抄録
Thrombin is a multifunctional serine protease in haemostasis and is a glycoprotein containing one carbohydrate chain, consisting of lactosamins and N-acetylneuraminic acid (NeuAc). The glycosylated site at Asn53 is located near the hydrophobic pocket flanked by the catalytic triad in an active site cleft of the thrombin molecule. Although the carbohydrate chain is binding near the important domain involved in thrombin protease activity, the function remains unknown. In this report, the enzymatic property of the carbohydrate chain moiety-modified thrombin was compared to that of intact thrombin in order to clarify the function of the carbohydrate chain. The NeuAc at the non-reductive terminal of the carbohydrate chain was removed by neuraminidase. Release of fibrinopeptides was measured by HPLC. The intact thrombin ([M+H]+=37,680, measured by a MALDI/TOF mass spectrometer) released fibrinopeptides A (FPA) and B (FPB) during fibrin clot formation. The NeuAc-lacking thrombin ([M+H]+=36,976) formed fibrin clot, however, FPA was primarily released while little FPB was released. The result suggests that access of the fibrinogen Bβ chain to the active site cleft was restricted by removing NeuAc. The modification of the carbohydrate chain thus may cause the structural alteration of the active site cleft of the thrombin molecule, and the carbohydrate chain may play an important role in thrombin protease activity, especially the recognition of a macromolecular substrate such as fibrinogen. [Jpn J Physiol 55 Suppl:S109 (2005)]
