Specific Clotting Activity Research Articles

Optimization of protease production from Rhizomucor miehei Rm4 isolate under solid-state fermentation

BackgroundProtease is one of the most important industrial enzymes. The importance of protease bioproduction comes from meeting the increasing demand for this enzyme especially in the cheese industry. Rhizomucor miehei protease is the preferred substitute for the traditional rennet. Solid-state fermentation (SSF) shows promising results in enzyme production. An optimization strategy was applied to optimize the production of Rhizomucor miehei protease in a solid medium. The components of the fermentation medium were screened by using the one-factor-at-a-time (OFAT) approach. The optimization process then was performed by using the response surface methodology (RSM) approach based on five factors (fermentation time, temperature, pH, moisture content, nitrogen concentration) at five levels. Specific milk clotting activity and milk clotting activity/proteolytic activity ratio were considered as response variables in the optimization process. ResultsAmong several combinations, wheat bran was selected as the best substrate. Casein was selected based on preliminary screening of nitrogen sources. The optimal conditions identified by RSM analysis were found to be 81.21 h, 41.11°C, 6.31, 80%, and 1.33% for fermentation time, temperature, pH, moisture content, and casein concentration, respectively. The performed fermentation process under the optimized conditions gave an enzymatic extract with the values of 5.11 mg/mL, 2258.13 Soxhlet unit/mL, 441.90 Soxhlet unit/mg, 1.14 protease unit/mg, and 388.66 for protein content, milk clotting activity, specific clotting activity, specific proteolytic activity, and milk clotting activity/proteolytic activity ratio, respectively. The aforementioned values were close to the predicted values. ConclusionThe high milk clotting activity and the relatively low proteolytic activity signify higher specificity of the produced enzyme, which is favorable in cheese making. The observed results reveal the efficiency of the applied statistical approaches in obtaining desired values of response variables and minimizing experimental runs, as well as achieving good predictions for response variables.

Human Prothrombin Variants with Modifications in the Active Site S4 Subpocket Demonstrate Resistance to Dabigatran

