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Mim8 is a novel, next-generation factor VIIIa mimetic in development for subcutaneous prophylactic treatment of patients with hemophilia A with and without inhibitors. In vitro and in vivo models indicate that Mim8 has a distinct hemostatic potential.To test the nonclinical safety and pharmacodynamics of Mim8.The Mim8 nonclinical safety program in cynomolgus monkeys consisted of three studies of 4-26 weeks in duration with Mim8 doses ranging from 0.3-60 mg/kg/week intravenously or subcutaneously. After sacrifice, macroscopic and microscopic pathological examinations were performed.Mim8 was well tolerated with no noteworthy clinical observations. No signs of excessive coagulation or pathological macroscopic or microscopic findings were observed at doses 0.3-3 mg/kg/week subcutaneous. Thrombosis-related findings were detected during histopathological examination in a small proportion of animals (16%) receiving doses ranging 6-20 mg/kg/week. Dose-dependent increases in factor X (FX) and factor IX (FIX) concentrations were observed. Shortening of activated partial thromboplastin time (APTT) and increased thrombin generation under ex vivo hemophilia A-like conditions were observed at all Mim8 dose levels.Thrombosis-related findings observed at doses above 6 mg/kg/week Mim8 may have been exaggerated pharmacological reactions to a procoagulant compound in normocoagulant animals. Increases in FX and FIX concentrations could be because of a half-life prolongation due to binding to Mim8, but were limited at clinically relevant exposure levels. Subcutaneous administration of up to 3 mg/kg/week (several fold greater than expected clinical exposure) for 26 weeks resulted in relevant pharmacodynamic effects, observed in thrombin generation and APTT, with no signs of thrombi or excessive coagulation activation.

Norwegian agriculture is challenged by increased production demand and climate change while being faced with tight restrictions to its environmental impact. Due to climate change, an increase in extreme weather events is expected. High intensity rainfall events lead to flooding and water-logged conditions, which have negative impacts on yield and operational conditions related to tillage and transport (trafficability of the soil). Two thirds of Norway's agricultural area is drained to prevent water logging, but at times these drained soils have problems with too high water content, leading to delayed tillage in spring time resulting in lower yields. About 10% of the cultivated land is considered poorly drained.Saturation and infiltration excess overland flow leads to sheet erosion. Erosion rates often follow a seasonal pattern with the highest soil losses during late autumn and early spring. For most of the total soil loss only a few runoff events are responsible each year. Soil loss from agricultural areas in Norway is not only harmful because of the loss of nutrient rich topsoil, but also because of off-site effects, especially in freshwater systems.Poorly drained soils are prone to deterioration of its structure. In areas where overland flow concentrates, this may lead to the development of gullies. Ephemeral gullies make up a considerable part of the sediment losses from agricultural areas. In addition, they are shortcuts for sediment transport, forming a connection between the hillslope and the surface water system.Seasonal saturation excess because of snow melt and rain also leads to high flow rates in Norway’s stream and river network. Flooding problems at the intersection between streams and roads occur even in first order streams.While many soil conservation and water retention measures complement each other, they sometimes affect each other adversely. Intensification of tile drainage, for example, may reduce sheet and gully erosion risk levels, but will have an adverse effect on peak flow rates and flood risk. Other measures, like buffer zones, serve both purposes. But when, how and under which circumstances water retention and soil conservation measures function remains a complex question.Understanding the spatio-temporal dynamics of water in the vadose and groundwater zones therefore is a key component of integrated agro-ecological management strategy at low (farm) and high (regional) levels. While the mechanics of overland water movement and infiltration are generally well understood, there are many significant challenges for system understanding at larger spatial scales, especially under increasingly non-normal weather conditions.NIBIO endeavors to reconcile measurements and observations with agrohydrological system understanding. Complexity and scale (time and space) are the main challenges in this endeavor. In this presentation we will present how NIBIO uses geophysics for understanding agrohydrological threats and solutions, with focus on drainage, erosion and buffer zones.

