PMID:1378406

Citation	Pintigny, D and Dachary-Prigent, J (1992) Aprotinin can inhibit the proteolytic activity of thrombin. A fluorescence and an enzymatic study. Eur. J. Biochem. 207:89-95
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 alpha-thrombin was analysed according to the Michaelis-Menten model and a dissociation constant of 30 x 10(-6) mol.l-1 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 microM and a Km of 1.72 microM. Aprotinin was a potent inhibitor of thrombin-induced aggregation. A Schild plot of the aggregation data yielded a slope of 0.97 +/- 0.12 and an apparent dissociation constant of 57.0 +/- 13.1 microM (mean +/- 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.
Links	PubMed
Keywords	Aprotinin/pharmacology; Benzamidines/pharmacology; Humans; Kinetics; Mathematics; Platelet Aggregation/drug effects; Platelet Aggregation Inhibitors/pharmacology; Spectrometry, Fluorescence/methods; Thrombin/antagonists & inhibitors; Thrombin/pharmacology
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
BOVIN:BPT1		GO:0070495: negative regulation of thrombin receptor signaling pathway	ECO:0000314:		P		Fig. 4: Aprotinin inhibits the ability of thrombin to induce a fibrin clot. The fibrinogen concentration was contant at curve a while the clotting time increased from curve b to e with the presence of aprotinin	complete CACAO 6983
BOVIN:BPT1		GO:0090331: negative regulation of platelet aggregation	ECO:0000314:		P		Fig. 5 shows the aggregation of human platelets was reduced by increasing concentration of aprotinin. Curve a was the control(without the presence of aprotinin)	complete CACAO 7006
BOVIN:BPT1	involved_in	GO:0090331: negative regulation of platelet aggregation	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete
BOVIN:BPT1	involved_in	GO:0070495: negative regulation of thrombin-activated receptor signaling pathway	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

References

See Help:References for how to manage references in GONUTS.