Chymotrypsin-like serine proteases are hallmarked by a protease domain comprising the catalytic triad residues His57, Asp102, and Ser195 (chymotrypsinogen numbering) situated in the active site cleft. While the catalytic triad in conjunction with the oxyanion hole residues regulate substrate cleavage, the active site subpockets (S1-4) control substrate recognition and binding. The high structural homology of the serine protease domains allows for analogous strategies in drug design, which is underscored by the direct oral anticoagulants (DOACs) for the prophylactic management of stroke in atrial fibrillation and prevention and treatment of venous thrombosis. DOACs inhibit coagulation serine proteases by reversibly engaging the active site with high affinity. To expand the repertoire of DOAC-specific reversal agents we have previously successfully modified the S4 active site subpocket of human factor Xa to prevent DOAC binding while preserving catalytic activity [Verhoef 2017 Nature Commun.]. To explore whether an analogous strategy can be applied to create DOAC resistance in the serine protease thrombin, specific substitutions or sequences in or around the dabigatran-binding S4 subsite derived from naturally occurring serine proteases or plasma proteins were introduced in prothrombin.A panel of prothrombin variants was generated and transfected into HEK293 cells to allow for stable protein expression. In some of the generated prothrombin variants comprising insertions in amino acid sequence 91-99 that is directly adjacent to the S4 subsite protein expression was severely impaired. This indicates that exchange of any surface-exposed serine protease or plasma protein region into the prothrombin protease domain is not necessarily compatible with protein expression. In contrast, exchange of the human prothrombin 91-99 sequence for that of human kallikrein 3 or targeted amino acid replacement of S4 subsite residue Ile174 resulted in prothrombin protein expression levels similar to wild-type prothrombin.Following expression, prothrombin variants were purified to homogeneity using the CaptureSelect tm affinity matrix that selects for fully gamma-carboxylated prothrombin. The specific prothrombin clotting activity analyses of the purified prothrombin variants KL3 (0.7 ± 0.2 U/mg), I174A (0.8 ± 0.2 U/mg), and I174F (0.8 ± 0.3 U/mg) demonstrated an overall ~10-fold reduced specific activity relative to wild-type prothrombin (7.5 ± 0.1 U/mg). As such, modification of the S4 subsite likely interferes with the binding and subsequent conversion of fibrinogen by thrombin. To determine whether the prothrombin variants supported tissue factor-initiated thrombin formation in human plasma, prothrombin-deficient plasma was supplemented with increasing plasma concentrations of prothrombin variant (90-180 ug/mL). Consistent with their reduced specific clotting activity, 180 ug/mL prothrombin variant was required to obtain substantial thrombin generation but with reduced thrombin generation parameters (peak thrombin, ETP) relative to supplementation with plasma concentrations of wild-type prothrombin (90 ug/mL). This calibrated automated thrombin generation assay set-up was used to assess the DOAC-resistance of the prothrombin variants. While thrombin formation reached half-maximum inhibition at 532 ± 58 nM dabigatran in wild-type prothrombin-supplemented plasma, addition of the prothrombin variants displayed a ~2-fold reduced sensitivity to dabigatran inhibition (IC50: 1186 ± 136 nM prothrombin-KL3; 851 ± 97 nM prothrombin-I174F; 772 ± 80 nM prothrombin-I174A). This demonstrates that the S4 subsite-modified prothrombin variants are able to support thrombin generation in the presence of physiological plasma concentrations of inhibitor.Collectively, our findings indicate that human prothrombin variants comprising a single point mutation at position Ile174 in the S4 subsite or at a region directly adjacent to the S4 subsite are able to generate thrombin in plasma inhibited by dabigatran. Hence, serine proteases with S4 subpocket modifications have the potential to bypass DOAC therapy and could provide a generic strategy in the development of novel DOAC-bypassing agents. [Display omitted] DisclosuresReitsma: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Patents & Royalties. Verhoef: VarmX. B.V.: Current Employment, Current holder of individual stocks in a privately-held company. Bos: VarmX B.V.: Research Funding; uniQure Biopharma B.V.: Research Funding.

Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (<13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Proteases from Calotropis gigantea stem, leaf and calli as milk coagulant source

Abstract Background Universal demand for cheese keeps the search for appropriate enzymes from plants mimicking animal rennet action in scientific focus. Objective To associate distribution of milk clotting potential and profile of whole/κ-casein hydrolysis by Calotropis gigantea stem, leaf and respective calli crude enzymes (CE). Materials and methods Milk clotting activity and index were assayed for CE. Caseinolytic activity (CA) was evaluated spectrophotometrically. 0.5 CA units of CE and Enzeco® were used for studying whole/κ-casein hydrolysis pattern by Tricine SDS-PAGE. Inhibition studies were performed for enzyme characterisation. Results Traditionally propagated (TP) stem and its callus CE exhibited high specific milk clotting activity (1297.30±0.2 U/mg of protein and 926.74±44.13 U/mg of protein, respectively) and milk clotting index (103.562±1.162 and 79.365±4.93, respectively). Comparison of whole casein hydrolytic pattern by 0.5 CA units of CE revealed closer resemblance between leaf callus and Enzeco®. However, κ-casein specificity analysis revealed TP leaf to be closely mimicking the performance by Enzeco®. Conclusion Study suggests CE from TP leaf to be a potential vegetable coagulant to work as an effective and low-cost milk clotting mediator in cheese industry.

Utility of Milk Coagulant Enzyme of Moringa oleifera Seed in Cheese Production from Soy and Skim Milks.

In this study, the potential use of Moringa oleifera as a clotting agent of different types of milk (whole, skim, and soy milk) was investigated. M. oleifera seed extract showed high milk-clotting activity followed by flower extract. Specific clotting activity of seed extract was 200 times higher than that of flower extract. Seed extract is composed by four main protein bands (43.6, 32.2, 19.4, and 16.3 kDa). Caseinolytic activity assessed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and tyrosine quantification, showed a high extent of casein degradation using M. oleifera seed extract. Milk soy cheese was soft and creamy, while skim milk cheese was hard and crumbly. According to these results, it is concluded that seed extract of M. oleifera generates suitable milk clotting activity for cheesemaking. To our knowledge, this study is the first to report comparative data of M. oleifera milk clotting activity between different types of soy milk.

