한국산 거머리인 Hirudo nipponia와 Whitmania sp.의 추출액으로 trypsin, factor Xa, thrombin에 대한 저해활성을 조사하였다. Hirudo nipponia로부터 얻은 추출액은 $20{mu}g/ml$ 농도에서 이 효소들의 활성을 완전히 저해하는 반면 Whitmania sp.의 추출액은 동일조건하에서 trypsin만을 저해하였다. 이 Hirudo nipponia의 추출물로부터 항thrombin 인자를 분리 정제하기 위하여 anion exchange chromatography, size exclusion chromatography 및 HPLC를 이용하였고 최종적으로 세 종류의 단백질(I-A,I-B,I-C)을 얻었다. 이 중 두 종류 단백질(I-A, I-B)의 아미노말단 서열을 부분적으로 분석한 결과 이 단백질이 주로 산성 아미노산잔기로 구성되어 있으며 heparin cofactor II의 일부분과 유사성이 있음이 발견되었다.
Two Korean leech species, Hirudo nipponia and Whitmania sp., were examined for the presence of anticoagulant activity. Crude extracts of the leech head parts were fractionated by ethanol and the anticoagulant activity of these extracts was assessed with respect to the inhibition of thrombin, factor Xa and trypsin using the chromogenic substrate assay. While the extract from Hirudo nipponia inhibited all three serine proteases employed in the assay, that of Whitmania sp. inhibited only trypsin, but not thrombin and factor Xa. Thus, the extract from Hirudo nipponia which shows antithrombin activity was subjected to further purification. Three steps of column chromatography including gel filtration on Superose 12 column, anion exchange on Mono-Q and reverse phase HPLC on C-8 column could resolve 3 antithrombin activity peaks from anti-trypsin and anti-factor Xa activities, indicating the presence of multiple forms of thrombin-specific inhibitors in Hirudo nipponia. N-terminal amino acid sequences of the two of these forms showed a limited degree of homology with a portion of heparin cofactor II.
