PICOMOLAR PLATELET-ACTIVATING-FACTOR MOBILIZES CA TO CHANGE PLATELET SHAPE WITHOUT ACTIVATING PHOSPHOLIPASE-C OR PROTEIN-KINASE-C - SIMULTANEOUS FLUOROMETRIC MEASUREMENT OF INTRACELLULAR FREE CA CONCENTRATION AND AGGREGATION

The purpose of this study was to investigate signal transduction mecha nisms activated by low and high concentrations of platelet-activating factor (PAF) in rabbit platelets and to contrast the responses to thos e induced by thrombin. We measured changes in intracellular free calci um ([Ca++](i)) with fura2, while monitoring light scatter simultaneous ly as a measure of shape change and aggregation in a dual-excitation d ual-emission spectrofluorometer. An abrupt 20% fall in light scatter, coincident with the peak of the [Ca++](i), indicated shape change in C a-containing or Ca-free medium and was blocked by BAPTA loading and 10 mu M cytochalasin B. A secondary decline in light scatter, indicating aggregation, occurred only in Ca-containing medium and only under con ditions favoring protein kinase C (PKC) activation. PAF at 10(-12) M d id not increase 1,4,5-inositol triphosphate content, which suggested P KC would not be activated. However, PAF at 10(-12) rapidly increased [ Ca++](i) to 900 nM in 7 sec seemingly by Ca influx through receptor-op erated channels inducing shape change. PAF at 10(-9) and 10(-8) M incr eased [Ca++](i) to 2 mu M in 12 sec and induced both shape change and aggregation. However, in platelets pretreated with 100 nM staurosporin e to inhibit protein kinases, 10(-9) M PAF did not cause aggregation e ven though [Ca++](i) still rose to 2 mu M, which indicated that PKC pl ays a role in aggregation but not in Ca++ mobilization . After 60 sec in Ca-free medium, [Ca++](i) rose to similar to 300 nM when stimulated by PAF or thrombin (1 U/ml), but this was sufficient to cause shape c hange except when Ca influx was critical, as it was for 10(-12) M PAF. In Ca-containing medium, thrombin invoked a sustained increase in [Ca ++](i) to 900 nM and a slower shape change in 30 sec before aggregatio n. Simultaneous measurements of fura2 and light scatter afforded new e vidence that 1 pM PAF opened receptor-operated Ca++ channels without a ctivating PLC or PKC such that [Ca++](i) rose to similar to 1 mu M to cause shape change but not aggregation. The number of signaling pathwa ys enlisted may increase as PAF receptor occupation increases.