Characterisation of general proteolytic, milk clotting and antifungal activity of Ficus carica latex during fruit ripening.

The physiological role of fig latex is to protect the plant from pathogens. Latex is a rich source of proteases, predominantly ficin. Fig latex also contains collagenolytic protease and chitinolytic enzymes. Our aim was to investigate changes in protein composition, enzyme and antifungal activities of fig latex during fruit ripening. Comparison of latex samples in different time periods showed a uniform increase of protein concentration in chronological order. The content of collagenolytic protease did not differ significantly in the latex samples, while the content of ficin decreased. Ficin-specific activity towards casein was the highest at the beginning of fruit development (about 80 U mg(-1)). Specific milk clotting activity increased as well as the abundance of casein band in the clots. Specific chitinolytic activity at the beginning of flowering was 6.5 times higher than the activity in the period when fruits are ripe. Antifungal activity is the most extensive in spring. Ficin forms with different casein specificities are present in different proportions during fruit ripening, which is of importance for applications in the dairy industry. The protection mechanism against insects and fungi, which relies on chitinolytic activity, is the most important in the early phases of flowering and is replaced with other strategies over time.

Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo

Using gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIX-TripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIX-WT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIX-R338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the low-dose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.

Oral Gene Delivery of Factor IX Muteins Formulated As Chitosan Nanoparticles for the Treatment of Hemophilia B

AbstractAbstract 2048 Background:Hemophilia B, a bleeding disorder caused by deficiency of coagulation factor IX (FIX), is well suited for a gene therapy approach since continuous plasma levels above 1% already prevent major long-term complications. Although viral vectors are more efficient, non-viral vectors become more and more attractive as gene delivery systems because of their excellent safety profiles. The transgene expression from those systems is relatively low but a repeated vector administration is possible to obtain durable transgene in a therapeutic range. In this study we explore oral gene delivery based on the cationic biopolymer chitosan and FIX variants with improved clotting factor function for gene delivery. The oral administration route is considered privileged not only because of the ease administration, but also because of the immunoprivileged presentation of antigens and the resulting potential to induce immune tolerance. Chitosan nanoparticles protect the encapsulated DNA from digestion in the gastrointestinal tract and enhance the uptake in the gut by improving intestinal trans- and paracellular permeability and its mucoadhesive nature. Methods:Nucleotide substitutions for protein modification were introduced by site-directed mutagenesis into the pcDNA3.1 vector and a minicircle system, which differ from standard plasmid vectors by the lack of plasmid bacterial sequences. All vectors consist of a FIX expression cassette driven by a CMV promoter. Vectors were formulated as chitosan nanoparticles for oral delivery to treat FIX-deficient mice. Protection of vector DNA in nanoparticle constructs was confirmed by DNase I digest and subsequent gel electrophoresis. Results:First, HEK293 cells were transfected with chitosan nanoparticles containing the reporter gene eGFP. At 24h post-transfection, cells yielded in eGFP expression visualized with fluorescence microscope. Transfection with nanoparticles containing wild-type FIX resulted in secretion of FIX protein up to 100 ng/ml (2% of normal plasma concentration). Encouraged by the in vitro findings, FIX-deficient mice were administerd eGFP formulated chitosan nanoparticles to confirm gene transfer in vivo. Immunofluorescent staining revealed eGFP expression in small intestine. Next, we treated groups of mice (n=4–5/group) with naked DNA or nanoparticles complexes containing FIX or mock DNA. Circulating plasma levels in all groups remained below the therapeutic range (<1%). However, RT-PCR confirmed a 30-fold higher FIX expression in the small intestine compared to naked DNA treated mice. To improve therapeutic efficacy, different amino acid substitutions known to increase FIX clotting activity were combined. One protein variant was identified with 1500% FIX specific clotting activity compared to the wild-type protein. Following oral gene transfer with variant FIX, FIX antigen expression levels remained under the detection limit but the clotting times of mice were shortened due to a FIX activity of 1–4%. Immune histochemical staining confirmed FIX accumulation in small intestine caused by FIX binding to its physiological binding partner collagen IV in the extracellular matrix of the gut. To overcome the physiological affinity and improve protein release into the circulation a second variant was generated with impaired binding to collagen type IV and with 2000% FIX specific clotting activity. FIX antigen levels still remained low, but FIX activity in the therapeutic range of 3–4% was measured in the plasma. Immunofluorescent staining indicated remaining extracellular collagen IV binding in the gut. Further, to improve the low in vivo transfection efficiency a minicircle system was used to produce smaller chitosan nanoparticles by reducing vector size. Treatment of FIX-deficient mice provided haemostasis with FIX activity of 2–14% of individual mice while antigen levels still remained undetected. No inhibitory antibodies against FIX were detected in any treated animal. Experiments with repeated FIX challenges prior and post gene transfer are ongoing. Conclusion:Oral gene transfer based on chitosan nanoparticles seems feasible but only results in low levels of FIX expression. Protein engineering for FIX and optimized expression vectors might provide a possibility to overcome this shortcoming and therefore help to develop a gene transfer strategy for prophylaxis of bleedings and/or inhibitory antibodies. Disclosures:No relevant conflicts of interest to declare.

