Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that plateletderived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the ad-

Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro-and anticoagulant activities. Nevertheless, their functional significance has remained unresolved.To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells.

Activation of platelets by agonists, such as collagen and thrombin, results in shedding of membrane microparticles (MP) 1 from their surface (1). Formation of MP results from an exocytotic budding process (2, 3) and may include both procoagulant (4, 5) and anticoagulant (6, 7) proteins when shed from platelets. Increased circulating concentrations of MP have been detected in vivo in syndromes of platelet activation (8, 9). It is likely that the shear forces in areas of disordered flow would favor MP formation (10, 11).MP might themselves evoke cellular responses in the immediate microenvironment of their formation. For instance, activation of endothelial cells results in MP shedding, which, in turn, activates neutrophils, enhancing their propensity to adhere to endothelium (12). Recently, we have demonstrated that arachidonic acid (AA) in platelet MP can influence platelet activation in a PKC-dependent manner and endothelial cell COX-2 expression via transcellular lipid metabolism (13). The molecular mechanisms by which MP induce such cellular responses are, however, unknown. The present studies were designed to delineate the signaling pathways involved in MPinduced COX-2 activation and prostaglandin formation.COX is the first rate-limiting enzyme in the synthesis of prostacyclin, prostaglandins, and thromboxane from arachidonic acid (14). Two isozymes of COX have been described (15,16), and both catalyze the cyclooxygenase-dependent transformation of prostaglandin G 2 from AA and the subsequent peroxidation of prostaglandin G 2 to prostaglandin H 2 (17). While COX-1 is expressed constitutively in most tissues, COX-2 is usually induced as an immediate early gene by mitogenic or inflammatory stimuli as well as by ligands that act via G protein-and PKC-mediated pathways (18). More recent findings link the prostaglandin biosynthetic pathways with activation of MAPK signaling cascades (19 -21).The MAPK pathway, of which there are three subgroups, is a conserved eukaryotic signaling cascade that is responsible for mediating the effects of extracellular stimuli on a wide variety of biological processes. The extracellular signal-regulated kinases (also termed p42 MAPK and p44 MAPK) and the stressactivated protein kinases (also termed c-Jun NH 2 -terminal kinase (JNK)) and p38 (22) are distinguished by activating signals, substrate specificity, and cellular responses (23). While

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.