The involvement of the cytoskeleton in regulating IP 3 receptor‐mediated internal Ca 2+ release in human blood platelets.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP 3 in regulating internal Ca 2+ release, and to measure IP 3 binding to its receptor. Our results indicate that platelet vesicles release Ca 2+ as early as 3 seconds after IP 3 addition. Using [ 3 H]IP 3 , we have found that platelets contain a single class of high affinity IP 3 binding sites with a Kd of ∼0.20 (± 0.01) nM. Immuno‐blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP 3 receptor. Immunofluorescence staining data indicate that the IP 3 receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP 3 binding and IP 3 ‐induced Ca 2+ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP 3 binding and IP 3 receptor‐mediated Ca 2+ release during platelet activation.