Phosphorus Labeling of Proteins and Phospholipids in Intact Platelets in Response to pH 5.3

Summary

Previous studies had shown that when gel-filtered or washed human platelets were incubated at pH 5.3, the cells secreted their granule-stored materials suggesting that low pH can act as a platelet activator. We determined here whether the effects of low pH on platelet protein phosphorylation and on platelet lipid metabolism were consistent with this view. When washed human platelets were incubated for 20 min at pH 5.3 and electrophoresed on SDS-PAGE, there was a great increase in ³²P-label in the 20,000 and 47,000 dalton protein bands. There was also an increase in the labeling of phosphatidic acid and a small decrease in phosphatidyl inositol. When the platelets were returned to pH 7.6, the ³²P labeling of the 20,000 and 47,000 dalton bands was greatly reduced, and that of phosphatidic acid reduced to the control value, while the labeling of phosphatidyl inositol was increased above control. Incubation at pH 5.3 for 60 min gave the same pattern, but return to pH 7.6 resulted in only partial reversal of labeling of the two protein bands and little decrease in the label associated with phosphatidic acid, but the radioactivity in phosphatidyl inositol was greatly increased. The changes in the ³²P-labeling of phospholipids and proteins after incubation of platelets at pH 5.3 may reflect an increase in cytoplasmic Ca⁺⁺ resulting from leakage of Ca⁺⁺ from intracellular storage sites, a process which becomes irreversible after longer time exposure to the low pH. The activation of human platelets at pH 5.3 is a slow process which may not be directly comparable to the fast events in the normal stimulation-response coupling, but which is accompanied by changes common to platelet activation. Most interesting in this respect is that a return to physiological conditions will reverse these events, although prolonged exposure will make them irreversible. These studies may therefore present a way to study the boundary between reversible and irreversible processes in platelets.

Key words

Phosphorus labeling - Platelet protein - Phospholipids - Platelet activation - Reversibility - pH 5.3

• References

• 1 Mürer EH, Stewart GJ. Insights into the mechanism of platelet activation through studies at pH 5.3. Thromb Haemostas 1977; 38: 1018-1029
• 2 Daniel JL, Molish IR, Holmsen H. Myosin phosphorylation in intact platelets. J Biol Chem 1981; 256: 7510-7514
• 3 Lapetina EG, Cuatrecasas P. Stimulation of phosphatidic acid formation precedes the formation of arachidonic acid and parallels the release of serotonin. Biochim Biophys Acta 1979; 573: 394-402
• 4 Lloyd JV, Mustard JF. Changes in the³²P-content of phosphatidic acid and phosphoinositides of rabbit platelets during aggregation induced by collagen and thrombin. Br J Haematol 1974; 26: 243-253
• 5 Mürer EH, Daniel JL. Fluoride-induced secretion from human blood platelets. Fluoride 1983; 16: 152-161
• 6 Hauser G, Eichberg J. Identification of cytidine diphosphate-diglyceride in the pineal gland of the rat and its accumulation in the presence of DL-propranolol. J Biol Chem 1975; 250: 105-112
• 7 Adelstein RS, Conti MA. Phosphorylation of platelet myosin increases the actin-activated myosin ATPase activity. Nature 1975; 256: 597-598
• 8 Lapetina EG. Metabolism of inositides and the activation of platelets. Life Sci 1983; 32: 2069-2082
• 9 Broekman MJ, Ward JW, Marcus AJ. Phospholipid metabolism in stimulated human platelets. Changes in phosphatidylinositol, phosphatidic acid and lysophospholipids J Clin Invest 1980; 66: 275-283
• 10 Broekman MJ, Ward JW, Marcus AJ. Fatty acid composition of phosphatidylinositol and phosphatidic acid in stimulated platelets. Persistence of arachidonyl-stearyl structure J Biol Chem 1981; 256: 8271-8274
• 11 Mürer EH, Stewart GJ, Daniel JL. Reversible and irreversible changes in platelets exposed to pH 5.3. Fed Proc 1981; 40: 808