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 mechanisms activated by low and high concentrations of platelet-activating factor (PAF) in rabbit platelets and to contrast the responses to those induced by thrombin. We measured changes in intracellular free calcium ([Ca++]i) with fura2, while monitoring light scatter simultaneously as a measure of shape change and aggregation in a dual-excitation dual-emission spectrofluorometer. An abrupt 20% fall in light scatter, coincident with the peak of the [Ca++]i, indicated shape change in Ca-containing or Ca-free medium and was blocked by BAPTA loading and 10 microM cytochalasin B. A secondary decline in light scatter, indicating aggregation, occurred only in Ca-containing medium and only under conditions favoring protein kinase C (PKC) activation. PAF at 10(-12) M did not increase 1,4,5-inositol triphosphate content, which suggested PKC 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-operated channels inducing shape change. PAF at 10(-9) and 10(-8) M increased [Ca++]i to 2 microM in 12 sec and induced both shape change and aggregation. However, in platelets pretreated with 100 nM staurosporine to inhibit protein kinases, 10(-9) M PAF did not cause aggregation even though [Ca++]i still rose to 2 microM, which indicated that PKC plays a role in aggregation but not in Ca++ mobilization.(ABSTRACT TRUNCATED AT 250 WORDS)
James-Kracke
M R
MR
Department of Pharmacology, School of Medicine, University of Missouri, Columbia.
Sexe
R B
RB
Shukla
S D
SD
D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D004789:Enzyme Activation; D005470:Fluorometry; D010972:Platelet Activating Factor / Q000494:pharmacology*; D015539:Platelet Activation / Q000187:drug effects*; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000502:physiology; D011493:Protein Kinase C / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D012542:Scattering, Radiation; D019311:Staurosporine; D013917:Thrombin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism*
D000470:Alkaloids; D015220:Calcium Channels; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013917:Thrombin; D019311:Staurosporine; D002118:Calcium