Stimulus-response coupling in human platelets. Changes evoked by platelet-activating factor in cytoplasmic free calcium monitored with the fluorescent calcium indicator quin2

Abstract

The role of changes in cytoplasmic free calcium, [Ca2+]i, in the responses to platelet-activating factor (PAF) was studied in human platelets loaded with the fluorescent calcium indicator, quin2. In the presence of 1 mM external calcium, PAF raised [Ca2+]i 8-10-fold in a few seconds to peak near 1 microM. [Ca2+]i then declined over several minutes towards the basal level. In the absence of external calcium there was a much smaller increase in [Ca2+]i of similar pattern. These findings suggest that PAF increases [Ca2+]i partly by discharge of internal Ca2+, but mainly by stimulated influx. Blockade of cyclo-oxygenase with aspirin only slightly reduced the [Ca2+]i changes, indicating that thromboxane A2 is not a major mediator of the calcium movements. In control conditions PAF could stimulate shape-change, aggregation and secretion. Aggregation and secretion were roughly halved by blockade of cyclo-oxygenase. Shape-change and secretion still occurred under conditions where the [Ca2+]i rise was small or suppressed, indicating a role for intracellular activators other than Ca2+. The possible involvement of products of phosphoinositide breakdown is discussed.

Characterization of platelet‐activating factor‐induced elevation of cytosolic free calcium concentration in eosinophils

“…However, this was not the case, since repeated application of different agonists produced a full response, The results support the concept that receptor-operated influx of Ca 2+ is necessary to obtain a maximal elevation of [Ca2+]i, However, our studies also show a small but consistent elevation of [Ca2+]i elicited by PAF both in the absence of Ca 2+ and in the presence of Ni 2+. These results also suggest that PAF, in addition to the stimulated influx, causes release of some Ca 2+ from intracellular stores as previously described in platelets, macrophages, and U937 [15][16][17]. Furthermore, an apparent increase in membrane permeability to Mn 2+, as seen in platelets in response to PAF [21,22], was not observed in eosinophils.…”

“…The importance of extracellular Ca 2+ ([Ca2+]e) in response to PAF has been demonstrated in platelets [15], macrophages [17], and U937 cells [16]. The effect of PAF on eosinophils also appears to involve a major influx of [Ca2+]e through receptor-mediated pores.…”

Elevation of pHi is not an essential step in calcium mobilisation in fura‐2‐loaded human platelets

“…Washed human platelets were prepared from blood freshly drawn from volunteers who gave informed consent, as described [8]. The washed platelet suspension (1 x lo9 cells/ml) was incubated with 0.4/1M fura-acetoxy methyl ester, and 2.0/cM BCECF acetoxy methyl ester and 1OOpM aspirin for 45 min at 37°C.…”

“…We used human platelets co-loaded with fura- [6] and BCECF [7] SO that [Ca2+]i and pHi could be monitored simultaneously by using dualwavelength excitation. Combining fluorescence measurement with absorbance [8] provided a simultaneous measurement of pHi with shape change and aggregation. Since the presence of Na+-H+ exchange in platelets is well established, as is the ability of thrombin and phorbol ester to activate this exchange [3,4], we did not make analysis of this mechanism a major focus of the work.…”

Stopped‐flow spectrophotometry monitoring the initial processes of platelet activation by platelet‐activating factor

“…This indicates that the chlortetracycline fluorescence change in fig. 1 b was attributable to intracellular calcium mobilization and not to calcium influx [16]. The release of calcium from internal stores occurred concomitantly with the platelet shape changes.…”