The tail cut bleeding model (CUT) is routinely used in factor VIII-deficient mice to assess pharmacodynamic effects of therapeutic strategies for haemophilia A. Results from this model are highly variable, many modifications to the model are reported and at times the animals' wellbeing may be compromised by recording survival as an endpoint. We therefore investigated if the ferric chloride carotid occlusion model (COM) used for thrombosis research can be applied to enhance data quality and animal welfare in haemophilia A research. Relative dose effects and relative dose variations were calculated for the CUT and COM. The requisite sample sizes were estimated and the importance of survival rates to assess rebleeds during recovery was evaluated by correlating initial blood loss to mortality. Relative dose effects increased with higher doses in both models. The COM was more sensitive at lower doses than the CUT, had up to 82% less variation across doses and clearly showed superior accuracy. Only 5% of the sample size required for the CUT would be needed to establish non-inferiority between a specific therapeutic dose in haemophilia A mice and healthy wild-type animals. A strong statistically significant correlation was found between initial blood loss and mortality within 24 h. Our findings clearly suggest that the COM is a valid tool for assessing haemophilia A treatment in vivo. The highly reproducible data means that significantly fewer animals are required and a more humane endpoint can be used by directly assessing clot stability instead of survival rate.

Galactomannans isolated from legume seed endosperms, including those of commercial interest, have been characterized by multidetection aqueous SEC. Galactomannans derived from seeds of the Faboideae subfamily had substantially higher Mw than those from Caesalpinioideae seeds (Mw,Fab = 2.4–3.1 × 106 g/mol, Mw,Caes. = 0.86–2.1 × 106 g/mol) and within the latter botanical subfamily, an apparent correlation between Mw and the degree of galactose substitution DG was found. The molar mass distributions were unimodal and differed primarily by a scale factor, with distributional widths narrower than a true Flory 'most-probable distribution'; good fits to Schulz–Zimm model were obtained. Across subfamilies no differences were found in the exponents of [η]–M and Rv–M relationships (0.61 ± 0.02, 0.54 ± 0.01, respectively), the Flory chain stiffness ratio (C∞ = 20 ± 1 (BSF analysis)), or the persistence length (Lp = 5.5 ± 0.2 nm) obtained from SEC fraction data. However, it was found that prefactors in the [η]–M and Rv–M relationships as well as the unperturbed parameter KΘ decrease in proportion to DG and therefore chain density. Generalized relationships incorporating galactose-dependent prefactors were therefore developed to model SEC fraction data of native galactomannans ([η]GM = (1800 ± 200) × Mo−1.61 × M0.61±0.02, Rv,GM = 0.63 ± 0.05 × Mo−0.54 × M0.54±0.01) as well as lower-M fractions obtained by ultrasonication ([η]GM = (730 ± 100) × Mo−1.71 × Mw0.71±0.02, Rv,GM = 0.49 ± 0.05 × Mo−0.57 × Mw0.57±0.01, M ≈ 1 × 105-native). As a consequence of this dependence and the observed patterns in molar mass variation, [η] varies within a narrow range for galactomannans as a whole despite substantial Mw differences.

The equilibrium aqueous solubility of a commercial carob flour was investigated by determining the percentage of soluble and insoluble components as a function of dissolution temperature. The cumulative polysaccharide yield was ca. 50% at 5°C, increasing approximately as a linear function of temperature to ca. 90% at 85°C. Overall molecular weight and intrinsic viscosity of the soluble polysaccharide were determined by size-exclusion chromatography. With increasing dissolution temperature, there was weak trend towards higher overall molecular weight and intrinsic viscosity: Mw from 1060 to 1150kg/mol, and [η] from 11.8 to 12.5dl/g, between 5 and 65°C, respectively. Broad, amorphous peaks were evident in the wide-angle X-ray scattering profiles, indicating no role for polysaccharide melting. The continuous increase of molecular weight and decrease of DSgal with dissolution temperature is interpreted as the result of thermodynamic partitioning based on a classical polymer-solvent fractionation, a mechanism that applies only for polysaccharide components with DSgal<0.35 within the accessible temperature range.