This is a Phase I application to develop new anti- thrombotic drugs for the treatment of thromboembolic disorders. In pilot studies, a coagulation inhibitor has been identified in an aqueous extract of Anodontia woodiana. Early chromatographic and spectral data suggest that the active principle(s) may be a small peptide with a molecular weight range between 2,000-4,000 Daltons. Active fractions from this preparation block thrombin activity and can also prolong the prothrombin and thrombin time measurements. The purpose of this application is to obtain support to further purify the active component(s) of the aqueous extract. Its physical, chemical and biological activities will then be evaluated with an eventual goal of elucidation of the mode of action of this new compound. In the Phase I study, total characterization of the active component of the extract is anticipated and this will be followed by Phase II development of the antithrombotic principle with eventual entry into clinical research. The major interest in Thrombo Diagnostic is to develop remedies from herbs and animal sources which have minimal side effects. Such compounds have been used in China for more than a thousand years. There is an extract from clam (A. woodiana) which has been used in China for thrombotic disorders. Clam shells are ground and extracted in acetic acid in the cold for 2 hours. After filtration, centrifugation and dialysis of the supernatant, a compound is obtained which prolongs the activated partial thromboplastin time (66 seconds as compared to a control of 26-30 seconds) (at a concentration of 50 ug/ml). Ion exchange chromatography, starting with QAE-Sephadex has been carried out and an active fraction eluted in the first peak. Further purification was done on a mono-Q column with FPLC. A UV spectrum shows a single or several related peptides. The fraction is trypsin-sensitive, but pepsin insensitive. In addition to activity in coagulation tests as mentioned, the compound inhibits alpha-thrombin. Thus, the major fraction may be a thrombin inhibitor. The compound is different from Hirudin and may represent a new class antithrombin-type anticoagulants. It is not thought to be a degradation product of another larger protein. In this Phase I application, a larger number of separation techniques will be used to purify the compound to homogeneity. This will involve additional types of chromatography which should resolve the material into a single component. The active fraction contains mostly polar molecules which are hydrophobic and is already quite pure. Thus, further chromatography may resolve the components. Structural studies are also anticipated, including nuclear magnetic resonance spectroscopy. Dr. Alfred Weinheimer, a medicinal chemist who is Chairman of the College of Pharmacy at the Unversity of Houston will act as a consultant as will Dr. Kenneth K. Wu, who is a world authority in the coagulation field. Questions to be asked include whether the compound binds to a site on thrombin which is similar to hirudin or to AT-III. Tests will also be carried out to learn whether the compound blocks activated factor X. If the final product contains more than one compound, these may have distinct anticoagulant properties. On the other hand, a single component may have multiple inhibitory properties. Specific coagulation factor assays will be carried out to determine the above mentioned details.
