Multiple Mechanisms Linked to Platelet Activation Result in Lysophosphatidic Acid and Sphingosine 1-Phosphate Generation in Blood

Abstract

Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (Sph1P) production was examined in vitro under conditions that simulated blood clotting. Several approaches were utilized to elucidate the metabolic pathways. 1) Platelet phospholipids were labeled using [ 32 P]orthophosphate, and the production of [ 32 P]Sph1P and LPA was examined. Thrombin stimulation of platelets resulted in rapid secretion of Sph1P stored within the platelet. In contrast, LPA was neither stored within nor secreted from platelets. Nonetheless, extracellular levels of LPA gradually increased following stimulation. 2) Stable-isotope dilution mass spectrometry was used to quantify the molecular species of LPA generated from platelets in vitro. Only 10% of the LPA generated following thrombin stimulation was associated with platelets, the remaining 90% was contained within the extracellular medium. The acyl composition of LPA produced by platelets differed depending on the presence or absence of plasma in the incubation. 3) The fate of exogenously added fluorescent phospholipid analogs was determined. Incubation of [(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl-(NBD)-labeled phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine with the supernatant fractions from thrombin-stimulated platelets yielded no LPA production. However, these lipids were converted to the corresponding lysolipids by released PLA 1 and PLA 2 activities. When incubated with plasma or serum the NBD-labeled lysophospholipids were readily converted to LPA. Inhibitors of lysophospholipase D and the biological activity of LPA were detected in plasma. These results suggest that the bulk of LPA produced through platelet activation results from the sequential cleavage of phospholipids to lysophospholipids by released phospholipases A 1 and A 2 and then to LPA by plasma lysophospholipase D.Lysophosphatidic acid (LPA) 1 and sphingosine 1-phosphate (Sph1P) are phospholipid mediators with pleiotropic growth factor properties that elicit their actions via the activation of G protein-coupled receptors encoded by the endothelial differentiation gene family (1, 2). Several investigators have identified platelets as the source of Sph1P and LPA. However, contradictions exist in the literature concerning the mechanisms by which these mediators are generated. Although some investigators found no Sph1P generation in thrombin-activated platelets (3), others reported as much as 0.5 M Sph1P in human serum (4). Although it is generally agreed that LPA is generated in thrombin-activated platelets (3, 5, 6), the rate of production found at 0.02 nmol/min/10 9 platelet cannot account for the 5-10 M concentration detected in human serum (7). During the first hour of blood clotting the concentration of LPA increases ϳ300 nM; however, its production continues and an additional 5 M is added to serum during the first 24 h, a time course that is hard to reconcile with that of platelet activation and consequently of platelet only origin. Gerrard and Robinson (6) quantified the molecul...

Identification of an intracellular receptor for lysophosphatidic acid (LPA): LPA is a transcellular PPARγ agonist

“…LPA is produced extracellularly by lipoprotein oxidation (31) or by the action of secretory phospholipases A 2 on microvesicles released from activated cells (39). LPA also is produced in plasma by thrombinactivated platelets (5) through the stimulated release of phospholipase A 1 (5,40), phospholipase A 2 (5), and lysophospholipase D (autotaxin) (41,42), which act on plasma lipids or secreted lysophosphatidylcholine (42). Whereas plasma contains undetectable amounts of LPA, its concentration in serum is several micromolar (28).…”

The early- and late stages in phenotypic modulation of vascular smooth muscle cells: Differential roles for lysophosphatidic acid

“…The total amount of LPA in plasma is in the low nanomolar range [70][71][72][73][74], and the portion derived from isolated platelets remains only a fraction of that generated ex vivo during blood clotting [70].In the course of blood clotting, large amounts of LPA are generated, reaching concentrations as high as 5-10 µM [70,71,75,76], and the molecular species are dominated by the 18:2 and 20:4 unsaturated acyl species (20% and 34% of total LPA, respectively) [70]. LPA 20:4 is a more potent activator of platelets than other saturated or mono-unsaturated acyl species [77][78][79].…”

Lipid Phosphate Phosphatases Regulate Lysophosphatidic Acid Production and Signaling in Platelets

“…Measurement of LPA Production by Platelets-LPA production by washed platelets was determined using minor modifications of a recently published procedure (10). In brief, gel-filtered platelets were washed and then resuspended in phosphate-free Tyrode's buffer containing 2 mCi of [ 32 P]PO 4 2Ϫ and 1 M prostaglandin E1 at 2 ϫ 10 9 platelets/ml.…”

“…37°C incubations in a gently shaking water bath were terminated by microcentrifugation. Lipids were extracted from the supernatant and separated by thin layer chromatography as described (10). [ 32 P]LPA was identified by co-migration with a radiolabeled standard run in parallel and quantitated using a STORM PhosphorImager system.…”