Immunogenicity causing development of anti-drug antibodies (ADAs) are major challenges in the treatment of haemophilia, as well as other diseases where proteins are used for treatment. Furthermore, it is a complication for preclinical testing of such therapies in animal models.To investigate if the immunosuppressive drug CTLA4 immunoglobulin (CTLA4-Ig) can induce tolerance in haemophilia A (HA) rats receiving recombinant human coagulation factor VIII (rhFVIII) treatment.Two different prophylactic rhFVIII compounds were given intravenously to HA rats for 4 weeks. Both rhFVIII compounds were co-administered with commercially available CTLA4-Ig or human IgG subclass 4 (hIgG4) as control, and blood samples were collected. To functionally test if pharmacological efficacy was retained, rats were subjected to a bleeding experiment under anaesthesia at end of study.The mean inhibitory level after 4 weeks in rats receiving rhFVIII and hIgG4 was 85.7 BU for octocog alfa and 37.4 BU for rurioctocog alfa pegol. In contrast, co-administration with CTLA4-Ig during rhFVIII therapy prevented the formation of ADAs (both binding and inhibitory) in 14/14 rats receiving octocog alfa and in 7/7 rats receiving rurioctocog alfa pegol. Moreover, we were able to show that the pharmacological efficacy of rhFVIII was preserved.In a rat model with spontaneous bleeding, co-administration of CTLA4-Ig with rhFVIII prevented antibody formation. No FVIII antibodies were detected, demonstrating that CTLA4-Ig co-administration can be applicable as a method to prevent immunogenicity, when evaluating human proteins in preclinical systems permitting continuous pharmacokinetic and pharmacodynamic assessment.

The occurrence of freeze–thaw cycles modifies water infiltration processes and surface runoff generation. Related processes are complex and are not yet fully investigated at field scale. While local weather conditions and soil management practices are the most important factors in both runoff generation and surface erosion processes, local terrain heterogeneities may significantly influence soil erosion processes in catchments with undulating terrain. This paper presents a field-based investigation of spatial and temporal heterogeneities in subsurface soil moisture and soil temperature associated with freezing, thawing, and snowmelt infiltration. The field setup consists of a combination of traditional point measurements performed with frequency domain reflectometry (FDR) and electrical resistivity tomography (ERT). The transect was approximately 70 m long and spanned an entire depression with a north-facing slope (average slope of 11.5%) and a south-facing slope (average slope of 9.7%). The whole depression was entirely covered with stubble. Observed resistivity patterns correspond well to the measured soil moisture patterns. During the observation period, the north facing slope froze earlier and deeper compared with the south facing slope. Freeze–thaw cycles were less pronounced in the north-facing slope than in the south-facing slope. There were also differences in soil temperature and soil moisture patterns between lower and upper parts of the monitored depression. These indicate that initiation and development of runoff related processes, and consequently soil erosion, in regions with freeze–thaw cycles may differ significantly depending on local terrain characteristics. Consequently, it indicates that spatial terrain heterogeneities, especially slope aspects, may be important when studying soil erosion processes, water flow and nutrient leaching in lowlands where patchy snowpacks and dynamic freeze–thaw cycles are predominating.

Degradation of organic chemicals in natural soils depends on oxidation-reduction conditions. To protect our groundwater resources we need to understand the degradation processes under anaerobic conditions . Available iron and manganese oxides are used as electron acceptors for anaerobic degradation and are reduced to the dissolved form of metallic cations in pore water . To monitor this process is a challenge, because anaerobic conditions are difficult to sample directly without introducing oxygen. A few studies have shown an impact of iron reduction on spectral induced polarisation (SIP) signature, often associated with bacterial growth. Our objective is to study the impact of iron and manganese oxide dissolution, caused by degradation of an organic compound, with spectral induced polarisation signatures. Twenty-six vertical columns (30 cm high, inner diameter 4.6 cm) were filled with a sand rich in oxides (manganese and iron) with a static water table in the middle. In half of the columns, a 2 cm high contaminated layer was installed just above the water table. As the contaminant degrades, the initial oxygen is consumed and anaerobic conditions form Every three days over a period of one month, spectral induced polarisation (twenty frequencies between 5mHz and 10 kHz) data were collected on six columns: three contaminated replicates and three control replicates. Chemical analysis was done on twenty columns assigned for destructive water sampling, ten contaminated columns and ten control. The results show an increase of the real conductivity associated with the degradation processes, independent of frequency. Compared with the pore water electrical conductivity in the saturated zone , the real conductivity measurement revealed the formation of surface conductivity before iron was released in the pore water. In parallel, we also observed an evolution of the imaginary conductivity in both saturated and unsaturated zones at frequencies below 1 Hz. Overall, the anaerobic reduction of iron and manganese oxide during the organic degradation increased both the conductive and polarisation component of the complex conductivity.

