Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles.
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 human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction, which it may then use to synthesize PGI2. Both PGE2 and iloprost, a stable PGI2 analog, evoke human umbilical vein endothelial cell COX-2 expression, albeit with kinetics that differ from the response to platelet microparticles. These studies indicate a novel mechanism of transcellular lipid metabolism whereby platelet activation may be amplified or modulated by concentrated delivery of arachidonic acid to adjacent platelets and endothelial cells.
Barry
O P
OP
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia 19104, USA.
Pratico
D
D
Lawson
J A
JA
FitzGerald
G A
GA
D000595:Amino Acid Sequence; D016718:Arachidonic Acid / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism* / Q000502:physiology*; D002478:Cells, Cultured; D051546:Cyclooxygenase 2; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism / Q000502:physiology*; D011464:Epoprostenol / Q000378:metabolism; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007527:Isoenzymes / Q000096:biosynthesis / Q000276:immunology; D008565:Membrane Proteins; D008830:Microbodies / Q000378:metabolism* / Q000502:physiology*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000276:immunology / Q000494:pharmacology; D054467:Phospholipases A2; D015539:Platelet Activation / Q000502:physiology*; D010974:Platelet Aggregation; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis / Q000276:immunology; D017482:Receptors, Thromboxane / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013928:Thromboxane A2 / Q000378:metabolism*; D014471:Umbilical Veins / Q000166:cytology
D007295:Inositol Phosphates; D007527:Isoenzymes; D008565:Membrane Proteins; D017482:Receptors, Thromboxane; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D013928:Thromboxane A2; D011464:Epoprostenol; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D010741:Phospholipases A; D054467:Phospholipases A2