Evidence of Thrombin-Independent Generation of Activated Protein C

• Published in: Blood 2016-12, Vol.128 (22), p.2568-2568
• Main Authors: Rühl, Heiko, Rossa, Janine, Berens, Christina, Winterhagen, Anna, Oldenburg, Johannes, Müller, Jens, Pötzsch, Bernd
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V128.22.2568.2568

Introduction: In a recent study, we performed autologous serum infusions to evaluate the elimination kinetics of hemostasis-related biomarkers in healthy human subjects. In order to monitor a serum-induced activation of coagulation, we measured free thrombin in the infused serum and in plasma samples taken during and after infusion, but did not detect any de novo thrombin formation [PLoS One. 2015; 10(12): e0145012]. To study if the low levels of free thrombin in the infused serum induce generation of activated protein C (APC) we additionally measured APC in samples drawn after autologous serum infusion and in vitro in a purified system. Methods: Autologous serum was infused (50 mL/30 min) into 19 healthy volunteers. Four of them were simultaneously receiving infusions of the thrombin inhibitor argatroban (1 µg/kg/min), initiated 1 h before and ceased 1 h after starting the infusion of serum. Thrombin and APC were measured in serum and in plasma samples drawn before and in 15-min intervals during the infusion of serum, using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) platform. In in vitro experiments, APC formation was induced by addition of purified thrombin or serum to buffer containing protein C and thrombomodulin in excess, and CaCl2 at physiological concentrations. The formation of APC was subsequently measured by OECA. Results: In the autologous serum median (interquartile range) concentrations of thrombin and APC were 6.68 (4.63 - 8.73) ng/mL and 9.17 (7.63 - 13.91) ng/mL, thus doses of 0.12 (0.07 - 0.15) ng/mL of thrombin and 0.16 (0.14 - 0.22) ng/mL of APC were infused per mL of the subjects' plasma volume. In the plasma of probands, that did not receive argatroban, peak thrombin levels of 0.04 (0.00 - 0.08) ng/mL were measured, indicating a rapid inactivation of thrombin by endogenous inhibitors present in the plasma. However, with 1.41 (0.76 - 2.97) ng/mL peak APC levels exceeded the infused APC doses by a multiple. This was also true for the plasma samples from the probands that received argatroban, in which peak levels of APC of 0.94 (0.79 - 1.22) ng/mL were measured despite thrombin inhibition indicated by prolongation of the aPTT of 42.9 (40.1 - 44.4) s and thrombin time of 78.3 (69.3 - 87.2) s. In the in vitro experiments addition of argatroban at the concentrations achieved in the probands completely abolished APC generation up to a thrombin concentration of 5 ng/ml. Addition of human serum as a source for thrombi