Thrombin receptor cleavage at the Arg41-↓-Ser42 peptide bond in the receptor's amino-terminal exodomain is necessary and sufficient for receptor activation. The rate of receptor cleavage at this site is a critical determinant of the magnitude of the cellular response to thrombin. These observations underscore the importance of defining the molecular basis for thrombin-receptor interaction and cleavage. We report that chimeric proteins bearing only thrombin receptor amino-terminal exodomain residues 36-60 are cleaved at rates similar to the wild-type thrombin receptor when expressed on the cell surface. A soluble amino-terminal exodomain protein was also cleaved efficiently by thrombin with a Km of 15-30 micro M and k(cat)of approximately 50s-1, with cleavage occurring only at the Arg41-↓-Ser42 peptide bond. In the context of previous studies, these data suggest that the receptor's LDPR cleavage recognition sequence and DKYEPF hirudinlike domain account for thrombin-receptor interaction. Because a P3 aspartate in protei n C's cleavage site inhibits cleavage by free thrombin, we investigated the role of the P3 aspartate in the receptor's LDPR sequence. Studies with mutant receptors revealed an inhibitory role for this residue only in the absence of the receptor's hirudin-like domain. These and other data suggest that the receptor's hirudin-like domain causes a conformational change in thrombin's active center to accommodate the LDPR sequence and promote efficient receptor cleavage. Taken together, these studies imply that the thrombin receptor's amino-terminal exodomain contains all the machinery needed for efficient recognition and cleavage by thrombin. Thrombin appears to bind and cleave this domain independently of the rest of the receptor, with one thrombin molecule probably activating multiple receptors.

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Macrophage scavenger receptors (MSR) have been implicated in atherogenesis via receptor mediated uptake of modified lipoproteins and are reportedly cation independent adhesion molecules on macrophages, but their major physiological functions remain obscure. Thus to elucidate their in vivo role, mice lacking both type I and type II MSR were created by gene targeting. Both the heterozygotes and homozygotes in the MSR mutation were normal in appearance and growth, and were fertile. The uptake of oxdized low density lipoprotein (LDL), acetyl LDL and advanced glycosylation end product (AGE) by peritoneal macrophages derived from homozygotes declined to between 25 and 40% as compared with those from the wild type. Numbers of adhesive MSR defected macrophages on a plastic surfaces were <50% compared with those of the wild type. Hepatic granulomas induced by zymosan injection in homozygotes were larger, diffuse and less circumscribed compared with those of the wild type. With regard to phagocytic clearance for apoptotic thymocytes, it has been clarified that MSR accounts for approximetely half of the phagocytic activity required for the uptake of apoptotic thymocytes.
These results indicate that the MRS not only functions to uptake modified LDL but also participates widely in the removal of foreign bodies and waste materials. Moreover, MSR appears to exert multifunctional roles of endocytosis, adhesion and phagocytosis in macrophages.

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To clarify the physiological role of CD36, we analyzed the gene abnormalities of CD36 deficient subjects and the role of CD36 as an oxidized LDL receptor by using monocyte-derived macrophages from normal and two CD36-deficient subjects. We further analyzed the expression of CD36 in human atherosclerotic tissues and the regulation of CD36 by oxLDL in vitro. Scatchard analysis of ¹²⁵I-OxLDL binding showed a linear plot and the maximum binding was lower by approximately 40% in the macrophages from subj ects with CD36 deficiency than those from normal controls. Competition studies showed that the uptake of ¹²⁵I-OxLDL was suppressed by OKM5, an antibody against CD36, by 53% in normal control macrophages, but not in the CD36-deficient macrophages. After incubation with OxLDL for 24h, cholesteryl ester mass accumlation was reduced in the macrophages from CD36-deficient subjects than those from normal controls. These results suggest that CD36 is one of the physiological receptors for OxLDL. In immunochemical analysis of human aortic tissues, the expression of CD36 is very low in normal aorta, while the expression of CD36 in atheromatous plaque, CD36 is highly expressed in macrophages. In vitro assay, CD36 is upregulated by the addition of oxidized LDL in medium after 24 hours incubation. From these observations, CD36 is suggested that it plays an important role on foam cell formation of macrophages in atherosclerosis.

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We have previously demonstrated that (1) vascular smooth muscle cells (SMC) isolated from atherosclerotic lesions express a characteristic gene of macrophages, macrophage colony-stimulating factor receptor (c-fms), and (2) a combination of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF)-BB induces stable expression of c-fms gene in normal vascular SMC to the level of that isolated from atherosclerotic lesions. To study the transcriptional regulation of c-fms expression in normal vascular SMC in response to PDGF and EGF, transcription activity of c-fms promotor was examined. In the deletion analysis of c-fms promotor, we demonstrated that PU. 1 binding element at -178 by to -165 by relative to the translation starting site, was required for full transcription activity in normal vascular SMC. Mutation in the PU. 1 binding element markedly reduced the promotor activity, suggesting that PU. 1 binding site plays an important role in the c-fms expression. Furthermore, we demonstrated that PU. 1 mRNA was induced in normal vascular SMC after treatment with PDGF and EGF, and that antisense oligonucleotide, targeted against 5' untranslated region including the ATG initiator codon of PU. 1 cDNA, inhibited growth factor-induced c-fms expression. These results indicated that multiple growth factors regulated the phenotypic transformation of medial smooth muscle cells to macrophage-like cells in atherosclerotic lesions by regulation c-fms expression through the interaction with PU. 1.

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Glycosaminoglycans (GAGs) on the surface of the endothelium and basement membrane have been suggested to be involved in vascular permeability, anticoagulation, cell movement and cell growth. In the present study, we demonstrated that fluid laminar shear stress enhanced syntheses of GAGs in porcine aortic endothelial cells, in vitro. Shear stress (15, 40dyn/cm²) for 24 hours significantly increased GAG synthesis and secretion, assayed by [³⁵S] sulfate incorporation, in “medium” and “trypsinated” fractions which include GAGs derived from the cell surface and from the solubilized matrix. The GAGs induced by shear stress (40dyn/cm²) in the trypsinated and medium fractions consisted mainly of heparan sulfate and chondroitin/dermatan sulfate, respectively. For both heparan and chondroitin/dermatan sulfate increases exposure of the cells to shear stress for more than 24 hours was required. Shear stressinduced increase in GAG synthesis was concomitant with a decrease in DNA synthesis and an increase in protein synthesis. Since DNA synthesis in static cells also was in inverse proportion to the GAG synthesis, GAGs on the surface of cells and/or extracellular matrix may involve cell growth. These findings indicate that relatively high shear stress may suppress atherogenesis by changing endothelial GAG synthesis.

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