Phospholipids released from activated platelets improve platelet aggregation and endothelial cell migration

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.