P203 PLASMA TRANSTHYRETIN FORMS IN PATIENTS WITH WILD–TYPE TRANSTHYRETIN CARDIAC AMYLOIDOSIS: INFLUENCED OF TAFAMIDIS TREATMENT

Abstract

Abstract Introduction Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol–binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR–CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR–CA patients, before and after administration of tafamidis, a TTR stabilizer. Material & Methods Plasma samples from patients affected by wild–type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex– and age–matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris–Gly polyacrylamide gel. Western blot analysis was performed with anti–TTR or anti–RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry. Results In both groups, the most represented forms were: TTR dimers or trimers (37–50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (>150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR–1, characterised by the presence of TTR tetramers (55kDa), and ATTR–2, characterised by its absence. The ATTR–1 group showed also higher levels of total TTR in comparison with ATTR–2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR–1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR–1 and –2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern. Conclusions The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.