In the platelet, the same enzyme, phospholipase, cPLA2α
(PLA2G4A), delivers AA both to COX-1 for formation of TxA2
and to 12-LOX for formation of 12-HETE, as was demonstrated by the virtual absence of agonist-induced biosynthesis of both of these eicosanoids in a patient with null mutations of cPLA2α
Thus, it was of considerable interest to find that PAR-induced signaling to activate the cPLA2α
coupled to COX-1 is quite different from PAR-induced signaling to the cPLA2α
coupled to 12-LOX. Three lines of evidence delineate the difference between these 2 cPLA2α
activation pathways in the platelet: (1) 12-HETE formation continues well after TxB2
biosynthesis has ceased, (2) PI kinases participate in the lipid signaling pathway for TxA2
production but not for 12-HETE, and (3) 1-butanol and propranolol inhibit PAR1-induced formation of TxB2
but do not block 12-HETE production.
The rate of PAR1-induced biosynthesis of TxA2
is markedly greater than that of 12-HETE. Maximal formation of TxA2
is reached in ≤15 seconds after the PAR1 peptide, whereas formation of 12-HETE increases gradually over 120 seconds. These differences between the rates of formation and the time at which maximal formation is achieved are very similar to those previously reported for the formation of TxA2
and 12-HETE following collagen-induced platelet activation.29
It is clear from the persisting 12-HETE formation long after TxA2
production has ceased that the AA substrate is not provided by the same mechanisms to COX-1 and to 12-LOX after PAR1 activation.
LY249002, a PI kinase inhibitor that blocks formation of both PI(4,5)P2
markedly inhibits PAR1 and PAR4-induced biosynthesis of TxA2
. In contrast, LY does not inhibit PAR1 or PAR4-induced formation of 12-HETE. Indeed, LY actually increases PAR1-induced 12-HETE formation. Also, our results indicate that LY does not have direct effects on the enzymatic activities of either COX-1 or TxA2
synthase. Thus, the results of the studies with LY also indicate that the PAR signaling that activates cPLA2α
coupled to COX-1 is different from the PAR signaling to the cPLA2α
coupled to 12-LOX.
Further evidence that the PAR signaling pathway activating the cPLA2α
coupled to COX-1 is discrete from that signaling to 12-LOX coupled cPLA2α
is derived from the results of the studies with 1-butanol and propranolol. Neither 1-butanol nor propranolol inhibits PI kinases.30,31
Thus, inhibition of the activation of the cPLA2
coupled to COX-1 by these agents occurs at a site in the signaling pathway different from the PI kinases. Importantly, neither 1-butanol nor propranolol inhibits PAR1-induced activation of the cPLA2
coupled to 12-LOX, thus providing additional evidence that this signaling pathway differs from that leading to activation of the cPLA2
coupled to COX-1.
The intracellular localization of COX-1 and 12-LOX is pertinent to a consideration of differential regulation of the cPLA2α
coupled to each of the enzymes. Platelet COX-1 is localized to the intracellular membrane complex with characteristics of the endoplasmic reticulum that is referred to as the dense tubular system.32
On the other hand, the 12-LOX is predominantly localized in the cytosol of rat platelets that have been isolated so as to minimize ex vivo activation, and it undergoes calcium- and thrombin-dependent translocation to a membrane fraction,33,34
from which it can be coimmunoprecipitated with cPLA2α
Thus, the differences between PAR-induced signaling to the cPLA2α
providing substrate to COX-1 and that coupled to 12-LOX may occur in the context of localization of the 2 oxygenases at separate sites in the platelet. cPLA2α
activity is regulated by several factors in cells, including interaction with membrane lipids and phosphorylation.35–40
Although these mechanisms have been well described in vitro, their relevance in vivo is still debated. Our results suggest that, in human platelets, a product of the PI kinases (likely PI[4,5]P2) participates in the activation of the phospholipase coupled to COX-1 (). By contrast, activation of the cPLA2α
coupled to 12-LOX does not appear to require signaling through a PI kinase. Together, our results support the hypothesis that regulation of cPLA2α
in human platelets determines the metabolic pathway of AA by translocating the lipase to separate pools of phospholipids, specifically coupling the lipase to either COX-1 or 12-LOX, or both.
Hypothetical model for PAR1-mediated formation of eicosanoids. cPLA2* indicates activated cPLA2; cPLA2, nonactivated cPLA2; PIP, phosphatidyl inositols; PL, phospholipids; PGH2, prostaglandin H2; 12-HpETE, 12-hydroperoxyeicosatetraenoic acid.
In contrast to the difference in the lipid signaling pathways that activate COX-1-coupled cPLA2α
but not 12-LOX-coupled cPLA2α
, calcium is required for the PAR-induced activation of the cPLA2α
coupled to both COX-1 and 12-LOX. Also, secondary signaling via the TP receptor contributes to the ultimate PAR1-induced activation of both COX-1-coupled cPLA2α
and 12-LOX-coupled cPLA2α
. TP-induced augmentation of the initial calcium response to PAR1 likely participates in this positive feedback loop regulating the extent of cPLA2α
activation. Previous research has shown that aspirin inhibits 12-HETE production by platelets activated by collagen41
and from that evidence, it was suggested that inhibition of the conversion of 12-hydroperoxyeicosatetraenoic acid to 12-HETE by a peroxidase accounted for this effect of aspirin. The present results indicate that TP receptor signaling contributes substantially to 12-HETE formation in response to PAR1. PAR4-induced 12-HETE formation is not affected by aspirin and is inhibited to a lesser extent by the TP receptor antagonist.
Both the TP receptor and PAR1 are Gq-coupled receptors that signal calcium release via the PLCβ pathway, which also leads to activation of PKC. Activation of PKC with PMA, however, produced platelet aggregation but did not lead to formation of either TxA2 or 12-HETE, thus excluding the PKC pathway as a mechanism for activation of cPLA2α.
We found that activation of the PAR4 receptor generated TxA2
more slowly than activation of PAR1 in this direct comparison of the agonists for the 2 receptors. These findings are consistent with previous evaluations in which the effects of PAR14
agonists were carried out in separate studies. Following stimulation of PAR4, the rate of 12-HETE formation is also less than that resulting from PAR1.
In conclusion, the PAR-induced signaling pathway that activates the cPLA2α that provides AA to COX-1 is different from that activating the cPLA2α that liberates substrate for 12-LOX. Thus, both COX-1 and 12-LOX are functionally coupled to a discrete subset of platelet cPLA2α that provides AA separately to each of these oxygenases.