The AcLDL uptake in THP-1 macrophage was not changed by glycerol (Figure S1). protein or tyrosine kinase inhibitor herbimycin A effectively blocked the effects of protein S on AcLDL uptake. Immunohistochemical analysis revealed that the level of protein S was substantially increased in human atherosclerotic arteries. Thus, human protein S can inhibit the expression and activity of SR-A through Mer RTK in macrophages, suggesting that human protein S is a modulator for macrophage functions in uptaking of modified lipoproteins. == Introduction == Protein S is a vitamin Kdependent plasma glycoprotein that serves as a key cofactor for the anticoagulant activity of activated MMP13 protein C.1In humans, protein S is synthesized mainly by hepatocytes, but also by megakaryocytes, endothelial cells, and vascular smooth muscle cells.2Protein S normally circulates in human plasma at a concentration of approximately 25 mg/L in free form and in complex with C4b-binding protein (C4BP). The critical antithrombotic role of protein S is revealed by the massive thrombotic complications suffered AC710 by infants homozygous for protein S deficiency.3Adult patients with mild heterozygous deficiencies in protein S are associated with AC710 risk for venous and arterial thrombosis,4,5ischemic stroke,6cerebral thrombophlebitis,7myocardial infarction, and vascular calcification.8,9Protein S infused in a murine model of AC710 ischemic stroke decreases infarction and edema volumes.10However, 2 epidemiologic studies have shown that human plasma protein S concentration is positively correlated with risk of coronary heart disease.11,12Clinical studies have also demonstrated that protein S is associated with triglyceride-rich lipoproteins of human plasma, binds with high-affinity to very low-density lipoprotein (VLDL) receptor on macrophages, and induces foam cell formation.13Immunohistochemistry experiments show that protein S localizes in atherosclerotic coronary lesions.14Although these findings suggest the involvement of protein S in atherosclerosis and atherothrombosis, direct evidence of a causal relationship between protein S and vascular disease is very limited, and the underlying mechanisms remain to be determined Protein S and its structural homologue, growth arrestspecific gene-6 (Gas6), may be the ligand for the Tyro3/Ax1 family of receptor tyrosine kinases (RTKs).15,16However, it is not fully understood what the cellular expression and functions of these RTKs are. Surprisingly, only limited studies have reported the nonanticoagulant functions of protein S.10,17,18Human protein S was shown to be a mitogen for vascular smooth muscle cells18and a stimulator for macrophage phagocytosis of apoptotic cells.17The physiologic relevance of human protein S interacting with macrophages or other tissue cells is important but remains largely unknown. Atherothrombosis, defined as atherosclerotic lesion disruption with superimposed thrombus formation, is the leading cause of mortality in the developed countries. In the atherosclerosis formation, the circulating monocytes are recruited to the arterial wall, wherein they transform into macrophages and foam cells through the increased uptake of modified LDL by scavenger receptors such as macrophage scavenger receptor A (SR-A).19SR-A plays critical roles in modified LDL uptake as well as in clearance of debris including necrotic and apoptotic cell fragments in the vessel wall.20However, the endogenous regulators of SR-A expression are not fully elucidated. In this study, we used an in vitro model to address whether protein S could affect SR-A function and expression, and which receptors, transcription factors, or signaling transduction pathways could be involved. We also performed immunohistochemistry analysis to detect the expression of protein S in human atherosclerotic and nonatherosclerotic arteries. This study demonstrates a new function of human protein S, which may play a critical role in the atherothrombotic disease. == Methods == == Chemicals and reagents == Human protein S was obtained from American Diagnostica (Stamford, CT). It was purified from fresh frozen human plasma via immunopurification and characterized by no protein S-C4BP complex, a single band at 69-kDa on 10% SDSpolyacrylamide gel electrophoresis (PAGE),.