Estimation of the phospholipid distribution in the human platelet plasma membrane based on the effect of phospholipase A2 from Naja nigricollis.

Human platelets in three physiological states were prepared. These states were the gel-filtered, the thrombin-induced shape-changed, and the thrombin-activated platelets. The phospholipid distributions in these three types of membrane were probed by using the basic phospholipase A2 of Naja nigricollis. This enzyme could penetrate through these membranes to hydrolyze all of their accessible phospholipids and to cause cell lysis. The hydrolytic time-courses displayed three phases. The state of platelet in each lipid hydrolytic phase was examined by: (1) measuring the leakage of lactate dehydrogenase; (2) analyzing the morphology by both scanning and transmission electron microscopy (scanning EM and transmission EM); and (3) estimating the hydrolysis of the [32P]phosphate-labeled platelets. The existence of these three hydrolytic phases may signify that the phospholipase A2 sequentially hydrolyzed its substrates in the membrane outer leaflet, in the inner one, and in the cytosol. The content and the distribution of each phospholipid class in the plasma membranes of the resting and of the shape-changed platelets were similar. These membrane surfaces consisted mainly of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Phosphatidylserine (PS) was not exposed on the surface of the shape-changed platelet. The content of each lipid class in the activated platelet membrane was 10% more than that in the resting platelet. PS was found on the activated platelet cell surface. This implies that PS is exposed only during platelet secretion.