Isolation and characterization of antistasin. An inhibitor of metastasis and coagulation

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.