Molecular analysis of novel platelet aggregation-inducing factor Aggrus

动脉粥样硬化性血管疾病、炎症、肿瘤转移和血栓形成之间存在明显的联系。因此，重要的是要确定血小板聚集的机制，具有病理生物学，预后和治疗相关性。我们以前已经确定Aggrus是一种新的血小板聚集因子，在许多人类癌症中表达。Aggrus中血小板聚集刺激结构域的测定通过建立稳定表达几种Aggrus点突变体的CHO克隆，我们测定了它们的血小板聚集活性。我们发现，O-糖基化的苏氨酸-34和苏氨酸-52在人类聚集所需的血小板聚集和转移促进活动。我们证实，苏氨酸残基转化为丙氨酸残基消除了体外血小板聚集诱导能力和体内转移促进能力。这些结果表明，聚集蛋白通过促进血小板聚集而有助于肺转移的建立。结论：1. Aggrus可作为一个理想的治疗靶点，用于肿瘤转移阻断药物的开发.一种新的Aggrus结合蛋白的鉴定我们筛选了Aggrus结合蛋白，发现四跨膜蛋白家族成员CD 9与细胞表面的Aggrus形成复合物。异位表达CD 9抑制肿瘤细胞的肺转移。我们发现，CD 9形成了一个复杂的聚集，通过跨膜结构域1和2（TM 1和2）和定位在四跨膜蛋白网络。由于缺乏TM 1和2的CD 9突变体不能抑制Aggrus诱导的血小板聚集，也不能抑制Aggrus介导的肺转移，因此我们得出结论，CD 9通过减弱Aggrus的血小板聚集能力来中和Aggrus的肿瘤转移促进能力。这些结果表明，我们可以通过调节Aggrus结合蛋白（如CD 9）来间接控制Aggrus功能。

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.