Involvement Of Calmodulin In Platelet Reaction

Calmodulin is a ubiquitous Ca²⁺-binding protein, modulating a number of Ca²⁺-dependent cellular reactions. In blood platelets, involvement of calmodulin has been demonstrated in the activation of myosin light chain kinase and of phospholipase A₂. In the present study, involvement of this protein in platelet reaction was thoroughly investigated, utilizing phenothiazines(chlorpromazine and trifluoperazine) known as selective inhibitors of calmodulin.

Phenothiazines in micromolar concentration completely inhibited platelet aggregation and secretion induced by ADP epinephrine, collagen, thrombin or Ca²⁺-ionophore. They also completely inhibited the aggregation triggered by arachido- nate or by the mixture of thromboxane A₂ and prostaglandin endoperoxides, which is probably due to the inhibition of activation of myosin light chain kinase. As another possible involvemnt of calmodulin in the reaction, the effect of phenothiazines on the release of arachidonate from phospholipids was studied in stimulated platelets to which radioactive arachidonate had been incorporated. The release was dose-dependently inhibited by either of phenothiazines. Then, their effect on the hydrolysis of phosphatidylinositol by platelet sonicates was investigated, using ³H-myoinositol phosphatidylinositol as substrate. Either of phenothiazines suppressed the hydrolysis in the micromolar concentration, indicating their inhibition on phosphatidylinositol specific phospholipase C.

These observations suggest involvement of calmodulin in at least two steps of platelt reaction: one is the step of the release of arachidonate from phospholipids (the activation of phospholipase C in addition to phospholipase A₂) and the other is the step of contraction of platelet actomyosin after the formation of thromboxane A₂ (the activation of myosin light chain kinase).