TRANSCELLULAR ACTIVATION OF PLATELETS AND ENDOTHELIAL-CELLS BY BIOACTIVE LIPIDS IN PLATELET MICROPARTICLES

Microparticles are released during platelet activation in vitro and ha ve been detected in vivo in syndromes of platelet activation. They hav e been reported to express both pro- and anticoagulant activities, Nev ertheless, their functional significance has remained unresolved. To a ddress the mechanism(s) of cellular activation by platelet micropartic les, we examined their effects on platelets and endothelial cells, Act ivation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; col lagen, 4 mu g/ml; and the calcium ionophore A23187, 1 mu M) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA(2) evokes a dos e-dependent increase in platelet aggregation, intracellular [Ca2+] mov ement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by a rachidonic acid isolated from them, However, platelet activation requi res prior metabolism of microparticle arachidonic acid to thromboxane A(2). Thus, pretreatment of platelets with the cyclooxygenase (COX) in hibitor, indomethacin (20 mu M), the thromboxane antagonist SQ29,548 ( 1 mu M), or the protein kinase C inhibitor GF109203X (5 mu M) prevents platelet activation by microparticles. However, platelet microparticl es fail to evoke an inositol phosphate response directly, via either o f the cloned thromboxane receptor isoforms stably expressed in human e mbryonic kidney (HEK) 293 cells, Prelabeling platelets with [H-2(8)] a rachidonate was used to demonstrate platelet metabolism of the micropa rticle-derived substrate to thromboxane. Platelet microparticles can a lso induce expression of COX-2 and prostacyclin (PGI(2)) production, b ut not expression of COX-1, in human endothelial cells, These effects are prevented by pretreatment with actinomycin D (12 mu M) or cyclohex imide (5 mu g/ml). Expression of COX-2 is again induced by the micropa rticle arachidonate fraction, which it may then use to synthesize PGI( 2). Both PGE(2) and iloprost, a stable PGI(2) analog, evoke human umbi lical vein endothelial cell COX-2 expression, albeit with kinetics tha t differ from the response to platelet microparticles. These studies i ndicate a novel mechanism of transcellular lipid metabolism whereby pl atelet activation map be amplified or modulated by concentrated delive ry of arachidonic acid to adjacent platelets and endothelial cells.