Abstract
The purpose of this study was to purify and characterize the agent responsible for the antimetastatic activity of an extract of the salivary glands (SGE) of the Mexican leech Haementeria officinalis. When administered intravenously in mice on the same day as the intravenous inoculation of T241 sarcoma cells, SGE markedly reduces the number and size of lung tumor colonies. In designing a purification protocol for the antimetastatic agent, we postulated that the antimetastatic agent would also display anticoagulant activity. Thus, we discovered that heparin affinity chromatography followed by anion-exchange chromatography results in a fraction highly enriched in both potent anticoagulant activity and potent antimetastatic activity. Approximately, 200-300 micrograms of purified protein is isolated from 150 mg of SGE. As little as 15 micrograms of this material inhibits tumor cell metastasis to the same extent as 1.0 mg of the unfractionated SGE. When analyzed on sodium dodecyl sulfate gels the active fraction consists mainly of one polypeptide band having an apparent molecular weight of approximately 17,000 under either reducing or nonreducing conditions. The protein has a pI of approximately 9.5 and a molecular weight of approximately 17,000 under nondenaturing conditions. A specific antiserum prepared against the 17,000-dalton protein indicated that this protein is the major anticoagulant and antimetastatic agent of leech salivary gland extract. We have termed this anticoagulant, antimetastatic agent "antistasin." We hypothesize that antistatin inhibits coagulation via factor Xa, and not thrombin, since factor Xa, but not thrombin, is rapidly inactivated upon addition of antistasin. The mechanism of antistasin's antimetastatic activity is currently under investigation.
Highlights
From the SLankenau Medical Research Center, Philadelphia, Pennsylvania 19151and the
Purification of the Antimetastatic Actiuity-We hypothesized that protein fractionspossessing anticoagulant activity would alsopossess antimetastatic activity. This hypothesis was based on preliminary observations from early attempts at purification of the antimetastatic agenwt hich showedthat fractions possessing antimetastaticactivity alsodisplayed anticoagulant activity
Antimetastatic activity was purified by a combination of heparin-agarose chromatography followed by anion-exchange chromatographyusing FPLC
Summary
From the SLankenau Medical Research Center, Philadelphia, Pennsylvania 19151and the The purposeof this study was to purify and charac-the European leech, such as the thrombin-specific inhibitor terize the agent responsible for the antimetastatic ac- [2] hirudin, and low molecular weight antiproteases [3], the tivity of an extractof the salivary glands(SGE) of the eglins. Other leech anticoagulant proteins have been identi-. 200-300 pg of purified protein is the active antimetastatic agent from one of these leech speisolated from 150 mg of SGE. A specific antiserum prepared gel electrophoresis were obtained from Bio-Rad. Reagents for fast against the 17,000-dalton protein indicated that this protein liquid chromatography were purchased from Pharmacia P-L protein is the major anticoagulant and antimetastatic Biochemicals.The chromogenic substrates N-benzoyl-L-isoleucylgluagent of leech salivary gland extract.We have termed tamylglycyl-L-arginine-p-nitroanilihdyedrochloride (S-2222)and H-
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