Effects of cytochalasin H, a potent inhibitor of cytoskeletal reorganisation, on platelet function

Platelets contain a well-developed and dynamic cytoskeleton composed mainly of actin and actin-associated proteins. Upon platelet activation there is rapid polymerisation of actin and a marked reorganisation of the platelet c ytoskeleton. Cytochalasins are agents that interfere with the polymerisatio n of actin, and it has recently been discovered that cytochalasin H (CyH) i s particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (AD P). Here we have used CyH to inhibit platelet cytoskeletal reorganisation a nd to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or who le blood obtained from human volunteers. PRP was treated with 10 muM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorgani sation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeleto ns and quantitated by densitometry. Platelet aggregation and aggregate stab ility in PRP were measured by monitoring changes in light absorbance; aggre gation was measured in whole blood via platelet counting. Shape change, P-s electin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha -actinin and actin-b inding protein into the cytoskeleton that occurred following ADP activation , and incorporation of myosin was markedly reduced. Aggregation was only pa rtially inhibited but, more dramatically, the rate of disaggregation follow ing addition of certain agents that interfere with fibrinogen binding to gl ycoprotein IIb/IIIa on the surface of platelets was markedly increased. The ADP-induced shape change was also inhibited. CyH had no effect on calcium mobilisation. Curiously, expression of P-selectin was potentiated by CyH, s uggesting a modulatory role of the cytoskeleton in platelet secretory activ ity. The results suggest that cytoskeletal reorganisation plays an importan t role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.