"In vitro" correction of the severe factor V deficiency-related coagulopathy by a novel plasma-derived factor V concentrate.

INTRODUCTION: Severe congenital factor V (FV) deficiency is a rare bleeding disorder characterized by very low/undetectable levels of FV. Fresh frozen plasma is the standard treatment for bleeding manifestations. Recently, a novel plasma-derived FV concentrate has been developed. AIM: To evaluate the "in vitro" ability of the novel FV concentrate to normalize clotting times and generate normal amount of thrombin in plasma collected from patients with severe FV deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (aPTT), FV activity and antigen levels and thrombin generation were measured pre- and postspiking of plasma samples of 10 patients with increasing doses of FV concentrate (from 0 to 100 IU/dL). RESULTS: Prothrombin time and activated partial thromboplastin time ratios as well as all thrombin generation parameters were fully corrected by the addition of FV concentrate at a final concentration of 25 IU/dL. However, the addition of FV at a concentration of 1-3 IU/dL was already sufficient to correct peak height and endogenous thrombin potential (but not lag time and time to peak) after activation with 5 pmol/L tissue factor. FV activity and antigen levels showed a linear response to supplementation with the novel FV concentrate. CONCLUSION: The novel plasma-derived FV concentrate was effective to correct "in vitro" severe FV deficiency in patients. The optimal FV concentration to fully normalize both global clotting times and thrombin generation parameters using the novel plasma-derived FV concentrate was 25 IU/dL.

Progress in large-scale purification of factor VIII/von Willebrand factor concentrates using ion-exchange chromatography.

BACKGROUND AND OBJECTIVES: We investigated and optimized the parameters of a chromatographic process suitable for industrial scale to obtain a highly purified factor VIII (FVIII)/von Willebrand factor (VWF) concentrate. MATERIALS AND METHODS: Several chromatographic runs were performed on the same production intermediate using different anion-exchange supports. The best matrix was selected and the final product was characterized. Once the chromatographic medium was chosen, the other parameters were evaluated to obtain the highest purified product and to modulate the VWF content in the FVIII/VWF complex. RESULTS: Fractogel EMD TMAE was the best support among those tested. It was the only one maintaining good results either with standard or double loading and flow rate conditions with respect to a typical industrial process. The chromatographic recovery of FVIII co-purified with VWF was at least 86% with a specific activity not lower than 140 IU/mg. The FVIII/VWF complex obtained is highly pure and, with the exception of immunoglobulin M (IgM), all investigated contaminant proteins are under the detection limit. Different concentrates characterized by variable FVIII/VWF ratios were purified by varying the chromatographic conditions. CONCLUSIONS: Several highly purified products, suitable for haemophilia A and von Willebrand disease management, can be obtained, through the same chromatographic process, on an industrial scale.

Effect of chitosan and of N-carboxymethylchitosan on intraocular penetration of topically applied ofloxacin.

The effects of chitosan hydrochloride (Ch-HCl) and of N-carboxymethylchitosan (CMCh), formulated in ophthalmic solutions, on the ocular pharmacokinetics of ofloxacin were studied in rabbits. The carboxymethylation of a chitosan of high molecular mass (1460 kDa) and deacetylation degree (89.9%) introduced 0.84 N-carboxymethyl groups per repeating unit. Aqueous solutions containing 1% (w/v) of either polymer showed a pseudoplastic rheologic behaviour, and, when instilled in rabbit eyes, produced no irritation. The kinetics of drug disappearance from tear fluid and the profiles of drug concentration in the aqueous humour versus time were determined and interpreted in the light of a pharmacokinetic model and of drug-polymer binding. Ch-HCl significantly enhanced intraocular drug penetration with respect to an isoviscous drug solution containing poly(vinyl alcohol) and to commercial ofloxacin eyedrops. This effect, which resulted in about 190% increase of the peak concentration in the aqueous, was ascribed to an increased corneal permeability. The polyanionic CMCh failed to enhance intraocular drug penetration. It nevertheless increased precorneal drug retention in virtue of its viscosity and of ofloxacin binding. Consequently, the residence time at concentrations higher than the MIC90 and the bioavailability of the antibiotic in the aqueous were increased by about 150 and 240%, respectively, with respect to the reference vehicle.

Analytical methods for the characterization of surface finishing in bricks.

The recent restoration works of Santo Stefano Church Façade (XV century) in Venice have shown traces variously saved of different kind of surface finishes. These finishes were found on the brick's surface both in the masonry and in the decorative elements. Different brick's surface and decorative tile samples were investigated using several techniques: optical microscopy, scanning electron-microscopy, thermal analysis, infrared spectroscopy and reflectance Fourier transform infrared microspectroscopy. The evaluation of the reached results was used to understand the decorative techniques and to recognize the material employed.