Biochemical characterization of isolated cholesterol-phospholipid particles continuously released from rat and human...

Platelets are a source of lipid that facilitates the coagulation process and that potentially accumulates in cells within atherosclerotic lesions. With this in mind, we have examined the release of lipid-containing particles from activated rat and human platelets. When washed platelets were activated with thrombin and incubated, cholesterol and phospholipid were continuously released for a period up to 2 hours. The amount of cholesterol released was approximately 20% of the platelet cholesterol content. Cholesterol release was also stimulated by strong platelet agonists such as collagen and the calcium ionophore A23187 but not by the weak agonist ADP. The release of cholesterol did not simply result from lysis of platelets since lactate dehydrogenase and cholesterol release could be dissociated. Colchicine substantially inhibited release of cholesterol but did not substantially inhibit release of lactate dehydrogenase. Cholesterol-phospholipid particles were isolated from platelet-release supernatants by centrifugation, microfiltration, and gel filtration chromatography. The particles, which eluted in the void volume, were composed of 57% protein, 32% phospholipids, 8% cholesterol, and 3% triglyceride. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of particle-associated protein showed a major protein component of Mr 42,000, presumably actin. The density of the particles in a sucrose density gradient was 1.16 g/ml. The cholesterol to phospholipid molar ratio of isolated particles from rat and human platelets was about 0.6, a value typical of plasma membranes, whereas the cholesterol to phospholipid molar ratio in particles released from platelets of rats fed a high-cholesterol diet increased to 1.0. Electron microscopic analysis of the particles showed them to be spherical or irregular in shape with sizes ranging from 50 to 550 nm and with projections that extended from their surfaces. We suggest that these cholesterol-phospholipid particles released from platelets may represent a mechanism by which the membrane becomes dispersed following platelet activation.