We examined the influence of 3,5-diisopropylsalicylic acid (3,5-DIPS) and calcium(II)3 (3,5-diisopropylsalicylate)6 (H2 O)6 [Ca(II)3 (3,5-DIPS)6 ], a new activator of calcium-dependent calmodulin-triggered nitric oxide synthase, on thrombin-induced platelet P-selectin expression. Citrated whole blood samples were incubated with either ethanol vehicle, 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6. These whole blood samples were also co-incubated with thrombin receptor activating peptide (TRAP) or adenosine diphosphate (ADP), to up-regulate P-selectin (CD62P) on platelets. Both TRAP and ADP up-regulated P-selectin on platelets compared with platelets in whole blood samples that were not incubated with either platelet activator. Co-incubation of whole blood samples with TRAP, ADP together with 3,5-DIPS, or Ca(II)3 (3,5-DIPS)6 revealed that Ca(II)3 (3,5-DIPS)6 caused a decrease in platelet P-selectin expression for TRAP, ADP, and no-activator co-incubated samples of whole blood. Incubation of platelets with 3,5-DIPS also caused a decrease in ADP-induced up-regulation of P-selectin but failed to affect TRAP or no-activator-treated platelets. Incubation of whole blood with Ca(II)3 (3,5-DIPS)6 induced some hemolysis. We found that hemolysis increases basal P-selectin expression on platelets. We therefore conclude that Ca(II)3 (3,5-DIPS)6 decreased not only basal, but also hemolysis-induced P-selectin expression on platelets. In contrast, incubation of haemolysed whole blood with SIN-1 (standard nitric oxide-releasing drug) had no effect on P-selectin expression. In summary, Ca(II)3 (3,5-DIPS)6, a new calmodulin-dependent nitric oxide synthase activator, decreases P-selectin expression of human platelets in response to thrombin receptor activation. Improved calcium-dependent calmodulin activators may become useful drugs for the treatment of disorders associated with platelet activation, and P-selectin may decrease expression due to hemolysis.