The prostacyclin receptor (IP), a G protein-coupled receptor, mediates the actions of the prostanoid prostacyclin and its mimetics. IPs from a number of species each contain identically conserved putative isoprenylation CAAX motifs, each with the sequence CSLC. Prostacyclin (prostaglandin (PG)1 I 2 ) is a labile metabolite of arachidonic acid, which is synthesized by the sequential actions of PGH 2 endoperoxide synthases 1 and 2 and prostacyclin synthase (1). The actions of prostacyclin generally counteract those of thromboxane A 2 , and thus the relative level of these two prostanoids in the circulation are important in the local control of vascular tone and platelet aggregation (2, 3). The main physiologic roles of thromboxane A 2 and prostacyclin are their contribution to the maintenance of vascular hemostasis: thromboxane A 2 , synthesized primarily by platelets, induces platelet shape change and aggregation and constriction of bronchial and vascular smooth muscle, whereas prostacyclin, mainly synthesized by the vascular endothelium, is a potent inhibitor of platelet aggregation and induces vasodilation (4 -7). Moreover, prostacyclin has been reported to confer a cytoprotective effect against tissue injury in acute myocardial ischemia or following hypoxic exposure of vascular endothelial cells (8). Imbalances in thromboxane A 2 or prostacyclin have been reported to be a major contributing factor in the development of a number of cardiovascular disorders including thrombosis, myocardial infarction, unstable angina, stroke, and atherosclerosis (9 -13). In addition to its central role in the cardiovascular system, prostacyclin may be important in the regulation of renal blood flow (14); it also acts as a negative feedback regulator of histamine secretion from mast cells (15) and acts as a lipolytic agent, antagonizing the antilipolytic effect of PGE 2 , in adipocytes (16).The actions of prostacyclin are mediated via interaction with a specific cell surface receptor, termed the prostacyclin receptor or IP (17). The major intracellular signaling pathway used is stimulation of adenylyl cyclase leading to increases in intracellular cAMP (18,19), a pathway thought to be relevant to inhibition of platelet aggregation and vascular smooth muscle relaxation (3). However, recent evidence indicates that IP agonists may couple to multiple signaling pathways including activation and inhibition of adenylyl cyclase, via G s and G i , respectively, stimulation of phosphoinositide metabolism, and changes in [Ca 2ϩ ] i concentrations (20,21). Evidence also exists to indicate that iloprost, a stable carbacyclin analogue of prostacyclin, can stimulate opening of ATP sensitive K ϩ channels resulting in hyperpolarization and relaxation of canine carotid artery (22).Molecular cloning of the human (23, 24), mouse (25), and rat

By use of six highly purified exoglycosidases with well-defined specificity, the oligosaccharide units of human plasma beta 2-glycoprotein I (beta 2I) were modified by sequential enzymatic degradation. The released monosaccharides (NeuAc, Gal, GlcNAc, and Man) were quantified, and the carbohydrate compositions of the resulting glycoprotein (gp) derivatives were determined. The gp was found to be both partially sialylated and galactosylated. These findings which are in agreement with earlier reports suggest that the carbohydrate moiety of beta 2I possesses more bi- than tri-antennas, probably three of the former and two of the latter carbohydrate units. Circular dichroic (CD) spectra of native beta 2I and its derivatives were measured in aqueous buffer and 2-chloroethanol (2-CE). Analysis of these spectra for elements of secondary structure showed beta 2I and most of the derivatives to contain predominantly beta-sheet and beta-turn structures. The lack of alpha-helical structures in aqueous buffer was noted. Removal of a large portion of the carbohydrate moiety did not alter the CD spectra or secondary structure of beta 2I in either aqueous buffer or in 2-CE. However, after enzymatic removal of approximately 96% of the carbohydrate moiety, large significant changes in the spectra and secondary structures were observed. In aqueous buffer a shift in the wavelength minimum occurred, accompanied by an increase in the magnitude of the molar ellipticity and the amount of beta-turn, with a reduction in random coil. One-third of the amino acids which were originally in random coil conformation assumed beta-turns after removal of 96% of the carbohydrate moiety.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidant stress leads to covalent oxidative modification of several plasma proteins, chief among which is fibrinogen. Aspirin can nonenzymatically acetylate fibrinogen's lysine residues, the functional groups most susceptible to oxidative modification. Because oxidation of fibrinogen may occur in the atheromatous environment, we studied the effects of oxidative modification on fibrinogen function and the consequences of acetylation by aspirin on fibrinogen's susceptibility to oxidation and functional properties. We exposed fibrinogen to Fe(3+) ascorbate for 1 hour and showed that the carbonyl/protein molar ratio increased from 0.71 +/- 0.18 to 2.86 +/- 0.50 mol carbonyl/mol protein (P < 0.02) with an accompanying reduction in the alpha-helical content of the protein from 34% to 29%. Exposure of fibrinogen to aspirin led to acetylation of lysine residues and inhibition of oxidation. Oxidized fibrinogen was more readily able to form fibrin, and acetylation prevented this enhancement of clot formation. Oxidized fibrinogen also supported platelet aggregation better than did native, unoxidized fibri ogen, and aretylation of fibrinogen prior to oxidation prevented the enhanced platelet aggregation. Oxidized fibrinogen was less effective in stimulating plasminogen activation by tissue-type plasminogen activator (tPA), with a catalytic efficiency that was reduced by 88% compared with native, unoxislized fibrinogen; metylated fibrinogen, by contrast, enhanced plasminogen activation by t-PA with a catalytic efficiency that was increased by 18% compared with native, anoxidized fibrinogen (P < 0.05) and was increased by 51% compared with oxidized fibrinogen (P < 0.05). Acetylation prevented the reduction in catalytic efficiency induced by oxidation. These data show that oxidized fibrinogen manifests prothrombotic effects that can be prevented by acetylation and suggest that inhibition of fibrinogen oxidation may be an additional antithrombotic benefit of aspirin therapy.

The structure of the glycans of the A-chain of human plasma alpha 2HS-glycoprotein was established from the chemical compositions of its derivatives prepared by sequential enzymatic degradation of the carbohydrate moiety, from the determination of the kind and amount of the monosaccharides liberated after each step of the enzymatic digestion, and from the distinct specificity of the highly purified exoglycosidases. The exoglycosidases were three sialidases (Vibrio cholerae, fowl plague virus, and Arthrobacter ureafaciens), two beta-galactosidases (Streptococcus pneumoniae and bovine testis), one alpha-N-acetylgalactosaminidase, one beta-N-acetylglucosaminidase, and one alpha-mannosidase. Utilizing sialidases with different cleavage specificities, the number of alpha 2-3- and alpha 2-6-linked sialic acid residues could be separately determined. As to the beta-galactosidases, the enzyme isolated from S. pneumoniae cleaves only beta 1-4-linked galactose residues, whereas the bovine testes enzyme acts on both the beta 1-4- and beta 1-3-linked galactose residues. Jack bean beta-N-acetylglucosaminidase cleaves beta 1-2, beta 1-4, and beta 1-6 GlcNAc with higher activity for the beta 1-2. Jack bean alpha-mannosidase cleaves alpha 1-2, alpha 1-6, and alpha 1-3 Man with greater activity for alpha 1-2 and alpha 1-6. Bovine liver alpha-N-acetylgalactosaminidase cleaves O-linked GalNAc. On the basis of these results, the A-chain of alpha 2 HS-glycoprotein was found to possess two biantennary N-glycans and two O-linked trisaccharides.

This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.