few years specific methods and strategies have been developed to prepare active proteins from these inclusion bodies. These methods include (i) isolation of inclusion bodies after disintegration of cells by mechanical forces and purification by washing with detergent solutions or low concentrations of denaturant, (ii) solubilization of inclusion bodies with high concentrations of urea or guanidine-hydrochloride in combination with reducing reagents, and (iii) renaturation of the proteins including formation of native disulphide bonds. Renatured and native disulphide bond formation are accomplished by (a) either air oxidation, (b) glutathione reoxidation starting from reduced material, or (c) disulphide interchange starting from mixed disulphides containing peptides. The final yield of renatured proteins can be increased by adding low concentrations of denaturant during renaturation.

Comparison of fibrin-mediated stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA) and fibrin-dependent enhancement of amidolytic activity of t-PA.

Studies in the past 10 years have shown that there are two different, but related pathways for the acceleration of tissue-type plasminogen activator (t-PA) catalysis: (1) fibrin-dependent enhancement of t-PA amidolytic activity by fibrin binding; (2) fibrin-mediated stimulation of plasminogen activation by t-PA via the formation of a ternary complex of fibrin, t-PA and plasminogen. The common characteristic of both phenomena is the affinity of t-PA for fibrin, which is realized by the same enzyme binding site. However, a comparison of the kinetic data, the participating fibrin structures and the differences between single-chain and two-chain t-PA (sct-PA and tct-PA, respectively) shows that both phenomena have different causes. Fibrin-mediated stimulation of plasminogen activation involves both sct-PA and tct-PA and different fibrinogen derivatives such as fibrin, fibrinogen cyanogen bromide fragment FCB-2, fibrin alpha-chain and poly-lysine. This mechanism is described by a marked apparent decrease in the KM value. In contrast, fibrin-dependent enhancement of t-PA activity against low molecular weight peptides is exclusive to sct-PA and is characterized by an increase in the kcat value and, depending on the nature of the substrate, by an increase in kcat and a decrease in KM. Thus, sct-PA activity modulation depends strictly on the correct three-dimensional folding of fibrin and is not mediated by fibrinogen fragment FCB-2 or isolated fibrin chains.(ABSTRACT TRUNCATED AT 250 WORDS)