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

Phin Cohen; M. Johan Broekman; Arie Verkley; Johannes W. W. Lisman; Arie Derksen

doi:10.1172/JCI106547

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

Phin Cohen, M. Johan Broekman, Arie Verkley, Johannes W. W. Lisman, and Arie Derksen

Published April 1, 1971 - More info

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Abstract

Platelets are a rich source for the study of inositol lipids in man. The substitution of an EDTA-KCl 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-³²P into platelet phospholipids was further simplified by separating eight ³²P-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 ³²P 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 ³²P 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.

These data show that drastic alterations of ionic environment can sharply influence the platelet's ability to incorporate orthophosphate-³²P into its phospholipids.

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

Phin Cohen, M. Johan Broekman, Arie Verkley, Johannes W. W. Lisman, and Arie Derksen

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

Phin Cohen, M. Johan Broekman, Arie Verkley, Johannes W. W. Lisman, and Arie Derksen

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