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By using both megakaryocytes and platelets to study effects of adenosine diphosphate (ADP), we hope to understand the ionic events underlying the shape changes induced by this nucleotide. Guinea pig bone marrow megakaryocytes were isolated to 50-90% purity by modifications of published procedures, and cultured for 12-48 hours. In response to ADP (l-10μM) megakaryocytes develop a ruffled border and spread in 15-30 minutes to 2-3 times their original diameter and adhere strongly to the substrate. Several other diphosphonuc1eotides or adenosine monophosphate have no effect. Spreading occurs in calciummagnesium free Hank’s salt solution, but is blocked when extracellular sodium was replaced by potassium, choline, or lithium. Amiloride, an inhibitor of sodium uptake, inhibits the extent of spreading 90% at 10-4M, but allows partial spreading in 60% of the cells. Spreading can be mimicked by incubating the cells with 2μM A-23187 combined with either 5mM methylamine or 1μM monensin, but not by A-23187 alone.  The rate of platelet shape change was measured spectro- photometrical1y at 37°C with 5mM EGTA both in platelet rich plasma (PRP) or after gel filtration in phosphate buffered saline. Amiloride inhibits shape change completely at 1mM and 50-60% at 0.1mM, but only if present during gel filtration. A rapid drop in buffer pH from 7.4 to 6.9 causes a 50% inhibition of rate of shape change in PRP or after gel filtration, but the inhibition spontaneously reverses within three minutes incubation at the lower pH.  These results are compatible with a model in which ADP causes a sodium influx which is linked to a change in intracellular pH. This may be a necessary but not necessarily sufficient step in ADP induced shape change.

Effects Of Altered pH And Sodium Concentration On The Response Of Megakaryocytes And Platelets To ADP