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.

Characterisation of factor IX with a glycine-to-valine missense mutation at residue 190 in a patient with severe haemophilia B

A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg¹¹⁶-Leu¹¹⁷, Lys²⁶⁵-Tyr²⁶⁶, Arg³²⁷-Val³²⁸, and Arg³³⁸-Ser³³⁹, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.

Generation of a novel factor IX with augmented clotting activities in vitro and in vivo.

Hemophilia B is an X-linked inherited disorder caused by the lack of functional factor IX (FIX). Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. In an effort to reduce factor usage and cost, we investigated the potential use of FIX variants with enhanced specific clotting activity. Seven recombinant FIX variants using alanine replacement were generated and assayed for their activity in vitro and in vivo. One variant containing three substitutions (V86A/E277A/R338A, FIX-Triple) exhibited 13-fold higher specific clotting activity and a 10-fold increased affinity for human FVIIIa compared with FIX-wild-type (FIX-WT) and was thus investigated systematically in vivo. Liver-specific FIX-Triple gene expression following hydrodynamic plasmid delivery revealed a 3.5-fold higher specific clotting activity compared with FIX-WT. Human FIX-Triple and FIX-WT knock-in mice were generated and it was confirmed that FIX-Triple has 7-fold higher specific clotting activity than FIX-WT under normal physiological conditions. Protein infusion of FIX-Triple into hemophilia B mice resulted in greater improvement of hemostasis than that achieved with FIX-WT. Moreover, tail-vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple in hemophilia B mice demonstrated a 7-fold higher specific clotting activity of FIX-Triple than FIX-WT. Our results indicate that the FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to FVIIIa, as demonstrated both in vitro and in vivo. Therefore, FIX-Triple is a good candidate for further evaluation in protein replacement therapy as well as gene-based therapeutic strategies.

Purification and characterization of a new recombinant factor VIII (N8)

A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.

