Signaling-mediated Functional Activation of Inducible Nitric-oxide Synthase and Its Role in Stimulating Platelet Activation

Jasna A Marjanovic,Aleksandra Stojanovic,Viktor M Brovkovych,Randal A Skidgel,Xiaoping Du

doi:10.1074/jbc.m801646200

Abstract

Nitric oxide (NO) is a short lived secondary messenger, synthesized by nitric-oxide synthases (NOS). It is believed that the activity of inducible NOS (iNOS) is regulated primarily at the transcription level by inducing expression of iNOS mRNA and protein, which then continuously produces NO, until its degradation. Platelets do not have the nuclear transcriptional regulatory mechanisms of the iNOS gene and are believed to generate NO in response to agonist stimulation via endothelial NOS (eNOS). However, here we show that agonist-induced NO production is only partially eNOS-dependent and is also mediated by iNOS. Platelet agonist-induced NO production is significantly reduced in iNOS-knockout platelets. Platelet NO production occurs within seconds after agonist addition and is not accompanied by changes in iNOS protein levels, indicating a signaling-mediated functional activation mechanism of iNOS. Importantly, iNOS knock-out and iNOS inhibitors reduce agonist-induced platelet secretion and aggregation and cGMP levels, indicating that iNOS activation is important in stimulating platelets via the newly identified NO-cGMP-dependent platelet secretion pathway. Furthermore, iNOS knock-out mice have prolonged bleeding time, suggesting that this novel mode of regulation of iNOS activity plays a physiologically relevant role in hemostasis.

Highlights

Expressed in cells, and the enzymatic activities of endothelial nitric-oxide synthases (NOS) (eNOS) and nNOS are regulated by intracellular Ca2ϩ levels and protein phosphorylation [3]. inducible NOS (iNOS) is currently described as a Ca2ϩindependent enzyme that is not normally expressed in resting cells [2]
We have recently shown that platelet agonists induce activation of phosphoinositide 3-kinase and Akt, which activates eNOS [12] and that eNOS plays an important role in Nitric oxide (NO) synthesis during platelet activation and in stimulating cyclic guanosine monophosphate-dependent platelet secretion and secretion-dependent platelet aggregation [13, 14]
Role of iNOS in Agonist-induced Platelet NO Production—To investigate which NOS isoform is responsible for agonist-induced NO production in platelets, wild-type or eNOSϪ/Ϫ platelets were stimulated with thrombin and analyzed for NO release in real time using an electrochemical detection method

Summary

EXPERIMENTAL PROCEDURES

Materials—Protein synthesis inhibitor puromycin was from Sigma. iNOS inhibitors, 1400W and aminoguanidine, and cGMP analog 8-bromo-cGMP were from Calbiochem. Materials—Protein synthesis inhibitor puromycin was from Sigma. INOS inhibitors, 1400W and aminoguanidine, and cGMP analog 8-bromo-cGMP were from Calbiochem. Human ␣-thrombin was purchased from Enzyme Research Laboratories. Luciferin/luciferase reagent and collagen were purchased from Chronolog. INOS- and eNOS-deficient mice, backcrossed for more than 10 generations to C57BL background, and C57BL/6 control mice were obtained from Jackson labora-.

Measurement of Platelet cGMP

RESULTS

The data presented in this study