CONICET | Buscador de Institutos y Recursos Humanos

Introduction: ADAMTS13 cleaves von Willebrand factor and it is absent or diminished in patients with thrombotic microangiopathy (TMA). ADAMTS-13 revealed a 4.7-kb mRNA transcript in liver and a 2.4 kb mRNA in placenta. Suzuki (2004) described the presence of mRNA in platelets (plt) and only performed a relative quantification of cDNAs in six healthy volunteers Objective: To develop a method of absolute quantification of the ADAMTS13 mRNA in plt and endothelial cells (ECs). Methods: The RNA extraction from plt and ECs was performed with Trizol and the Real Time PCR using SYBR Green system. The standard curve was obtained by serial dilutions of the plasmid pCMV6-XL5 containing the ADAMTS13 cDNA insert. The plasmid was amplified in two replicates for each standard dilution point. The standard curve was measured repeatedly on different days. For the amplification and data analysis system LightCycler was used. Samples: Plt from 11 normal donors and 1 patient with TMA (37 U/dL of plasma ADAMTS13, normal range 46-184 U/dL) without mutations in exon 3, 6, 20, 27, 28); HUVEC and HMVEC-L. Results: the inter-assay and intra-assay coefficients of variation of the Cp values were < 3.65% and < 2.65% respectively. The method had a good sensitivity, 3.103 copies. The amplification efficiency (E=10-1/Slope) was 1.95. The normal range obtained in the quantification of the ADAMTS13mRNA was 7-76x10-8 mRNA ADAMTS13/RNA total in normal donors. The TMA patient: 9 x10-8 mRNA ADAMTS13/RNA total. HUVEC and HMVEC-L: 7x10-6 and 5x10-6 mRNA ADAMTS13/RNA total, respectively. Discussion: The plasma ADAMTS13 activity is slightly diminished but it would not be associated with quantitative mRNA defects. Conclusion: We achieve the absolute quantification of the ADAMTS13mRNA in plt and ECs with a reproducible and accurate method