Half-life extension through albumin fusion technologies

Haemophilia B is characterized by a deficiency of coagulation factor IX (FIX), a protein that is important in the process of haemostasis and normal blood clotting. Recurrent bleeding into joints and soft tissues is the hallmark of severe haemophilia B. The goal of treatment is to prevent and manage haemorrhage and thereby reduce disabling joint and tissue damage, improve quality of life, and extend life expectancy. Current treatment with FIX replacement concentrates often requires repeated, frequent infusions, owing to the relatively short terminal half-life of FIX in the circulation. We have developed a unique technology for improving the biological characteristics of FIX in vivo. For this approach, recombinant FIX (rFIX) was genetically fused to albumin via a cleavable peptide linker. Constructs of the fusion protein were expressed in mammalian cells and characterized following purification. In vitro activation studies demonstrated that cleavage of the linker occurred in parallel with FIX activation. The molar specific clotting activity of the cleavable fusion protein (rIX-FP) was 10- to 30-fold greater than that of the fusion protein with non-cleavable linkers. In rats, rabbits, and FIX-deficient mice, the pharmacokinetics of rIX-FP were significantly improved compared with rFIX. Using the tail-clip bleeding model in FIX-deficient mice, rIX-FP effectively corrected the bleeding time and blood loss. These findings suggest that rIX-FP may be a promising therapy for the treatment of patients with haemophilia B.

Genetic fusion to albumin improves the pharmacokinetic properties of factor IX

Haemophilia B is a X-chromosome linked disease characterised by a deficiency of functionally active coagulation Factor IX (FIX). Patients with severe haemophilia B at risk of recurrent bleeding are treated approximately twice a week in a prophylactic setting by application of FIX concentrates. To increase convenience and compliance of the therapy it is desirable to reduce the dosing frequency by improving the pharmacokinetic properties of FIX. Here a concept of rFIX (recombinant factor IX) albumin fusion proteins (rIX-FPs) with cleavable linker peptides derived from the FIX activation sequence is presented. Constructs of the genetic fusion of FIX to albumin via cleavable linkers were expressed in mammalian cells and characterised after purification. In vitro activation studies with FXIa demonstrated that cleavage of the linker and the activation peptide proceeded comparably well. In a clotting assay the rIX-FPs with cleavable linker showed a 10- to 30-fold increase in the molar specific clotting activity compared to fusion proteins with non-cleavable linkers. Furthermore, in-vivo recovery, terminal half-life and the AUC of rIX-FPs in rats and rabbits as determined by FIX antigen measurements were significantly increased compared to rFIX (BeneFIX). In FIX deficient (FIX(-/-)) mice the in-vivo recovery and the AUC were also significantly increased. The efficacy in reducing bleeding time was shown in FIX(-/-) mice by a tail tip bleeding model. The results suggest that rIX-FPs with a cleavable linker between FIX and albumin are a promising concept that may support the use of the albumin fusion technology to extend the half-life of FIX.

Identification of a novel thrombin-like phospholipase A2 from Gloydius ussuriensis snake venom

The coagulant effects of phospholipase A2 with Gln at 49 sites (Gln49-PLA2), purified from Gloydius ussurensis snake venom, were investigated on human citrated plasma and fibrinogen. Gln49-PLA2 clotted human plasma dose-dependently from 180.67 +/- 1.86 s to 19.00 +/- 0.58 s, and reduced the re-calcification time from 7.46 +/- 1.17 to 0.75 +/- 0.33 min and the prothrombin time from 12.4 +/- 0.29 s to 6.95 +/- 0.20 s, but it could not activate factor XIII, and the procoagulant effects were inhibited by heparin. The specific clotting activities of Gln49-PLA2 were equivalent to 1100 NIH thrombin U/mg on human fibrinogen, and the specific arginine esterase activity on the substrate BAEE was 1747 U/mg. Gln49-PLA2 hydrolyzed fibrinopeptide A faster than fibrinopeptide B, and the fibrinongenolytic ability was inhibited by the serine protease inhibitor phenyl-methylsulphonyl fluoride, but not by the metalloprotease inhibitor ethylenediamine tetraacetic acid. This finding demonstrates that Gln49-PLA2 is consistent with thrombin-like properties, and therefore should be a new thrombin-like serine protease.

