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 cytoskeleton. Cytochalasins are agents that interfere with the polymerisation of actin, and it has recently been discovered that cytochalasin H (CyH) is particularly effective as an inhibitor of the cytoskeletal reorganisation that occurs in platelets following activation by adenosine diphosphate (ADP). Here we have used CyH to inhibit platelet cytoskeletal reorganisation and to determine its effects on various aspects of platelet function. Experiments were performed in hirudinized platelet-rich plasma (PRP) or whole blood obtained from human volunteers. PRP was treated with 10 microM CyH or vehicle, then activated by ADP. The effect of CyH on cytoskeletal reorganisation was determined by SDS-PAGE of the Triton X-100 insoluble cytoskeletons and quantitated by densitometry. Platelet aggregation and aggregate stability in PRP were measured by monitoring changes in light absorbance; aggregation was measured in whole blood via platelet counting. Shape change, P-selectin expression and changes in intracellular calcium were measured using flow cytometry. CyH prevented the normal incorporation of actin, alpha-actinin and actin-binding protein into the cytoskeleton that occurred following ADP activation, and incorporation of myosin was markedly reduced. Aggregation was only partially inhibited but, more dramatically, the rate of disaggregation following addition of certain agents that interfere with fibrinogen binding to glycoprotein 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, suggesting a modulatory role of the cytoskeleton in platelet secretory activity. The results suggest that cytoskeletal reorganisation plays an important role in platelet shape change and aggregation and contributes in a major way to the stability of the aggregates that form.
Natarajan
P
P
Centre for Vascular Research, University of Nottingham, UK.
May
J A
JA
Sanderson
H M
HM
Zabe
M
M
Spangenberg
P
P
Heptinstall
S
S
D000244:Adenosine Diphosphate / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects* / Q000502:physiology; D002118:Calcium / Q000378:metabolism; D048429:Cell Size / Q000187:drug effects; D003572:Cytochalasins / Q000494:pharmacology*; D003598:Cytoskeletal Proteins / Q000187:drug effects / Q000502:physiology; D003599:Cytoskeleton / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019007:P-Selectin / Q000187:drug effects / Q000378:metabolism; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology
D003572:Cytochalasins; D003598:Cytoskeletal Proteins; D019007:P-Selectin; D010975:Platelet Aggregation Inhibitors; C034891:cytochalasin H; D000244:Adenosine Diphosphate; D002118:Calcium