Molecular analysis of novel platelet aggregation-inducing factor Aggrus

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There exists a clear link between atherosclerotic vascular disease, inflammation, tumor metastasis, and thrombosis. Thus, it is important to identify the mechanisms of platelet aggregation that have pathobiologic, prognostic, and treatment-related relevance. We have previously identified Aggrus as a novel platelet-aggregating factor expressed on a number of human cancers.1. Estimation of the platelet aggregation-stimulating domains in AggrusBy establishing stable CHO clones expressing several Aggrus point mutants, we estimated their platele-aggregating activities. We found that O-glycosylated threonine-34 and threonine-52 in human Aggrus were required for platelet-aggregating and metastasis-promoting activities. We confirmed that conversion of threonine residues to alanine residues eliminated the platelet aggregation-inducing capability in vitro and metastasis-promoting ability in vivo. These results indicate that Aggrus contributes to the establishment of pulmonary metastasis by promoting platelet aggregation. Aggrus could serve as an ideal therapeutic target for drug development to block metastasis.2. Identification of a novel Aggrus-binding proteinWe screened Aggrus binding protein (s) and found that a four transmembrane-domain protein of the tetraspanin family member, CD9, formed a complex with Aggrus on the cell surface. Ectopic expression of CD9 suppressed pulmonary metastasis of tumor cells. We found that CD9 formed a complex with Aggrus, via transmembrane domains 1 and 2 (TM1 and 2) and localized in the tetraspanin web. Because the CD9 mutant lacking TM1 and 2 could not suppress Aggrus-induced platelet aggregation nor Aggrus-mediated lung metastasis, we concluded that CD9 neutralized tumor metastasis-promoting ability of Aggrus by attenuating Aggrus's platelet aggregating ability. These results suggest that we can indirectly control Aggrus function by regulating Aggrus-binding protein, such as CD9.