Characterization of the milk-clotting properties of extracts from artichoke ( Cynara scolymus, L.) flowers

Extract of artichoke ( Cynara scolymus, L.) flowers have been investigated as a source of enzymes to be used in cheese making as an alternative or in addition to calf rennet. Coagulation activity was highly dependent upon milk pH and temperature. The rennet strength (RS) of this extract increased hyperbolically with increasing concentrations of calcium, and the concentration was saturated at 50 m m . However, the effect of sodium chloride on RS was not significant. Additionally, the properties of individual cynarases obtained from artichoke flowers were also studied. Purification led to a decrease in the specific coagulant activity relative to that of the crude extract in the case of cynarases A and C, whereas cynarase B increased its specific clotting activity. Moreover, whereas the cynarases A and C showed a slight increase in specific peptidase activity relative to the initial extract, the specific peptidase activity of cynarase B was much higher.

Milk-clotting activity of enzyme extracts from sunflower and albizia seeds and specific hydrolysis of bovine κ-casein

Milk-clotting activity found in ammonium sulfate-precipitated protein extracts from Albizia lebbeck and Helianthus annuus seeds was studied. Specific clotting activity of albizia seed extract was 15 times higher than that of sunflower seed extract. Zymogram analysis revealed several proteolytic bands in albizia seed extract and one diffuse proteolytic band for sunflower seed extract. Whole bovine casein was incubated with the plant seed extracts or chymosin and some breakdown products were characterized by reversed-phase high-performance liquid chromatography and electrophoresis. Similar to chymosin, the two seed extracts exhibited proteolytic activity toward κ-casein, α s-casein and β-casein, with the highest activity observed for the albizia seed extract. Mass spectrometry analysis showed that the sunflower extract hydrolyzed κ-casein at the Phe 105–Met 106 bond, as does chymosin. The albizia extract also displayed activity on κ-casein, but the Lys 116–Thr 117 bond was its preferred target.

Role of Hirudin-like Factor Va Heavy Chain Sequences in Prothrombinase Function

Proexosite I on prothrombin has been implicated in providing a recognition site for factor Va within prothrombinase. To examine whether hirudin-like sequences (659-698) on the cofactor contribute to this interaction, we expressed and purified two-chain FVa derivatives that were intracellularly truncated at the C terminus of the heavy chain: FVa709 (des710-1545), FVa699 (des700-1545), FVa(692 (des693-1545), FVa678 (des679-1545), and FVa658 (des659-1545). We found that FVa709, FVa699, FVa692, and FVa678 exhibited specific clotting activities that were comparable with plasma-derived and recombinant FVa. Additionally, kinetic studies using prothrombin revealed that the Km and kcat values for these derivatives were unaltered. Fluorescent measurements and chromatography studies indicated that FVa709, FVa699, FVa692, and FVa678 bound to FXa membranes and thrombin-agarose in a manner that was comparable with the wild-type cofactors. In contrast, FVa658 had an approximately 1% clotting activity and reduced affinity for FXa membranes (approximately 20-fold) and did not bind to thrombin-agarose. Surprisingly, however, FVa(658) exhibited essentially normal kinetic parameters for prothrombin when the variant was fully saturated with FXa membranes. Overall our results are consistent with the interpretation that any possible binding interactions between prothrombin and the C-terminal region of the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.

Purification and properties of a coagulant thrombin-like enzyme from the venom of Bothrops leucurus

A thrombin-like enzyme from Bothrops leucurus venom, named leucurobin (leuc), was purified by gel filtration, affinity and ion exchange chromatographies. Physicochemical studies indicated that the purified enzyme is a 35 kDa monomeric glycoprotein on SDS-PAGE under reducing conditions, which decreased to 29 kDa after deglycosylation with N-glycosidase F (PNGase F). The amino acid sequence of leuc was determined by automated sequencing of the intact native protein and peptides produced by digestion of the S-pyridyl-ethylated protein with trypsin. The protein sequence exhibits significant similarities with other serine proteases reported from snake venoms, and contains two potential sites of N-linked glycosylation. The proteinase split off fibrinopeptide A (FPA) rapidly from human fibrinogen; however, only negligible traces of fibrinopeptide B (FPB) were observed. In addition, the enzyme released the N-terminal peptide (Mr=4572) containing the first 42 residues from the Bbeta-chain. Leuc could neither activate factor XIII nor release kinins from heat-treated bovine plasma. Its specific clotting activity was equivalent to 198 NIH thrombin U/mg on human fibrinogen. Kinetic properties of leuc were determined using representative chromogenic substrates. The enzyme evoked the gyroxin syndrome when injected into the tail veins of mice at levels of 0.143 microg/g mouse. The inhibitory effects of PMSF and benzamidine on the amidolytic activity suggest that leuc is a serine proteinase, and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Antibothropic serum, SBTI and EDTA had little or no effect on its amidolytic activity. However, the clotting effect of the enzyme was strongly inhibited by antibothropic serum. A Dixon plot showed that the hydrolysis of Bz-L-Arg-pNA by leuc was competitively inhibited by benzamidine (Ki=1.61+/-0.25 mM).

