Changes in Phosphoinositides in Rabbit Platelets during Clot Formation. Comparison of Platelets Stimulated by ADP or by Thrombin in the Presence of Polymerising Fibrin

Abstract

Platelet phosphoinositide metabolism was examined during platelet-fibrin clot formation stimulated by ADP (10 μM) plus reptilase, or by thrombin (1 U/ml), for 120 s in the presence of fibrinogen, to determine which changes are specifically associated with this process. Stirring at 200 rpm was used to minimise the contribution of aggregation to the platelet changes. Under these conditions, thrombin caused extensive release of the contents of platelet granules; ADP plus reptilase did not. The presence of fibrinogen decreased the amount of extractable phosphatidylinositol 4,5-bisphosphate (PIP(2)) by 46.4±5.5% when thrombin was the stimulus, and by 47.4±5.5% when the platelets were stimulated by ADP plus reptilase. Fibrinogen did not decrease the extraction of other phospholipids. The amount of phosphatidylinositol 4-phosphate (PIP) increased when platelets were stimulated in either the presence or absence of fibrinogen. These increases were greater in the presence of fibrinogen and the thrombin-induced increase was smaller than the increase induced by ADP plus reptilase; with ADP plus reptilase, the increase in PIP more than accounted for the loss of extractable PIP(2). In platelets prelabelled with [(3)H]inositol, the decrease in PIP(2) labelling induced by fibrinogen with ADP plus reptilase as the stimulus was accounted for by the increase in PIP labelling; the decrease induced by fibrinogen with thrombin as the stimulus was not. With thrombin, 46.5% of the decrease in PIP(2) labelling, caused by fibrinogen, was accounted for by label that remained with the interfacial protein after lipid extraction; with ADP plus reptilase, the amount of label with this protein was the same with or without fibrinogen. Only thrombin increased the amount of label in inositol trisphosphate (IP(3)) and the amount of phosphatidic acid (PA); these changes were not increased by fibrinogen. Thus, the results with ADP plus reptilase indicate that clot formation is not dependent on release of granule contents, formation of detectable IP(3) or PA (and hence does not require activation of phospholipase C) or association of [(3)H]inositol-labelled compounds with protein. Clot formation is associated with a shift in the PIP(2)-PIP equilibrium toward PIP.