THE COMPOSITION OF THE PLATELET CYTOSKELETON FOLLOWING ACTIVATION BY ADP - EFFECTS OF VARIOUS AGENTS THAT MODULATE PLATELET-FUNCTION

Platelet activation by adenosine diphosphate (ADP) results in changes in the composition of the large cytoskeletal fragments that can be iso lated following solubilization of platelets with Triton X-100 and low speed centrifugation, Here we have used several different agents that modify platelet responses to investigate some of the factors that affe ct these cytoskeletal changes, All the experiments involved use of hir udinized platelet-rich plasma in which TXA(2) synthesis and release of dense body constituents does not occur following platelet activation with ADP, ADP alone caused a significant and sustained increase in the cytoskeletal content of actin binding protein (ABP), myosin, alpha-ac tinin, a 66K protein and actin, and a significant decrease in a 31K pr otein, In the presence of MK-852 or GR 144053 (GpIIb/IIIa antagonists) , in samples merely left unstirred and in Glanzmann's thrombasthesenia , ADP produced no increase in ABP or the 66K protein and no decrease i n the 31K protein, The increase in myosin and alpha-actinin became rev ersible but there was still incorporation of actin into the cytoskelet on, In the presence of ARL 66096 (a P-2T purinoceptor antagonist that inhibits aggregation but not shape change) there was no increase in AB P or the 66K protein and no decrease in the 31K protein, ARL 66096 als o prevented incorporation of alpha-actinin and actin, As with MK-852, myosin incorporation became reversible, IIoprost inhibited all the cyt oskeletal changes, the effects of MgCl2 were similar to those of MK-85 2, and acetylsalicylic acid (ASA) had no effect, In some experiments M K-852, ARL 66096, iloprost or MgCl2 were added 0.5 min after the ADP. They all produced disaggregation and this was accompanied by reversal of the changes in the composition of the cytoskeleton that had occurre d initially on stimulating the platelets with ADP, The results suggest that: (1) myosin is incorporated into the cytoskeleton transiently du ring shape change; (2) ADP interaction with the P-2T receptor leads to incorporation of alpha-actinin and actin into the cytoskeleton as wel l as platelet aggregation; (3) further incorporation of alpha-actinin and myosin and incorporation of ABP and the 66K protein occur conseque nt to fibrinogen binding and platelet aggregation; (4) displacement of the 31K protein from the cytoskeleton is also a consequence of fibrin ogen binding and platelet aggregation; (5) platelet disaggregation is accompanied by reversal of any cytoskeletal changes that have already occurred; (6) continuous occupation of the P-2T receptor is required f or maintenance of the cytoskeletal changes; (7) cAMP inhibits and reve rses cytoskeletal inhibits and reverses cytoskeletal assembly; and (8) MgCl2 acts similarly to a GpIIb/IIIa antagonist under these experimen tal conditions.