The objectives were to determine whether the permeability-decreasing activity of platelet-conditioned medium (PCM) is associated with a lipid bound to albumin and whether lysophosphatidic acid (LPA) is present in the PCM. A decrease in permeability was assessed by an increase in electrical resistance across endothelial cell monolayers derived from bovine pulmonary arteries and microvessels. The Sephacryl S-200 fraction of PCM that contained albumin, the albumin immunoprecipitate from the PCM, and the methanol extract from the albumin immunoprecipitate all increased endothelial electrical resistance. Increased electrical resistance induced by PCM was not abolished by boiling and was mimicked by 1-oleoyl-LPA and 1-palmitoyl-LPA. Analysis of a methanol-chloroform extract of one sample of PCM by electrospray mass spectrometry revealed many fatty acids, ceramide, diacylglycerol, phosphatidic acid, and palmitoyl-LPA, but analysis of a second sample of PCM and the methanol extract of its albumin immunoprecipitate revealed no LPA, only lipids. These findings indicate that a bioactive lipid(s), possibly LPA, released from platelets and subsequently bound to albumin forms an active complex that decreases endothelial permeability.

Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels. Using a desensitization protocol with cAMP/protein kinase A (PKA)-enhancing agents and pharmacological inhibitors, we determined that the activity of PCM is independent of PKA and PKG. Genistein, an inhibitor of tyrosine kinases, prevented the increase in endothelial electrical resistance. Because lysophosphatidic acid (LPA) has been proposed to be responsible for this activity of PCM and is known to activate the G(i) protein, inhibitors of the G protein pertussis toxin and of the associated phosphatidylinositol 3-kinase (PI3K) wortmannin were used. Pertussis toxin and wortmannin caused a 10- to 15-min delay in the characteristic rise in electrical resistance induced by PCM. Inhibition of phosphorylation of extracellular signal-regulated kinase with the mitogen-activated kinase kinase inhibitors PD-98059 and U-0126 did not prevent the activity of PCM. Similar findings with regard to the cAMP protocols and inhibition of G(i) and PI3K were obtained for 1-oleoyl-LPA. These results demonstrate that PCM increases endothelial electrical resistance in vitro via a novel, signal transduction pathway independent of cAMP/PKA and cGMP/PKG. Furthermore, PCM rapidly activates a signaling pathway involving tyrosine phosphorylation, the G(i) protein, and PI3K.

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