Purposes of these studies were first; to determine whether or not Calcium(II)3 (3,5-
diisopropylsalicylate)6(H2O)6 [Ca(II)3(3,5-DIPS)6], a lipophilic calcium complex, could decrease
activated-platelet aggregation, and second; to determine whether or not it is plausible that
Ca(II)3(3,5-DIPS)6 decreases activated-platelet aggregation by facilitating the synthesis of Nitric
Oxide (NO) by Nitric Oxide Synthase (NOS). The influence of Ca(II)3(3,5-DIPS)6 on the initial
rate of activated-platelet aggregation was determined by measuring the decrease in rate of increase
in transmission at 550 nm for a suspension of Thrombin-CaCl2 activated platelets following the
addition of 0, 50, 100, 250, or 500 μM Ca(II)3(3,5-DIPS)6. To establish that the Ca(lI)3(3,5-
DIPS)6-mediated decrease in aggregation was due to activation of NOS, the effect of ʟ-NMMA, an
inhibitor of NOS, on the inhibition of platelet aggregation by Ca(II)3(3,5-DIPS)6 was determined
using a suspension of activated platelets contaimng 0 or 250 μM Ca(II)3(3,5-DIPS)6 without or
with 1 mM ʟ-NMMA. An in vitro Bovine Brain NOS reaction mixture, containing CaCl2 for the
activation of Phosphodiesterase-3' ,5'-Cyclic Nucleotide Activator required for the activation of
NOS, was used to determine whether or not Ca(II)3(3,5-DIPS)6 could be used as a substitute for the
addition of Ca. The decrease in absorbance at 340 nm, lambda maximum for NADPH, was
measured to determine NOS activity following the addition of NOS to the complete reaction
mixture containing either CaCl2, Ca(II)3(3,5-DIPS)6, or neither Ca compound. Increasing the
concentration of Ca(II)3(3,5-DIPS)6 caused a concentration related decrease in activated platelet
aggregation. The addition of ʟ-NMMA to activated platelets, in the absence of Ca(II)3(3,5-DIPS)6,
caused a 129% increase in initial rate of platelet aggregation. The initial rate of platelet
aggregation decreased 74% with the addition of 250 μM Ca(II)3(3,5-DIPS)6 and the addition of ʟ-NMMA plus 250 μM Ca(II)3(3,5-DIPS)6 caused a 197% decrease in initial rate of aggregation
compared to the initial rate observed width the presence of 1 mM ʟ-NMMA alone. There was only
a small, 27%, increase in initial rate of 0.4 mM NADPH oxidation when 0.9 mM CaCl2 was added
to the NOS reaction mixture in comparison to the initial rate of NADPH oxidation with no addition
of CaCI2. Addition of an equivalent amount of Ca in the form of Ca(II)3(3,5-DIPS)6, 333 μM,
caused a 37% increase in initial rate of NADPH oxidation compared to the addition of 0.9 mM
CaCl2. Addition of increasing concentrations of ʟ-NMMA plus 0.9 mM CaCl2 or 333 μM Ca(II)3(3,5-DIPS)6 to the NOS reaction mixture caused a concentration related increase in initial
rate of NADPH oxidation. Addition of ʟ-NMMA while expected to decrease NADPH oxidation
actually increased the rate of NADPH oxidation. Additions of 133 μM or 267 μM Ca(II)3(3,5-
DIPS)6 also caused concentration related increases in initial rate of NADPH oxidation in the
presence of 113 μM ʟ-NMMA. However, the addition of 533 μM Ca(II)3(3,5-DIPS)6 caused a
dramatic decrease in initial rate of NADPH oxidation by NOS. It is concluded that: 1) Ca(II)3(3,5-
DIPS)6 activates platelet NOS in preventing platelet aggregation, 2) in vitro NOS activity can be
observed spectrophotometrically by following the consumption of NADPH as a decrease in
absorbance at 340 nm, 3) Ca(II)3(3,5-DIPS)6 plays a role in enhancing Bovine Brain NOS activity
resulting in an increased rate of NADPH oxidation by NOS, 4) Ca(II)3(3,5-DIPS)6 is a useful form
of Ca in activating NOS and superior to CaCl2 with regard to the facilitation of a NADPH
oxidation, and 5) ʟ-NMMA stimulates Bovine Brain NOS activity rather than causing an inhibition
of this enzyme and must serve as a reducible substrate for Bovine Brain NOS.
Identification and characterization of a calmodulin-dependent nitric oxide synthase from GH3 pituitary cells