Copyrighr @ 1976 by Academic Press. Inc. A/i rights of reproduction in any fbrm reserved Experimental Cell Research 101 (1976) 41-46 MITOGENICITY OF THROMBIN AND SURFACE ALTERATIONS ON MOUSE SPLENOCYTES L. B. CHEN, N. N. H. TENG and J. M. BUCHANAN Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA SUMMARY Splenocyte cultures prepared from BALB/c mice can be stimulated to DNA synthesis by thrombin and trypsin but not by bovine serum. The stimulation by thrombin as a function of concentration of the enzyme is the same in the presence or absence of serum, whereas lower concentrations of trvusin are not mitoaenic if combined with serum. These observations indicate that the inactivating or neutralizing proteins for thrombin have been substantially reduced during the clotting process but that inhibitors of trypsin still remain. The stimulation of splenocytes to DNA synthesis is accompanied by a loss of a lactoperoxidase-iodinatable protein on the splenocyte cell surface. This nrotein is 45 000 D and corresnonds in size to a groun of lymphocyte surface proteins including H-2 ktigen. We have proposed-that thrombin may play i role in wound healing by stimulating proliferation of not only tibroblasts but also B lymphocytes, a process of possible physiological significance in defending the host against pathogens and during inflammations. The mechanisms involved in triggering cell division in fibroblasts and lymphocytes are currently under intensive investigation in many laboratories. Much recent attention has been focused on the initial action of mitogens on the cell surface since such events are believed to play an essential role in cell proliferation [ 11. Whereas most tibro- blast mitogens are polypeptide hormone- like substances such as Cohen’s epidermal growth factor [2], Gospodarowicz’s fibro- blast growth factor [3], somatomedin [4], etc., mitogens for lymphocytes are either specific antigens or non-specific stimulants such as phytohemagglutinin (PHA), con- canavalin A (ConA), pokeweed mitogen (PWM) and lipopolysaccharides (LPS) [5]. All these mitogens appear to exhibit their mitogenicity through binding onto certain cell surface receptors. Therefore, a critical step in investigating mitogenicity is the characterization of cell surface receptors. Unfortunately, the above mentioned mito- gens have been used with only limited suc- cess in the identification of cell surface re- ceptors either because the binding complex frequently cannot survive treatment with detergents such as sodium dodecyl sulfate (SDS) and Triton X-100, or because the solubilized receptors can no longer be re- cognized by the mitogen. Therefore, de- spite the fact that many mitogens have been well characterized, their interactions with the cell surface remain obscure. We wish to suggest that use of a highly specific protease might aid in understanding the relationship between surface perturba- tion and cell division. Recently we have demonstrated that thrombin, a highly spe- cific serine protease that plays a central role Exp Cell Res 101 (1976)