Diagnostic X-ray spectrometry (DXS), based on X-ray fluorescence, was used to quantitate directly the multiple elemental composition of washed, intact human platelets (n = 16), with the following results: K = 3.08 +/- 1.00 mg/g, Ca = 1.18 +/- 0.29 mg/g, Zn = 35 +/- 9 micrograms/g. These values show that washed platelets contain significant pools of K, Ca, and Zn, the latter some 30-60-fold higher than plasma levels. Dialysis of whole platelets against cation exchange resin (Chelex-100) did not extract Ca(II) and Zn(II) sequestered within whole cells. To identify the subcellular locale of the elements, platelet lysate was subjected to 30-70% sucrose gradient ultracentrifugation and subcellular enriched fractions were obtained. Fractions were analyzed by DXS (for elements), electron microscopy (for dense granules), and subcellular markers fibrinogen and von Willebrand factor. In contrast to Ca and K, which accumulate in the dense granules and the cytoplasm, respectively, Zn appears to be distributed in the alpha-granules (40%) and the cytoplasm (60%). The subcellular distribution of Zn(II) is discussed within the context of the sensitivity of platelet response to the availability of Zn(II) and the platelet release reactions following stimulation.
The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.
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