.1oirmal zyxwvutsrqponmlk o/ zyxwvutsrqponmlk Neurorlieinistry zyxwvutsrqponm Raven Press, zyxwvutsrqponm Ltd., New York zyxwvutsrqp (cl 1993 International Society for Neurochemistry Rapid Communication Protein S, an Antithrombotic Factor, Is Synthesized and Released by Neural Tumor Cells Donald J. Phillips, "Judith S. Greengard, *Jose A. Fernandez, Maria Ribeiro, Bruce L. Evatt, *John H. Griffin, and W. Craig Hooper zyxw Hemalologic Diseases Branch, Division zyxwvutsr of HI V/AIDS, Centerfor Infectious Diseases, Centersfiir Disease Control and Prevention, Atlanta, Georgia; and *Department of Molecular and Experimental Medicine and the C4)mmittee on Vascular Biology, The Research Institute of Scripps Clinic, La Jolla, California, U.S.A. zy Abstract: Protein S, an anticoagulant factor in the protein C anti- thrombotic pathway, was found to be synthesized and released by six tumor cell lines of neural origin by western blotting and ELISA. The rate of synthesis ranged from three- to 11-fold higher than that of a microvascular endothelial cell line and 36-144% that of a hepatoma cell line. The secreted protein S displayed specific anticoagulant activity similar to that of purified plasma protein S, implying that it was fully y-carboxylated. Ten primary brain tumor tissues also expressed protein S antigen, as shown by western blot analysis. Expression of anticoagulantly active protein S by neural cells raises important questions concerning possible physiologic roles for this multidomainprotein beyond its function in control of thrombosis. Key Words: Protein S-Brain tumor-Coagulation-y-Carboxylase. J. Neurochem. 61,344-347 (1 993). Protein S is a 75-kDa multidomain vitamin K-dependent plasma protein produced in vivo mainly in the liver. Its presence has also been demonstrated in platelets (Schwarz et al., 1986)and megakaryocytes (Ogura et al., 1987), as well as hepatoma and vascular endothelial cells (Fair and Mar- lar, 1986; Fair et al., 1986; Stern et al., 1986). Normal plasma levels of total protein S range from I9 to 35 kg/ml (Griffin et al., 1992). About 60% of plasma protein S circu- lates in a reversible stoichiometric complex with a negative regulator of the complement cascade, C4b-binding protein (C4bBP). The complexed form is inactive as the cofactor for activated protein C (aPC). Free protein S acts as an anti- thrombotic agent both independently of aPC (Heeb et al., 1993)and as a cofactor for aPC that inactivates the coagula- tion cofactors Va and VIIIa, thus decreasing thrombin gen- eration. Decreased levels of protein S are associated with an increased incidence of thrombosis (Comp and Esmon, 1984; Schwarz et al., 1984; Gladson et al., 1988). Recent observations may portend important physiologic functions for protein S outside of the coagulation system. Protein S may protect against inflammatory damage by localizing C4bBP on the neutrophil surface (Furmaniak-Kazmierczak et al., 1993). Osteopenia was reported in association with two cases of thrombosis due to congenital protein S defi- ciency (Pan et al., 1990), prompting the discovery that pro- tein S is synthesized in human osteosarcoma cell lines and adult osteoblast-like cells and is a component of bone ma- trix (Maillard et al., 1992). Protein S is also known to be produced by certain other human tumor cells, including lung epidermoid carcinoma, melanoma, and breast and co- lon adenocarcinomas (Al-Mondhiry and Wallin, 1989). The importance of protein S for host defense was conclu- sively shown in a baboon model of Escherichia coli shock, where neutralization of endogenous protein S converted a nonlethal dose of bacteria to a lethal dose (Taylor et al., 1991). Many tissues are known to express y-carboxylase activity, although their endogenous substrate(s) remain largely un- identified. Brain tissue has not been shown to express this activity directly but has recently been reported to express prothrombin mRNA (Dihanich et al., 1991). To explore whether protein S may also be produced by transformed neural tissue, we examined brain tumor cells for the pres- ence of protein S transcripts, antigen, and activity. We re- port here that functionally active protein S is constitutively expressed in brain tumor cell lines as well as in primary brain tumors. MATERIALS AND METHODS Cell lines Tumor cell lines were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.) and main- tained by the Tissue Core Facility, Division of Scientific Services, Centers for Disease Control. The microvascular endothelial cell line HMEC- 1 has been described (Ades et al., 1992). Cells were propagated as suspension or mono- layer cultures, and fluids were removed at various times for protein S quantification by western blot, ELISA, or anticoag- ulant assay. To determine the amount and rate of protein S ~ ~ Received March 3, 1993; accepted March 26, 1993. Address correspondence and repnnt requests to Dr. W. C Hooper at MS-DO2, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, U.S.A Abbrevrafrans twd aPC, activated protein C; C4bBP, C4b-bind- ing protein, HRP, horseradish peroxidase. 344