Monocytes appear to be central to atherogenesis both as the progenitors of foam cells and as a potential source of growth factors mediating intimal hyperplasia, but the chemical messages which stimulate the influx of monocytes into human atheroma remain unknown. Monocyte chemoattractant protein-i (MCP-1) is a recently described molecule with powerful monocyte chemotactic activity expressed by monocytes, vascular endothelial cells, and smooth muscle cells in culture. To begin to address the role of MCP-1 in vivo, we examined 10 normal arteries and 14 diseased human arteries for MCP-1 expression by in situ hybridization. MCP-1 mRNA was detected in 16% of 10,768 cells counted in human carotid endarterectomy specimens with highest expression seen in organizing thrombi (33%) and in macrophage rich areas bordering the necrotic lipid core (24%) as compared to the fibrous cap (8%) and the necrotic lipid core itself (5%). Based on immunohistochemical staining of serial sections and on cell morphology, MCP-1 mRNA appeared to be expressed by vascular smooth muscle cells (VSMC), mesenchymal appearing intimal cells (MICs), and macrophages. By contrast, few cells expressing MCP-1 mRNA were found in normal arteries (< 0.1%). These data suggest a potential role for MCP-1 in mediating monocytic infiltration of the artery wall. (J.

We used PCR to amplify proteinase activated receptor-2 (PAR-2) from human kidney cDNA. The open reading frame comprised 1191 bp and encoded a protein of 397 residues with 83% identity with mouse PAR-2. In KNRK cells (a line of kirsten murine sarcoma virus-transformed rat kidney epithelial cells) transfected with this cDNA, trypsin and activating peptide (AP) corresponding to the tethered ligand exposed by trypsin cleavage (SLIGKV-NH2) induced a prompt increase in cytosolic calcium ion concentration ([Ca2+]i). Human PAR-2 (hPAR-2) resided both on the plasma membrane and in the Golgi apparatus. hPAR-2 mRNA was highly expressed in human pancreas, kidney, colon, liver and small intestine, and by A549 lung and SW480 colon adenocarcinoma cells. Hybridization in situ revealed high expression in intestinal epithelial cells throughout the gut. Trypsin and AP stimulated an increase in [Ca2+]i in a rat intestinal epithelial cell line (hBRIE 380) and stimulated amylase secretion in isolated pancreatic acini. In A549 cells, which also responded to trypsin and AP with mobilization of cytosolic Ca2+, AP inhibited colony formation. Thus PAR-2 may serve as a trypsin sensor in the gut. Its expression by cells and tissues not normally exposed to pancreatic trypsin suggests that other proteases could serve as physiological activators.

Thrombin is a multifunctional serine protease generated at sites of vascular injury. A host of thrombin actions on vascular endothelial cells, smooth muscle cells, and macrophages has been defined in cell culture systems, but the in vivo significance of these activities is unknown. We have defined the expression of the recently identified receptor for thrombin in human arteries by both in situ hybridization and immunohistochemistry. In normal-appearing arteries, thrombin receptor was expressed almost exclusively in the endothelial layer. By contrast, in human atheroma, the receptor was widely expressed, both in regions rich in macrophages and in regions rich in vascular

Abstract. We recently isolated a cDNA clone encoding a functional platelet thrombin receptor that defined a unique mechanism of receptor activation. Thrombin cleaves its receptor's extracellular amino terminal extension, unmasking a new amino terminus that functions as a tethered peptide ligand and activates the receptor. A novel peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, suggesting that this unusual mechanism accounts for platelet activation by thrombin. Does this mechanism also mediate thrombin's assorted actions on non-platelet cells? We now report that the novel W E recently isolated a cDNA clone encoding a functional thrombin receptor by expressing cloning in Xenopus oocYtes (23) . The library used was made from Dami cells, a megakaryocyte-like cell line, and the clone was shown to be expressed by platelets . The clone encodes a seven transmembrane domain receptor with a one hundred-residue extracellular amino terminal extension . This extracellular extension contains a putative thrombin cleavage site (LDPR/S ; / represents the point of cleavage) resembling the known thrombin cleavage site found in the thrombin-activated zymogen protein C (LDPR/I). Structureactivity studies with the cloned receptor (10, 22, 23) strongly suggest a unique mechanism of receptor activation. Thrombin cleaves its receptor at the LDPR/S cleavage site unmasking a new amino terminus beginning with the sequence SFLL . . . ; this new amino terminus then functions as a tethered peptide ligand, binding to an as yet undefined site in the body of the thrombin receptor, effecting receptor activation. A peptide mimicking this new amino terminus was a full agonist for platelet secretion and aggregation, supporting the model of receptor activation proposed above and suggesting that this unusual mechanism accounts for platelet activation by thrombin (22) .In addition to its critical role in activating platelets to effect hemostasis and thrombosis (8), thrombin has potent actions on a variety of non-platelet cell types (reviewed in 16) . For example, thrombin has potent chemotactic activities for monocytes (1) and is mitogenic for several cell types (3, 4), © The Rockefeller University Press, 0021-9525/92/02/827/6 $2 .00 The Journal of Cell Biology, Volume 116, Number 3, February 1992 827-832 thrombin receptor agonist peptide reproduces thrombininduced events (specifically, phosphoinositide hydrolysis and mitogenesis) in CCL-39 hamster lung fibroblasts, a naturally thrombin-responsive cell line. Moreover, these thrombin-induced events could be recapitulated in CV-1 cells, normally poorly responsive to thrombin, after transfection with human platelet thrombin receptor cDNA . Our data show that important thrombininduced cellular events are mediated by the same unusual mechanism of receptor activation in both platelets and fibroblasts, very likely via the same or very similar receptors .actions that may play important roles in inflammatory and proliferative processes in vivo. Are these asso...

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