IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
STUDY OF ADAMTS13 ISOFORMS IN HUVEC
Autor/es:
CARRIVALE MA; ZAPATA VA; KELLER L; SÁNCHEZ-LUCEROS A; KEMPFER AC; LAZZARI MA
Lugar:
Boston-USA
Reunión:
Congreso; XXII Congress of the International Society on Thrombosis and Haemostasis; 2009
Institución organizadora:
International Society on Thrombosis and Haemostasis
Resumen:
Introduction: Three isoforms (ISO) have been described of ADAMTS 13 in cDNA library of lung or liver. ISO-1 corresponds to complete gene, ISO 2 and 3 show two deletions: 1) 3847-4014pb, corresponding to 55 aa between the eighth trombospondin domain-1 and the first CUB domain, which share the ISO2 and ISO3. 2) 1272-1365pb involves 14aa C-terminal of metalloprotease domain and 16aa in the extreme N-terminal of desintegrins domain in ISO 3. It has been reported that the mRNA of ADAMTS13 is found in platelets and HUVEC. Objective: To identify the ISO 2 and 3 of ADAMTS13 in HUVEC. Methods: RNA extraction of HUVEC was performed with Trizol. Real Time PCR using SYBR Green. The characterization of the products was carried out by agarose electrophoresis (1.5%). We planned two strategies for the design of primers (P): 1- sense P matches sequences common to the three ISO and antisense P matches sequences originated by deletions. 2- The P flanks the deletions and the products obtained had different lengths for each ISO. Both strategies were tested in different reaction conditions; HUVEC RNA was used as a template. Results: the recovery of RNA from HUVEC (6x106 cells)  was 80 <img src="/images/math/gl-mu.gif" align="bottom">g. Products have not been detected using the strategy 1. With the second strategy, we detected  products from ISO 1 in HUVEC. Conclusions: It is possible that the strategy 1 was not successfully because the deletions involve the region rich in GC.  This study implies that the ISO2 and ISO3 from HUVEC do not exist or that the method used might not have sufficiently low detection limit