Sustained Pharmacological Activation of Protein C (PC) in Baboons.

The introduction of two mutations at the specificity sites S3-S4 of thrombin (W215A and E217A) results in a mutant thrombin (WE) with more than 1000-fold reduced activity towards fibrinogen and PAR1. WE retains its antithrombotic activity via selective activation of the circulating PC pool and elevation of activated PC (APC) levels when administered as a bolus to primates (Gruber et al. Blood 2002). We investigated whether increased APC levels can be sustained and/or the PC pool exhausted by a 4 to 6 hour long infusion of WE in 15 baboons. In order to assess whether intravascular thrombomodulin availability is rate limiting in pharmacological PC activation, the effect of high dose WE administration on circulating APC (enzyme capture assay) and PC activity (Protac-activatable PC) levels were tested with or without co-administration of equimolar doses of recombinant human soluble thrombomodulin (rhsTM). Infusion of a very high dose of WE alone (110 ug/kg bolus followed by 110 ug/kg/h) gave a peak APC concentration of 201 ng/mL by 10 min. The endogenous APC concentration then sharply decreased with an apparent initial half life (T1/2) of 25 min - similar to that of exogenous human APC in baboons and almost returned to baseline (8 ng/mL) by the end of the treatment despite continued infusion of high dose WE. Thrombin-antithrombin (TAT) complex levels gradually increased reaching an apparent equilibrium at 104 ng/mL by the 4th hour. D-Dimer levels increased throughout the treatment and reached a level that was 20-fold of the baseline value by the end of the treatment. Co-administration of rhsTM (180 ug/kg bolus + 180 ug/kg/h infusion) with the high dose WE also resulted in rapid PC activation and an early APC peak (240 ng/ml at 10 min). The enzyme peak was followed by a decline in APC concentration with an apparent initial T1/2 of 51 min to an average equilibrium concentration of 40 ng/mL by the end of the treatment. TAT levels sharply increased from the 3.5 ng/mL baseline level, reaching greater than 240 ng/ml by the 10th min of the treatment, and remained outside the range of the test. There was a nonsignificant increase in D-Dimer levels towards the end of the WE+rhsTM treatment. Approximately 40% and 80% of the Protac-activatable circulating PC pool was consumed by administration of WE and WE+rhsTM, respectively. Based on the findings we propose that: 1) there is sufficient PC substrate for a sustained pharmacological increase in endogenous APC levels; 2) either complex formation of WE with antithrombin is significantly accelerated by rhsTM or clearance of TAT is inhibited by rhsTM; 3) the existing endogenous soluble and/or endothelial thrombomodulin pool appears to be transiently depleted upon WE infusion; and 4) the circulating PC pool might not be homogeneous such that part of the PC pool is not activatable in vivo by infused WE+rhsTM. In summary, significant and sustained levels of endogenous circulating APC can be generated in baboons by infusion of rhsTM with the highly selective WE thrombin mutant.