The Contribution of Hirudin-Like Acidic Sequences within the Factor Va Heavy Chain to Prothrombinase Function.

The molecular mechanism governing the ability of activated factor V (FVa) within prothrombinase to accelerate prothrombin activation is not well understood. It is possible that FVa provides a docking surface for prothrombin, and/or causes a conformational change in FXa which exposes an exosite that enhances the presentation of the substrate to the active site of the enzyme. While there is indirect evidence that FVa binds prothrombin via (pro)exosite I, it is not entirely clear whether this interaction directly leads to the enhanced function of prothrombinase. It has been speculated that the corresponding binding site for (pro)exosite I on FVa is contributed by hirudin-like acidic sequences at the C-terminus of the heavy chain (659–709). In support of this, peptide inhibition studies indicate that residues Asp695 to Tyr698 are required for optimal prothrombinase function and likely provide a binding site for prothrombin. To directly assess the role of this region in prothrombin activation we exploited a novel strategy in which recombinant FVa (rFVa) variants truncated at the C-terminus of the heavy chain were produced. The entire B-domain and heavy chain sequences were removed and a PACE-furin cleavage site was introduced between these deleted residues and the light chain generating: FVa659 (des660–1545), FVa679 (des680–1545), FVa693 (des694–1545), FVa700 (des701–1545), and FVa709 (des710–1545). These single-chain proteins were intracellularly processed and secreted into the media as two-chain proteins with appropriate truncations to the heavy chain. Factor Va709 served as a control as it is chemically identical to plasma-derived (PD)-FVa and rFVa. Following stable expression and purification in high yield, SDS-PAGE and N-terminal sequence analysis suggested the derivatives were correctly processed. Factor Va709, rFVa and PD-FVa had high specific activities (~1500 U/mg) in a one stage PT-based clotting assay. Surprisingly, we found that FVa679, FVa693, and FVa700 exhibited specific clotting activities comparable to the wild-type cofactors. Detailed kinetic studies using prothrombin and saturating amounts of cofactor revealed that both the Km and kcat values for each of these variants were essentially equivalent to wild-type FVa. We observed minor differences (<4-fold) in the kinetic parameters when using prethrombin-1. Direct fluorescent and kinetic measurements also indicated that these variants bound FXa-membranes with high affinity. An exception to these findings was FVa659, which only had a minor reduction in the kcat for prothrombin (~1.5-fold), but had ~1–2% clotting activity and an increased Km (4-fold) and decreased kcat value for prethrombin-1 (>13-fold). Direct binding fluorescent measurements indicated that this variant had a marked reduction in the binding affinity for FXa-membranes (>15-fold). Additionally, the maximal fluorescent signal was reduced (~2-fold), further suggesting that this derivative interacts with FXa-membranes differently compared to wild-type FVa. Surprisingly, these data demonstrate that deletion of residues 679–709 does not impair cofactor function towards the physiological substrate prothrombin. Additionally, deletion of residues 659–709 only appears to significantly compromise FXa binding, a finding that likely explains the reduced functional activity of FVa659. Our results suggest that any possible binding interactions between prothrombin and the C-terminal acidic region on the FVa heavy chain do not contribute in a detectable way to the enhanced function of prothrombinase.