Autologous serum infusion induces no thrombin formation but generation of activated protein C

C. Berens, H. Rühl, J. Rossa, A. Winterhagen, J. Oldenburg, J. Müller, B. Pötzsch (Bonn, Germany)

Bleeding disorders, coagulation and fibrinolytic factors
Date: 17.02.2017,
Time: 17:15 - 18:15

Objective: We demonstrated previously that infusion of autologous serum does not induce generation of quantifiable amounts of thrombin, even when monitored using a highly-sensitive oligonucleotide-based enzyme capture assay (OECA) which detects thrombin in the low picomolar range. Objective of the present study was to evaluate if the small amounts of free thrombin present in the serum induce generation of activated protein C (APC).

Methods: 15 healthy subjects received infusions of 50 mL of autologous serum over 30 min without accompanying medication, while the thrombin inhibitor argatroban was infused into four volunteers simultaneously with the serum at a dose of 1 µg/kg/min. APC and thrombin were measured before, during and after serum infusion using an OECA platform. In in vitro experiments, APC formation was induced by addition of serum or purified thrombin to buffer containing CaCl2 at physiological concentrations. and protein C as well as thrombomodulin in excess. APC generation was subsequently measured by OECA.

Results: Median (interquartile range) concentrations of thrombin and APC were 185.56 (128.61 - 242.50) pmol/L ( and 163.75 (136.25 – 248.21) pmol/L in the autologous serum, thus doses of 3.33 (1.94 – 4.17) pmol/L of thrombin 2.86 and (2.50 – 3.93) pmol/L of APC were infused per mL of the probands’ plasma volume. Peak thrombin levels of 1.11 (0.00 – 2.22) pmol/L were measured in plasma of the subjects without anticoagulation, indicating a rapid inactivation. APC peak levels in plasma exceeded the infused APC doses by a multiple (25.18 (13.57 – 53.04) pmol/L), even in samples from probands that received argatroban (16.79 (14.11 – 21.79) pmol/L). 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 138.89 pmol/L. Addition of human serum as a thrombin source in the same purified system consistently induced formation of greater amounts of APC than expected on the basis of the amount of thrombin present in the serum samples.

Conclusion: The data presented here suggests a mechanism of APC generation that does not depend on thrombin. We hypothesize that further experiments with endothelial cells will shed more light on this alternative way of APC generation.