Aprotinin can inhibit the proteolytic activity of thrombin

Abstract

Aprotinin has been shown to reduce blood loss and blood requirement when administered prior to surgery and this therapeutic benefit appears to be related to its specificity as a protease inhibitor. The inhibition of plasmin by aprotinin is well characterized, but little is known of its effect on thrombin. In preliminary experiments, we showed that aprotinin can prevent platelet aggregation induced by thrombin. Follow-up studies have now been performed in order to clarify the effect of aprotinin on thrombin. A fluorescence study of the direct binding of aprotinin to human cr-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x mol . 1-l was determined. Aprotinin can displace p-aminobenzamidine, a fluorescent-probe molecule which binds to the active site of serine proteases, showing that the active site of thrombin was involved. Aprotinin also inhibited the ability of thrombin to induce a fibrin clot from purified fibrinogen and to induce the hydrolysis of the chromogenic substrate H-D-phenylalanylpipecolylarginine-p-nitroanalidehydrochloride (S-2238). With S-2238, double-reciprocal plots show that the inhibition is competitive with a Ki of 61 pM and a K , of 1.72 pM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 f 0.12 and an apparent dissociation constant of 57.0 13.1 pM (mean f SEM). Thus, the inhibition of thrombin-induced platelet aggregation by aprotinin fits a model of competitive inhibition. Conclusions are that, in addition to a possible direct effect of aprotinin on platelets, the inhibition of thrombin-induced platelet activation by aprotinin can be also explained, in part, by a direct effect of the inhibitor on the thrombin molecule itself. This supports the concept that a proteolytic step is involved in the platelet response to thrombin. Finally, evidence is in favour of the participation of Trp245 in the fluorescence response of thrombin on binding to aprotinin.Aprotinin [l], a well-known basic proteinase inhibitor isolated from bovine tissues, is of particular interest because of its broad inhibitory specificity. It is a reversible and competitive inhibitor of various proteinases, the active site of the enzyme being blocked by an equimolar complex formation. Besides trypsin and chymotrypsin, it strongly inhibits plasmin, kallikrein and protein C [l, 21. This specificity accounts for its initial therapeutic use, particularly in pancreatisis, various states of shock or hyperfibrinolytic hemorrhage [I]. More recently, the administration of aprotinin during cardiac surgery has been shown to reduce the peroperative and postoperative bleeding, leading to a reduction in the transfusion requirement [3 -61.The beneficial effect of aprotinin on hemostasis was ascribed to protection of the plasma proteins cascade from activation and to platelet preservation [6 -81. Platelet preservation can be ensured by the protection of platelet receptors from exposure and/or degradation by agonists ...

Clinical Inhibition of the Seven-Transmembrane Thrombin Receptor (PAR1) by Intravenous Aprotinin During Cardiothoracic Surgery

“…2,3 However, the obvious hypothesis, that aprotinin as a serine protease inhibitor directly inhibits the catalytic activity of thrombin in solution, is unlikely, because the systemic concentration of aprotinin achievable during bypass is Ϸ60-fold below the inhibitory constant (K i ) for thrombin. 15 The PAR1-sparing mechanism described here places aprotinin into a distinct category of PAR1 antagonists, with implications beyond its primary use as a hemostatic agent during cardiac surgery. The present study demonstrates that PAR1 activation by thrombin can be inhibited therapeutically; however, we are not advocating extending the therapeutic role of aprotinin beyond its current license.…”

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

“…However, this method indirectly measured APC activity, and therefore the interaction between aprotinin and thrombin needs to be considered. Although aprotinin is a poor inhibitor of thrombin (K i ¼ 61 lmol/l) (Pintigny & Dachary-Prigent, 1992), it reduced the peak TG in a dose-dependent manner (but was not statistically significant) in both normal (Figs 1 and 2) and PC-depleted plasma samples (Fig 3). This is consistent with other reports (Poullis et al, 2000;Day et al, 2004;Khan et al, 2005).…”

Tick-derived Kunitz-type inhibitors as antihemostatic factors

“…(Ascenzi et al, 1988;Broze et al, 1990;Pintigny and Dachary-Prigent, 1992)). This resistance of the major procoagulant proteinase to inhibition by Kunitz modules is due to its narrow active-site cleft, which does not allow insertion of the rather bulky reactive-site loop of a canonically binding Kunitz domain ( (Bode et al, 1989(Bode et al, , 1992; see also Fig.…”