This study was undertaken to isolate phospholipids released from activated platelets and to investigate their biological activities. Freshly washed platelets were activated with freezing/thawing, thrombin, ionophore 23187, and arachidonic acid. Thrombin was incubated with platelet-rich plasma to promote synthesis and release of phospholipids from platelets. Phospholipids in supernatants of activated platelets were extracted with butanol and separated by thin-layer chromatography. Release of phosphatidylserine (PS) and phosphatidic acid (PA) increased when platelets were treated with freezing/thawing, ionophore, and thrombin. The lysophosphatidyl ethanolamine (LPE) appeared not to be induced with freezing/thawing, but increased significantly by thrombin, ionophore, and arachidonic acid. The effects of platelet phospholipids on hemostasis and angiogenesis were studied with platelet aggregation and endothelium chemotaxis. Phospholipids isolated from thrombin-stimulated platelet-rich/platelet-poor plasmas were used as synergistic agonists in platelet aggregation and as chemotactic agents in endothelial cell migration. Several phospholipids increased chemotaxis and platelet aggregation; these were PS, PA, LPE, and sphingosine-1-phosphate. Also, chemotaxis of those phospholipids increased when combined with charcoal-stripped fetal bovine serum, suggesting that cofactors in serum enhanced phospholipid-induced cell migration. These observations suggest that activated platelets release biologically active phospholipids into the blood stream, where they may play an important role in thrombosis and angiogenesis.
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