CLIN. EXPL. METASTASIS, 1983, VOL. 1, NO. 4, 297-307 Involvement of a cathepsin B-like cysteine proteinase in platelet aggregation induced by tumor cells and their shed membrane vesicles PHILIP G. CAVANAUGHt, BONNIE F. SLOANE~:§, ANDREW S. BAJKOWSKI§, GABRIEL J. GASIC¶, TATIANA B. GASIC¶ and KENNETH V. HONNt§II Departments of Biological Sciencest, Pharmacology{, Radiation Oncology§ and Radiologyll, Wayne State University, Detroit, MI 48202, U.S.A. and Pennsylvania Hospital¶, Philadelphia, PA 19107, U.S.A. (Received 1 June 1983; accepted 6 July 1983) Murine 15091A mammary adenocarcinoma cells and membrane vesicles sponta- neously shed from these tumor cells in culture can induce aggregation of washed human platelets. A spectrum of proteinase inhibitors was tested for their ability to inhibit 15091A induced platelet aggregation. Of the inhibitors tested the most effective were those selective for cysteine proteinases. The effect of the spectrum of p roteinase inhibitors on 15091A induced platelet aggregation was compared to the effect on cathepsin B-like cysteine proteinase activity in homogenates of 15091A tumor cells and their spontaneously shed vesicles. The results suggest that there is a correlation between activity of a cathepsin B-like proteinase in 15091A cells and vesicles and the ability of these cells and vesicles to induce aggregation of washed human platelets. Introduction Patients with malignant neoplasms often have abnormalities in hemostatic function which can include alterations of both platelet aggregation and coagulation [35]. The relationship among these is not yet completely understood; however, the interaction between host platelets and circulating tumor cells is believed to facilitate metastasis [20]. In in vivo studies Gasic et al. [12] have shown that thrombocytopenia reduces the number of lung tumor colonies formed after i.v. injection and that this reduction can be prevented by infusion of platelets. Aggregation of platelets in vitro can be induced by tumor cells, by membrane vesicles spontaneously shed from tumor cells in culture and by a membrane-derived fraction of the tumor cells [5, 8, 11, 15, 20]. Platelet aggregation can also be induced in vitro by ADP, collagen, thrombin, trypsin, papain, platelet activating factor, etc. We had previously shown that the tumor cell activities of a cathepsin B-like cysteine proteinase correlate with the lung colonization potential (tail vein meta- stasis) of murine B16 melanoma variants [30] and that B16 tumor cells in culture release a cathepsin B-like proteinase [31]. Since there is 100 per cent sequence homology at the active site and active site groove between papain and cathepsin B [33] and since papain has been shown to induce platelet aggregation [1], we speculated that one tumor cell principle which might be responsible for inducing platelet aggregation is a cathepsin B-like proteinase. Recently we demonstrated that