The effect of aprotinin on activated protein C‐mediated downregulation of endogenous thrombin generation

Abstract

SummaryThrombin plays a central role in coagulation and haemostasis. Binding of thrombin to thrombomodulin generates activated protein C (APC), which exerts a negative feedback on thrombin formation. Aprotinin, a natural proteinase inhibitor is used extensively during cardiac surgery because this procedure is often associated with profound activation of coagulation and inflammatory pathways. Some in vitro evidences suggest that aprotinin inhibits APC, but the clinical relevance is unclear. The recombinant human soluble thrombomodulin (rhsTM)-modified thrombin generation (TG) assay was used to investigate the effects of aprotinin on APC in plasma samples obtained from healthy volunteers, aprotinin-treated cardiac surgical patients and in protein C (PC)-depleted plasma. Based on the results of in vitro TG assay, addition of rhsTM (0AE75-3AE0 lg/ml) to volunteer or patient plateletpoor plasma significantly reduced (70AE8 ± 21AE9 and 95AE3% ± 4AE6%, respectively) thrombin formation when compared with PC-depleted plasma (8AE3% ± 5AE2%). Aprotinin (100-200 KIU) caused a small, statistically insignificant decrease in the peak thrombin formation in normal and PC-deficient plasma (12AE0 ± 6AE1%). In cardiac surgical patients, levels of functional PC, factor II, antithrombin and platelet significantly decreased after cardiopulmonary bypass (CPB). Soluble thrombomodulin concentrations were increased after CPB (3AE5 ± 2AE2 to 5AE0 ± 2AE2 ng/ml), but they were still within the normal range for human plasma. Our results showed that, even though endogenous PC level is decreased after CPB, it retains its activity in the presence of thrombomodulin, and aprotinin has limited inhibitory effect on APC generation.

Enhanced Thrombin Generation After Cardiopulmonary Bypass Surgery

“…Similar to our results, Tanaka et al 30 demonstrated a reduced thrombin generation compared with prebypass values by using the Thrombin Generation and Cardiac Surgery method described by Hemker et al, 45 which was also confirmed by others. 32,52 The postoperative period has not yet been investigated.…”

Effects of recombinant activated factor VII on thrombin-mediated feedback activation of coagulation

“…The reduction of bleeding shown with aprotinin and lysine analogs after cardiopulmonary bypass illustrates the importance of antifibrinolytic activity in the presence of reduced thrombin generation [2]. Interestingly, aprotinin -clinically used as an antifibrinolytic agent -is a potent FXIa inhibitor (inhibitor constant ¼ 0.89 mmol/l; therapeutic level, 4.3 mmol/l) [26].…”

“…A bleeding tendency is clinically observed in hemophilia or after cardiopulmonary bypass when thrombin generation is slow or insufficient [1,2]. The initial steps of coagulation are mediated by the extrinsic pathway (tissue factor, activated factor VII, activated factor X); however, intrinsic pathway enzymes, activated factor XI (FXIa), tenase (activated factor IX, activated factor VIII) and prothrombinase (activated factor X, activated factor V) are vital to the propagation steps of thrombin generation (Fig.…”

Interaction between thrombin mutant W215A/E217A and direct thrombin inhibitor

“…Using the Thrombinoscope system that continuously monitors thrombin generation based on the hydrolysis of fluorogenic substrate, Z-Gly-Gly-AMC (Bachem Bioscience, King of Prussia, Pennsylvania, USA), we showed that in plasma the combination of W215A/E217A (5µg/ml~135 nmol/l) and thrombomodulin (0.75µg/ml; Asahi Kasei Pharma, Oh-hito, Japan) [12], but not W215A/E217A alone exerted anticoagulant effects based on the reduced peak levels of thrombin generation (Fig. 2a).…”