Quantification of human platelet inositides and the influence of ionic environment on their incorporation of orthophosphate-32P

Abstract

A B S T R A C T Platelets are a rich source for the study of inositol lipids in man. The substitution of an EDTAKCl solution for the water component of the Bligh and Dyer procedure permitted quantitative extraction of polyphosphoinositides. The latter, with monophosphoinositide, were found to comprise, on a molar basis, 6.7% of total platelet phospholipids. Study of the incorporation of orthophosphate-'P into platelet phospholipids was further simplified by separating eight 'P-labeled lipids, including the inositides, with a single chromatographic development on formaldehyde-treated paper. Particular attention was paid to the influence of ionic environment on the pattern and degree of labeling.In 300 mOsm media major phospholipids other than the inositides were not labeled. Small amounts of label appeared in certain trace phospholipids, notably phosphatidic acid. In 150 mOsm media, labeling of inositides was moderately increased, that of trace phospholipids enormously so. The increased labeling was not solely due to thrombocytolysis since (a) platelet disruption by sonication or freeze-thawing abolished 'P incorporation into phospholipids and (b) in timed studies, restoration of osmolarity to 300 mOsm by addition of hypertonic sorbitol blunted the enhancement effect of previous 150 mOsm exposure. Lowering K and compensatorily increasing Na concentration of 300 mOsm media also stimulated aP labeling of inositides and, to a lesser extent, the trace phospholipids. However, the pattern and degree of stimulation were not as strikingly altered as in the osmolarity studies.

Phospholipid Metabolism in Stimulated Human Platelets

“…The phosphorus content of the LPE area ( Fig. 1) rose rapidly from barely detectable levels to a plateau value of 5 nmol, a statistically significant change ( Fig. 2 and Table II).…”

“…Lipids were extracted as previously described (5,11,12). Aggregation did not interfere with this procedure.…”

“…As in many secretory tissues (1), platelet stimulation increases the turnover of phosphatidylinositol (PI)' and related phospholipids, such as phosphatidic acid (PA) and di-and triphosphoinositide (2)(3)(4). The metabolism of these phospholipids is also relatively active in unstimulated platelets (5,6). The turnover of ethanolaminephosphoglycerides (PE), phosphatidylcholine (PC), and sphingomyelin was reported to be very low in both stimulated and unstimulated platelets (5,6).…”

“…The metabolism of these phospholipids is also relatively active in unstimulated platelets (5,6). The turnover of ethanolaminephosphoglycerides (PE), phosphatidylcholine (PC), and sphingomyelin was reported to be very low in both stimulated and unstimulated platelets (5,6). However, platelet stimulation increases de nlovo synthesis of phosphatidyl serine (7) and PI (8).…”

Differential activation by fMet‐Leu‐Phe and phorbol ester of a plasma membrane phosphatidylcholine‐specific phospholipase D in human neutrophil

“…EDTA (2.6 mM final) and cold PA (lO,cg/sample) were also added. Products of [3H]alkylacyl-GPC hydrolysis were separated by TLC as described by Cohen et al [19]. Plates were scanned for radioactivity using an automatic TLC linear analyzer (Berthold LB 2848).…”