Assessing redox conditions in soil and groundwater is challenging because redox reactions are oxygen sensitive, hence, destructive sampling methods may provide contact with air and influence the redox state. Furthermore, commonly used redox potential sensors provide only point measurements and are prone to error. This paper assesses whether combining electrical resistivity (ER) and self-potential (SP) measurements can allow the mapping of zones affected by anaerobic degradation. We use ER imaging because anaerobic degradation can release iron and manganese ions, which decreases pore water resistivity, and produces gas, which increases resistivity. Also, electrochemical differences between anaerobic and aerobic zones may create an electron flow, forming a self-potential anomaly. In this laboratory study, with four sand tanks with constant water table heights, time-lapse ER and SP mapped changes in electrical/electron flow properties due to organic contaminant (propylene glycol) degradation. Sampled pore water mapped degradation and water chemistry. When iron and manganese oxides were available, degradation reduced resistivity, because of cation release in pore water. When iron and manganese oxides were unavailable, resistivity increased, plausibly from methane production, which reduced water saturation. To bypass the reactions producing methane and release of metallic cations, a metal pipe was installed in the sand tanks between anaerobic and aerobic zones. The degradation creates an electron surplus at the anaerobic degradation site. The metal pipe allowed electron flow from the anaerobic degradation site to the oxygen-rich near surface. The electrical current sent through the metal pipe formed an SP anomaly observable on the surface of the sand tank. Time-lapse ER demonstrates potential for mapping degradation zones under anaerobic conditions. When an electrical conductor bridges the anaerobic zone with the near surface, the electron flow causes an SP anomaly on the surface. However, electrochemical differences between anaerobic and aerobic zones alone produced no SP signal. Despite their limitations, ER and SP are promising tools for monitoring redox sensitive conditions in unsaturated sandy soils but should not be used in isolation.

Energy metabolism and reproduction are closely linked and reciprocally regulated. The detrimental effect of underweight on reproduction complicates the safety evaluation of anti-obesity drugs, making it challenging to distinguish pathological changes mediated through the intended drug-induced weight loss from direct drug effects on reproductive organs. Four-weeks dosing of normal weight Sprague Dawley rats with a glucagon-like peptide 1 (GLP-1)/glucagon receptor co-agonist induced a robust weight loss, accompanied by histological findings in prostate, seminal vesicles, mammary glands, uterus/cervix and vagina. Characterization of the hypothalamus-pituitary-gonadal (HPG) axis in male rats revealed reduced hypothalamic Kiss1 mRNA levels and decreased serum luteinizing hormone (LH) and testosterone concentrations following co-agonist dosing. These alterations resemble hypogonadotropic hypogonadism typically seen in adverse energy deprived conditions, like chronic food restriction. Concomitant daily administration of kisspeptin-52 from day 21 to the end of the four-week co-agonist dosing period evoked LH and testosterone responses without normalizing histological findings. This incomplete rescue by kisspeptin-52 may be due to the rather short kisspeptin-52 treatment period combined with a desensitization observed on testosterone responses. Concomitant leptin treatment from day 21 did not reverse co-agonist induced changes in HPG axis activity. Furthermore, a single co-agonist injection in male rats slightly elevated LH levels but left testosterone unperturbed, thereby excluding a direct acute inhibitory effect on the HPG axis. Our data suggest that the reproductive phenotype after repeated co-agonist administration was driven by the intended weight loss, however, we cannot exclude a direct organ related effect in chronically treated rats.

