Biomedical subjects

U Schlokat

Publications and source records attributed to U Schlokat.

At least 19 recordsLinked to original sources

PubMed2001

'Shed' furin: mapping of the cleavage determinants and identification of its C-terminus.

The human endoprotease furin is involved in the proteolytic maturation of the precursor molecules of a wide variety of bioactive proteins. Despite its localization in the membranes of the trans-Golgi system by means of a transmembrane domain, it has repeatedly been reported to form a C-terminally truncated, naturally secreted form referred to as 'shed' furin. In order to identify the cleavage site, internal deletion mutants of increasing size, N-terminal to Leu(708), and subsequently individual amino acid substitutions were introduced, and Arg(683) was identified as the prime determinant for shedding. MS analysis determined Ser(682) as the C-terminus of shed furin, suggesting that monobasic cleavage may occur N-terminal to Arg(683). Alteration of Arg(683) directs the shedding mechanism to alternative cleaving sites previously unused.

Amino Acid Sequence↗

PubMed2001

Recombinant von Willebrand factor: preclinical development.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

Animals↗

PubMed2000

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.

Animals↗

PubMed1999

Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Animals↗

PubMed1999

Preclinical evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease (vWD) have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of vWD in humans. We used this canine model of vWD to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with an rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 hours in one dog and 22.1 hours in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 hours, and LMW-rvWF, 9 hours. The in vivo recovery of vWF:Ag after administration of rvWF was 59, 64 and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78, 110 and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Animals↗

PubMed1999

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.

Binding Sites↗

PubMed1998

High expression of a B-domain deleted factor VIII gene in a human hepatic cell line.

The expression of a modified human coagulation factor VIII cDNA in a human liver-derived cell line is described. A B-domain deleted FVIII (rVIIIdB928) cDNA controlled by a strong viral promoter/enhancer was linked to a dominant selection-/amplification marker and transfected into the human hepatic cell line SK-HEP-1. By means of this system, up to 3.5 U rFVIIIdB928/10(6) cells x 24 h could be detected immediately after selection without gene amplification. This level is orders of magnitude higher than that obtained in Chinese hamster ovary (CHO) f1p4s under the same conditions. Efficient expression of rFVIIIdB928 in SK-HEP-1 cells was temperature dependent, a 4-fold higher level of activity was achieved in culture supernatants at decreased incubation temperatures of 28 degrees C. This system allows the production of high amounts of recombinant rFVIIIdB928 without time and labour consuming gene amplification procedures.

Cell Line↗

PubMed1998

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.

Dimerization↗

PubMed1998

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Animals↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Effects of human recombinant, plasma-derived and porcine von Willebrand factor in pigs with severe von Willebrand disease.

The effects of the infusion of a human recombinant von Willebrand factor (vWF) preparation in pigs homozygous for von Willebrand disease (vWD) were evaluated on serial measurements of von Willebrand factor antigen and activity, FVIII activity, vWF multimer analysis, in-vivo bleeding time and platelet adhesion and thrombus formation on collagen at high shear rates in an ex-vivo model of experimental thrombosis. Plasma-derived human and porcine vWF were used for comparison. Before infusion, the pigs were characterized by undetectable plasma vWF levels, a low level of FVIII, prolonged bleeding time, severely impaired platelet adhesion and thrombus formation. After infusion of the human recombinant vWF, in-vivo recovery of vWF activity ranged from 58% to 82%, depending on the dose infused, and its half-life was longer than for the plasma-derived concentrates. The highest-molecular-weight forms of human recombinant vWF were removed from the circulation gradually. Infusion of the three vWF concentrates produced inconsistent effects on bleeding time and moderate improvement of platelet adhesion and thrombus formation. After infusion, a prolonged increase of FVIII (> 48 h) was observed, suggesting that human recombinant vWF is able to bind and to stabilize porcine factor VIII and that porcine vWD is a good model for studying such interactions.

Animals↗

PubMed1997

In vivo characterization of recombinant von Willebrand factor in dogs with von Willebrand disease.

Hereditary von Willebrand factor (vWF ) deficiency in Dutch Kooiker dogs, which have undetectable levels of vWF, causes spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. Therefore, we used this canine model to study the in vivo effects of a new recombinant von Willebrand factor (rvWF ) preparation containing all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF ) and with a plasma-derived factor VIII/vWF concentrate (pdvWF ). In the vWF-deficient dogs, the half-life of vWF:Ag was 21.6 and 22.1 hours for rvWF, 7.7 hours for pdvWF, and 9 hours for LMW-rvWF; in vivo recovery of vWF:Ag was 59%, 64%, and 70% for rvWF, 33% for pdvWF and 92% for LMW-rvWF; in vivo recovery of RCoF was 78%, 110%, and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII, which were sustained even when vWF:Ag had decreased to nearly undetectable levels and only monomeric or dimeric species were detectable on agarose gels. At the dosages used, no effect was seen on bleeding time, but the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Animals↗

PubMed1997

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.

Animals↗

PubMed1996

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.

Animals↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Overexpression of human prothrombin in permanent cell lines using a dominant selection/amplification fusion marker.

Human prothrombin was overexpressed in transformed eukaryotic cells using a dominant bifunctional selection and amplification marker. The marker consists of the murine wild-type dihydrofolate reductase (dhfr) cDNA and the Escherichia coli hygromycin phosphotransferase gene fused in frame. The gene of interest is connected by the encephalomyocarditis virus 5' untranslated region to the fusion marker gene, forming a dicistronic transcription unit. The human prothrombin gene (FII) was used to monitor expression after initial selection for hygromycin B resistance and DHFR activity. In Chinese hamster ovary (CHO) cells, 5-15 mU prothrombin/10(6) cells per 24 h was obtained; in human 293 kidney cells levels of 20-50 mU/ 10(6) cells per 24 h were obtained. Methotrexate-mediated amplification of the foreign gene in CHO cells resulted in a more than 10-fold increase in FII expression, while in the presence of methotrexate, 293 cells expressed 200-250 mU/10(6) cells per 24 h. The use of this fusion marker within a dicistronic transcription unit allowed efficient dominant selection of cell clones and amplification of the gene of interest. Stably transfected cell lines that were able to secrete high levels of processed gamma-carboxylated human prothrombin were thus obtained.

Animals↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

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.

Animals↗