IMEX   05356
INSTITUTO DE MEDICINA EXPERIMENTAL
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Human umbilical vein endothelial cells, platelets and human tumor cell lines express isoforms 2 and 3 of ADAMTS13
Autor/es:
KEMPFER AC; POWAZNIAK Y; CALDERAZZO JC; PAIVA-PALOMINO J; ALONSO DF; SÁNCHEZ LUCEROS A; LAZZARI MA
Lugar:
Amsterdam
Reunión:
Congreso; XXIV CONGRESS OF THE INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS; 2013
Institución organizadora:
INTERNATIONAL SOCIETY ON THROMBOSIS AND HAEMOSTASIS
Resumen:
Background: ADAMTS13 contains 29 exons and spans 37 kb on chromosome 9q34. It has 1427 amino acids, 180 kDa of molecular weight and consists of a signal peptide, a propeptide, a metalloprotease and a disintegrin-like domain, a thrombospondin type 1 (TSP1) motif, a cysteine-rich domain, seven TSP1 repeats and two CUB domains. ADAMTS13 appears to be synthesized in the liver, particularly in hepatic stellate cells. Recent reports have shown that ADAMTS13 is synthesized and released from human megakaryocytes and platelets. ADAMTS13 mRNA is detected in almost every organ tissue by reverse transcriptase-polymerase chain reaction (RT-PCR), suggesting that the vascular endothelium may also be the source of plasma ADAMTS13. Given the large surface area of vascular endothelial beds, even a few endothelial cells producing ADAMTS13 at any moment may produce a significant contribution to plasma levels of ADAMTS13 activity. Therefore, any small change at the site of ADAMTS13 syntheses may be crucial in the pathogenesis of TTP and possibly other thrombotic diseases. Aims: We analyzed alternative splicing of ADAMTS13 in Human Umbilical Vein Endothelial Cells (HUVEC), platelets and human tumor cell lines. Methods: Experiments were performed in primary HUVEC obtained with 0.1% collagenase type 1. Platelets were isolated by differential centrifugation from total blood of healthy donors. Human cancer cell lines: Hep3b hepatoma and breast cancer cell lines (MCF-7 and MDA-MB-231). RNA extraction and RT-PCR. Total RNA was extracted from cells using TRIzol reagent. The integrity of RNA was verified by 260/280 optical density ratios. The RNA sample was treated with DNase I, followed by phenol/chloroform extraction. The RNA was reverse transcribed into cDNAs using random primers. In our PCR experiments, we used the primers to amplify the portion of ADAMTS13 that is present in all the known isoforms, the expected size of ADAMTS13 isoform 1 was 610 bp, isoform 2 and 3 were 442 bp, and isoform IR25 was 1075 bp. We used Hep3b as a positive control for isoforms 2 and 3 mRNA expression. The ADAMTS13 and b-actin mRNA was amplified for 30 cycles in the same reactions. The PCR products were analyzed on an agarose gel containing SYBR Safe. Results: All PCR performed, except the controls, were positive for bactin, showing the presence of amplifiable cDNA. We have detected, in our view for the first time, isoforms 2 and 3 in HUVEC and human platelets. In both breast cancer lines isoforms 2 and 3 mRNA were found to be strongly expressed in comparison to those of HUVEC and platelets, although they didn?t reach the expression levels of Hep3b cells. Conclusion: The presence of isoforms 2 and 3 in HUVEC and platelets from normal donors that we detected will enable us to assess the importance of the presence of ADAMTS13 alternative splicing products in tumor line cells and whether there is a correlation between the levels of expression of the isoforms and thrombotic events.