Role of α-1 Anti Trypsin, A Constitutive Form of Nitric Oxide Synthase in Cyclic AMP Mediated Inhibition of Platelet Aggregation

Introduction: As reported before, the treatment of platelets with all currently known platelet aggregating agents was resulted in the reduction of nitric oxide (NO) synthesis through the inhibition of enzyme nitric oxide synthase (NOS). This reduction of NO in consequence released arachidonic acid from the platelet membrane ultimately leading to the aggregation of platelets. Aim: Experiments were carried out to identify the platelet NOS. Results: The amino acid sequence of the protein sample thus prepared was determined by mass spectrometry in which the platelet cytosolic NOS was identified to be α1 anti trypsin (AAT) when matched with the protein databank. The incubation of NOS/AAT with l-arginine resulted in the increase of NO synthesis to ~1nmol/h (P<0.01) compared to control measured by methemoglobin method. Similarly, addition of 80µM aspirin to NOS/AAT also stimulated the NO synthesis activity of the protein. In contrast, the treatment of the purified protein with platelet aggregating agents including 4µM ADP, 2µg/ml collagen, 5µM thrombin, 5µM epinephrine, 0.1µM dermcidin and 0.1mM l-NAME inhibited NOS activity of AAT. Additionally, the incubation of NOS with cAMP resulted in the binding of cAMP to the purified protein. Scatchard plot analysis of the binding of cAMP to AAT demonstrated Kd =15.54nM with 1.56 X 103 molecules of cAMP bound to each AAT molecule. It was found that the binding of cAMP to AAT resulted in the lack of inhibition of the NOS activity of AAT by the aggregating agents. Conclusion: The identification of the NOS as the AAT can be a therapeutic target for the treatment of acute coronary syndromes.