C0068 Haplotypes of the endothelial protein C receptor gene and circulating protein C levels

Abstract

Background: Activated protein C (APC) is an enzyme with anticoagulant and cytoprotective functions. Accumulative data suggest that the number of situations in which in vivo circulating APC will need to be measured will increase over the next few years. Therefore, the availability of a rapid, sensitive and simple assay for quantifying circulating APC is crucial. Available assays are difficult to apply in routine laboratory. Our previously reported assay required an excessive pre-analytical handling: drawing of blood into two citrate tubes; immediate addition of a fresh APC inhibitor to one tube in order to block complexation of circulating APC to protein C inhibitor (PCI), and of heparin to the other tube to force all circulating APC to bind to PCI, and measurement of APC:PCI complexes in both tubes by ELISA (method A). Here we describe a simplifiedmethod based on the measurement of APC:PCI in a unique blood tube anticoagulated with heparin (method B). Methods: We measured APC levels, with both methods, in 125 plasma samples, 69 from patients with venous thrombosis (VT) and 56 from healthy controls. Results: ThemeanAPC level in the 125 sampleswas 1.06±0.46 ng/ml (method A) and 2.21±0.85 ng/ml (method B). The coefficient of correlation between both methods was r=0.849 (Pb0.001). The mean APC level in the 69 VT patients wase 0.84±0.38 (method A) and 1.80± 0.67 (method B), significantly lower (as previously reported) than those in the 56 controls, 1.33±0.39 and 2.72±0.79, respectively, (Pb0.001). In both groups there was a significant correlation between the levels obtained by the two methods (VT, r=0.772; controls, r=0.777 (Pb0.001). Comment: These results show that both assays are equivalent, and confirm that the APC level is lower in VT patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories. (FIS PS09/00610, RECAVA RD06/0014/0004, Prometeo/ 2011/027, y Fundacion para la Investigacion Hospital La Fe; Pilar Medina is a Miguel Servet Researcher, ISCIII CP09/00065).