Summary Common redox potential sensors provide point measurements and are error prone. Destructive sampling can cause contact with air which will influence the redox state. Hence, assessing redox conditions in soil is challenging. Redox sensitive reactions like the degradation of organic contaminants will change water chemistry and geophysical properties. We explored geophysical techniques ERT and SIP to observe the degradation processes of Propylene Glycol (PG), a de-icing chemical used at airports. Two laboratory experiments were performed to monitor changes caused by PG degradation: 1) 3D ERT was measured, every week, in four sand tanks. One rich in oxides. 2) SIP was measured, every three days, in twenty-six oxides rich sand columns. In both experiments PG was applied just above the water table. ERT - When iron and manganese oxides were available, degradation reduced resistivity. When oxides were unavailable, the electrical resistivity increased, most likely due to methane production which reduced water saturation. SIP - An increase of the real conductivity was associated with the metal ions release but independent of the frequency. Also an increase of phase angle and imaginary conductivity at frequencies below 1Hz that precede the ion release was observed in the water.

A bioelectrochemical system was developed to facilitate biodegradation of an organic contaminant (propylene glycol) using a sandbox containing an iron bar that crossed the capillary fringe. In the days following the introduction of the organic contaminant, a strong negative electric potential anomaly (on the order of −35 to 50 mV) was observed at the top surface of the sandbox, evidencing the transport of electrons in the metallic bar and the degradation of the organic contaminant. The iron bar served to transmit electrons between the electron donor (i.e., biodegradation of the propylene glycol) and oxygen used as the terminal electron acceptor. Numerical modeling indicates that the source of current associated with the electric potential anomaly is at the position of the iron bar. The monitoring of this anomaly possibly can be used to monitor the amount of electrons passing through the electronic conductor and the radius of influence of the bioelectrochemical cells with respect to biodegradation of the organic contaminant.

Non-invasive spatially resolved monitoring techniques may hold the key to observe heterogeneous flow and transport behavior of contaminants in soils. In this study, time-lapse electrical resistivity tomography (ERT) was employed during an infiltration experiment with deicing chemical in a small field lysimeter. Deicing chemicals like potassium formate, which frequently impact soils on airport sites, were infiltrated during snow melt. Chemical composition of seepage water and the electrical response was recorded over the spring period 2010. Time-lapse electrical resistivity tomographs are able to show the infiltration of the melt water loaded with ionic constituents of deicing chemicals and their degradation product hydrogen carbonate. The tomographs indicate early breakthrough behavior in parts of the profile. Groundtruthing with pore fluid conductivity and water content variations shows disagreement between expected and observed bulk conductivity. This was attributed to the different sampling volume of traditional methods and ERT due to a considerable fraction of immobile water in the soil. The results show that ERT can be used as a soil monitoring tool on airport sites if assisted by common soil monitoring techniques.

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

During storage, erythrocytes undergo changes that alter their clearance and function after transfusion and there is increasing evidence that these changes contribute to the complications observed in transfused patients. Stored erythrocytes were incubated overnight at 37°C to mimic the temperature after transfusion. After incubation, several markers for erythrocyte damage were analysed. After overnight incubation, stored erythrocytes showed increased potassium leakage, haemolysis, PS exposure and vesicle formation, and all these effects increased with increasing storage time. Furthermore, we demonstrated that long-term stored erythrocytes develop decreased flippase activity and increased scrambling activity after overnight incubation, leading to PS exposure and the release of vesicles. Reduced intracellular potassium was identified as the cause of the decreased flippase activity. Lastly, we provide evidence that erythrocytes can return to a PS-negative state by shedding parts of their membrane as PS-containing vesicles and that these vesicles can serve as a platform for the coagulation cascade. These findings reveal that potassium leakage, a well-known phenomenon of prolonged erythrocyte storage, primes erythrocytes for PS exposure. PS exposure will lead to vesicle formation and might have an important impact on the post-transfusion function and side effects of stored erythrocytes.

