Polyphosphoinositide Changes in Rabbit Platelets Stimulated with Platelet Activating Factor During the Formation of Platelet-fibrin Clots.
Phosphoinositide metabolism in rabbit platelets prelabelled with [(32)P]phosphate and [(3)H]inositol was stimulated by platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) with stirring at 200 rpm for 120 s in the presence of polymerising fibrin produced by the action of batroxobin (B. atrox) (also referred to by the proprietary name Reptilase) on fibrinogen. Under these conditions platelet-fibrin clots formed and retracted around the stirring bar. Phosphoinositides were extracted with chloroform: methanol: HC1. The role of the secretion of platelet granule contents in the phosphoinositide changes was examined by comparison of the effects of 1 nM PAF which did not cause secretion, with 50 nM PAF which caused extensive secretion. Stimulation of platelets with PAF in the presence of polymerising fibrin caused a greater decrease in the amount and labelling of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) than was observed with platelets stimulated in the presence of fibrinogen. With 1 nM PAF, the decrease (1.26 ± 0.11 nmol/10(9) platelets) in amount of extractable PIP(2) when platelets were stimulated in the presence of polymerising fibrin compared with in the presence of fibrinogen was accounted for by an increase in the amount of phosphatidylinositol 4-phosphate (PIP). With 50 nM PAF, the decrease in amount of extractable PIP(2) (1.09±0.11 nmol/10(9) platelets) was not accounted for by an increase in the amount of PIP; the decrease in the amount of [(3)H]inositol label in PIP(2) in platelets stimulated in the presence of polymerising fibrin was accounted for by the sum of the increases in PIP labelling and the label associated with interfacial protein from the lipid extractions. When fibrin polymerisation was blocked with glycyl-L-prolyl-L-arginyl-L-proline (GPRP), the large decrease in extractable PIP(2) and the increase in the association of label with the interfacial protein did not occur. Thus, both the formation of a fibrin network, and the changes that accompany the secretion of granule contents, are necessary for the association of the (3)H-labelled material with interfacial protein. Blocking thromboxane A(2) formation had no effect on the changes in response to 50 nM PAF. Although PAF stimulated phospholipase C, resulting in increases in amount and (32)P-labelling of phosphatidic acid and (3)H-labelling of inositol bisphosphate and inositol phosphate, the increases were similar in the presence of polymerising fibrin or fibrinogen. Thus, further stimulation of phospholipase C does not occur in association with clot formation. The specific radioactivities of labelling with [(3)H]inositol of the phosphoinositides in unstimulated platelets differed (PIP(2)> phosphatidylinositol (PI) > PIP). Upon stimulation of the platelets with 1 nM PAF, the specific radioactivity of PIP rose above that of PI and toward that of PIP(2), indicating that the increase in PIP was due to degradation of PIP(2). Thus, the large decrease in extractable PIP(2) and increase in formation of PIP caused by the presence of polymerising fibrin appear to be due to increased degradation of PIP(2) to PIP.
Department of Pathology, Faculty of Health Sciences, McMaster University, Hamilton, Canada.
J D
JD
Vickers
R L
RL
Kinlough-Rathbone
M A
MA
Packham