The effect of thrombin inhibitors on coagulation activity and generation of activated protein C

The direct thrombin inhibitor inogatran was, in the TRIM trial (n=1209), compared with unfractionated heparin (UFH) concerning the effect on cardiovascular end-points in unstable coronary artery disease. The results in TRIM demonstrated a trend towards a benefit for UFH during the treatment phase, which was reduced after drug withdrawal. The first phase of this thesis investigates if the findings in the TRIM trial could be explained by patterns of molecular markers for coagulation activity, prothrombin fragment 1+2 (PF 1+2), thrombin-antithrombin (TAT) complexes, soluble fibrin and fibrin D-dimers, in a subset of patients (n=320). The relationship between treatment intensity measured by activated partial thromboplastin time (APTT) and clinical end-points was also investigated. Inhibition of thrombin anticoagulant activity, Le the generation of activated protein C (APC) by thrombin bound to thrombomodulin, could be a possible explanation for results in clinical trials with direct thrombin inhibitors. This concept was evaluated in the second phase of this thesis, which involved studies on the influence of different thrombin inhibitors on the generation of APC and fibrin formation, measured by release of fibrinopeptide A (FPA), in purified systems and on endothelial cells (EC).

During treatment with inogatran, PF 1+2, TAT complexes and D-dimer decreased to low levels, which lasted the entire infusion period. After cessation of treatment the markers returned to baseline levels. During treatment with heparin PF 1+2 and D-dimer rapidly decreased, but later increased above baseline levels. The increase continued after cessation of treatment. High APTT level during inogatran treatment was related to increased risk of cardiovascular events whereas high APTT level during heparin treatment was related to decreased risk of cardiovascular events. The in vitro experiments in this thesis demonstrated that clinically relevant concentrations of inogatran, melagatran, hirudin and AT, in the absence or presence of UFH or LMWH, inhibited thrombinthrombomodulin (TM)-mediated APC generation, in solution or on EC. The irreversible inhibitor PPACK, which lacks therapeutical potential, also inhibited thrombin-TM mediated APC generation on EC, but exaggerated concentrations of the hirudin derivative hirugen neither inhibited APC nor FPA generation. Furthermore the experiments indicated that the tested thrombin inhibitors inhibited FPA generation to the same extent as APC generation. Conclusions Treatment with inogatran or UFH in unstable coronary artery disease decreases markers of coagulation activity, but not in a consistent way. After drug withdrawal a reactivation in biochemical markers of coagulation activity is observed, which may be associated with the increase in cardiovascular events during the same period. Treatment intensity measured by activated partial thromboplastin time is not well defined for low molecular weight direct thrombin inhibitors. All tested thrombin inhibitors inhibited APC generation and fibrin formation approximately to an equal extent. These findings may offer an explanation of the lack of a dose-response effect in clinical trials but do not support the hypothesis that inhibition of APC generation is a likely cause for advantages or disadvantages of direct thrombin inhibitors in comparison to unfractionated heparin in clinical trials on patients with unstable coronary artery disease.

List of scientific papers

I. Linder R, Oldgren J, Egberg N, Grip L, Larson G, Siegbahn A, Wallentin L (1999). "The effect of a low molecular mass thrombin inhibitor, inogatran, and heparin on thrombin generation and fibrin turnover in patients with unstable coronary artery disease. " Eur Heart J 20(7): 506-18
https://pubmed.ncbi.nlm.nih.gov/10365287

II. Oldgren J, Linder R, Grip L, Siegbahn A, Wallentin L (1999). "Activated partial thromboplastin time and clinical outcome after thrombin inhibition in unstable coronary artery disease" Eur Heart J 20(22): 1657-66
https://pubmed.ncbi.nlm.nih.gov/10543929

III. Linder R, Blomback M, Egberg N, Grip L (1999). "Thrombin inhibitors suppress the thrombin-thrombomodulin-mediated generation of activated protein C. " Thromb Res 95(2): 117-25
https://pubmed.ncbi.nlm.nih.gov/10418800

IV. Linder R, Frebelius S, Jansson K, Swedenborg J (2002). "Inhibition of endothelial cell mediated generation of activated protein C by direct and antithrombin dependent thrombin inhibitors." (Submitted)

V. Linder R, Frebelius S, Grip L, Swedenborg J (2002). "The influence of direct and antithrombin dependent thrombin inhibitors on the procoagulant and anticoagulant effects of thrombin." (Submitted)