The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology.

A recent chemical footprinting study in our laboratory suggested that region 1803–1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803–1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803–1810 and 1811–1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811–1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811–1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803–1810 and FVIII/FV 1811–1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811–1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811–1818 contributes to FIXa binding. Both regions 1803–1810 and 1811–1818 contribute to FVIIIa stability. Background: Factor VIII (FVIII) region 1803–1818 may bind activated factor IX (FIXa) or contribute to the stability of activated factor VIII (FVIIIa). Results: Replacing 1803–1810 or 1811–1818 with the homologous regions of factor V decreases FVIIIa stability. FVIII variant F1816A exhibits reduced FIXa binding and FVIIIa stability. Conclusion: FVIII region 1803–1818 is critical for cofactor function. Significance: Novel insight is obtained about FVIII cofactor function.

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.

Abstract 3357 Previously, we identified a role for the lysine residue couple 1967/1968 in the stability of activated factor VIII (FVIIIa). Using tandem mass tags (TMT 126/127) in combination with mass spectrometry, we identified lysine residues involved in the interaction between the A2 domain and the rest of heterodimer (A1/A3-C1-C2) of FVIIIa (Bloem et al., J Biol Chem 2012;287:5575–83). Upon FVIII activation and A2 domain dissociation, the highest increase in surface exposure occurred for the lysine couple 1967/1968, and functional studies confirmed the role thereof in FVIIIa stability. In addition to lysines 1967/1968 also other lysines displayed an increased surface exposure, including those in positions 1804, 1808, 1813 and 1818. The A3 domain region 1803–1818 has previously been implicated in interactions with ligands such as activated factor IX (FIXa). As such, one might expect increased surface exposure due to FVIII activation. On the other hand, A2 domain dissociation may have rearranged the A3 domain surface in this region. The relation between FIXa assembly and A2 domain retention was therefore explored in the present study. To unravel the role of region 1803–1818 in FVIIIa stability and FIXa binding, either region 1803–1810 or 1811–1818 was replaced by the corresponding regions of the homologous factor V. Additionally, as Asn1810 is N-linked glycosylated and this glycan is maintained in both chimeras, a variant that lacks this glycan (N1810C) was investigated. Factor × activation kinetics were used to investigate the apparent FIXa binding affinity of the FVIII variants. FXa generation was assessed on 15% phosphatidyl serine (PS) containing membranes. FIXa titration experiments showed that the affinity for the 1811–1818 variant is reduced (apparent Kd from 1.3 nM to 2.4 nM). Removal of the glycan and substitution of 1803–1810 has little or no effect on the apparent FIXa binding. FVIIIa-FIXa assembly on membranes containing 15% PS was studied using lipid- coated glass beads (lipospheres). Lipospheres were incubated with fluorescein-labeled FIXa and different FVIIIa concentrations. FIXa did only display liposphere binding in the presence of FVIIIa. Therefore, the mean fluorescent intensity on the lipospheres at increasing FVIIIa concentration could be used as a measure for FVIIIa-FIXa assembly on lipids. Results from these experiments showed that the 1811–1818 variant fails to promote FVIIIa-FIXa assembly, whereas the other variants behave like WT. To investigate FVIIIa stability, and thereby the role of the mutations on A2 domain dissociation, the activity of the variants was followed over time. Results showed that the 1811–1818 variant has a decreased half life of 1.5 min, compared to 6.9 min for WT. Also substitution of region 1803–1810 results in a lower half life, although to a lesser extent (2.8 min), whereas the glycan lacking variant behaves like WT (6.8 min). Incubation of FVIIIa variants with FIXa is known to stabilize FVIIIa and leads to an increased half life for all variants. However, the 1803–1810 variant is most efficiently stabilized by FIXa, shown by a 3-fold increase in half life, instead of 1.6-fold as seen for both WT and N1810C. The 1811–1818 variant is stabilized by 2.2-fold and therefore remains significantly less stable than WT. Together these results show that the 1811–1818 region is not only involved in FIXa binding, but additionally plays a major role in A2 domain retention. Region 1803–1810 also plays a role in FVIIIa stability, although to a lesser extent. Remarkably, the glycan at position Asn1810 does not influence neither FIXa binding nor FVIIIa stability, and apparently serves some other function. Disclosures: No relevant conflicts of interest to declare.

Abstract 2248 Factor VIII (FVIII) circulates in a tight complex with its carrier protein von Willebrand factor (VWF). Activation of FVIII results in the dissociation of the FVIII-VWF complex after which FVIII can perform its role in the coagulation cascade. In the complex with VWF, FVIII is protected from rapid clearance from the circulation. Individuals with a mutation in VWF that impairs the ability of VWF to bind FVIII can therefore have a bleeding disorder caused by a low plasma level of FVIII. Mature VWF contains multiple domains of which the N-terminal D'-D3 domains have been shown to comprise the FVIII binding site. Detailed information about amino acid regions in VWF that contribute to the direct interaction with FVIII is, however, lacking. In the present study we have employed a chemical footprinting approach to identify amino acid regions of VWF that are involved in binding FVIII. To this end, the lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII. VWF was subsequently cleaved into peptides employing chymotrypsin. The identity of the peptides and whether or not they contained a modified lysine residue was assessed by nanoLC mass spectrometry. The results showed that the lysine residues of almost all identified peptides were modified to the same extent upon incubation of VWF with FVIII or activated FVIII. However, lysine residue 773 in the N-terminal peptide comprising the residues 766-SCRPPMVKL-774 was protected from chemical modification in the presence of FVIII. In addition, a peptide was identified in which the free amine group of serine 764 at the start of the D' domain was also differentially modified in the presence of FVIII or activated FVIII. We next studied the structure of a molecular model of the D' domain that was obtained by comparative homology modeling. Structure analysis revealed that the N-terminal region 764–773 is situated at the tip of the D' domain and that the amino acid residues Ser764 and Lys773 are in close proximity. This observation combined with the results obtained with the chemical footprinting approach implies that the residues Ser764 and Lys773 at the N-terminus of VWF are directly involved in the FVIII-VWF complex formation. Alternatively, upon binding of FVIII, there is a conformational change in this N-terminal region resulting into a differential accessibility of these residues for chemical modification. To further investigate on this issue, we constructed recombinant VWF variants in which the lysine residue 773 and the serine residue at position 764 were replaced by alanines. The variants Ser764Ala, Lys773Ala and WT-VWF were expressed in 293 cells and purified. The binding of Ser764Ala and Lys773Ala to FVIII was evaluated employing surface plasmon resonance (SPR) analysis. The data revealed that the N-terminal region of the VWF D' domain modulates the interaction with FVIII. The contribution of Ser764 and Lys733 was mainly reflected in the dissociation kinetics of the complex. We also assessed the association of the VWF variants to FVIII in a solid phase binding assay. In addition, we evaluated to what extent the VWF variants can compete with WT-VWF for binding FVIII. The results were in agreement with the findings obtained with SPR analysis, and demonstrated a modulatory role of the residues 764 and 773. Taken together, our data reveal that the residues Ser764 and Lys773 at the N-terminus of mature VWF contribute to the affinity of the FVIII-VWF complex. Disclosures: No relevant conflicts of interest to declare.

Summary Soil contamination of organic origin is a major environmental challenge. One of the most common reactions in nature is the electron exchange between chemical species, also known as a reduction-oxidation (redox) reaction. Redox reactions drive organic matter degradation in the ground. Monitoring these reactions is therefore needed to protect soil and groundwater. This work aims to provide non-invasive monitoring methods to study redox state changes that occur in the near-surface interface, at the boundary between aerobic and anaerobic conditions, as well as a potential remediation solution to attenuate the negative impact of anaerobic condition.