β ₂ -Adrenoceptors Activate Nitric Oxide Synthase in Human Platelets

Abstract

Nitric oxide (NO), generated by platelets through stimulation of nitric oxide synthase (NOS), limits platelet adhesion and aggregation after a prothrombotic stimulus. Platelet beta-adrenoceptors (betaARs) mediate inhibition of aggregation, but no direct link has been shown between these receptors and platelet adhesion or NO production. We examined NOS activity in human platelets from the conversion of L-[(3)H]-arginine to L-[(3)H]-citrulline, after betaAR stimulation or cAMP elevation. Basal NOS activity was 0.11+/-0.03 pmol L-citrulline/10(8) platelets. The betaAR agonist isoproterenol 1 micromol/L and the adenylyl cyclase activator forskolin 1 micromol/L each increased NOS activity, to 0.26+/-0.04 and 0.23+/-0.03 pmol L-citrulline/10(8) platelets, respectively (P<0.01 for each). Both responses were abolished by the adenylyl cyclase inhibitor SQ22536 50 micromol/L. NOS activation by isoproterenol or forskolin was not associated with a change in intracellular Ca(2+). In functional studies, isoproterenol inhibited U46619-induced platelet aggregation in a concentration-dependent manner, but this effect was not significantly diminished by NOS inhibition. In contrast, thrombin-stimulated platelet adhesion to cultured human umbilical vein endothelial cell monolayers was inhibited by isoproterenol, and this effect was abolished by NOS inhibition (1.3+/-0.2% versus 2.6+/-0.2% respectively; P<0.001). Effects of isoproterenol on NOS activity, platelet aggregation, and adhesion were mediated exclusively through beta(2)ARs, as determined by coincubation with betaAR subtype-selective antagonists. We conclude that beta(2)ARs activate platelet NOS by increasing cAMP, and that this activation is Ca(2+)-independent. beta(2)ARs may contribute to modulation of platelet aggregation and adhesion to endothelium, and our findings suggest that activation of the L-arginine/NO system mediates the effects of beta(2)ARs on adhesion but not aggregation.

Platelet-Derived Nitric Oxide Signaling and Regulation

“…␤ 2 AR stimulation inhibits platelet adhesion to human umbilical vein endothelial cell monolayers, an effect abolished by NOS inhibition. 20 The sole involvement of the AC-cAMP-PKA pathway in ␤ 2 AR-mediated NOS3 activation in platelets contrasts with the situation reported in human umbilical vein endothelial cells, where ␤ 2 AR-mediated NOS3 activation involves both PKA and protein kinase B (Akt). 24,25 We have recently demonstrated that platelet NOS3 binds to the globular, but not to the filamentous, form of ␤-actin, and the affinity of this binding is substantially increased by heat shock protein 90 (Hsp90).…”

“…␤-Adrenoceptors (␤AR), which are present on platelets and couple to adenylyl cyclase (AC), 22,23 can stimulate NOS3 activity and NO biosynthesis, by increasing cAMP (cyclic adenosine monophosphate) and protein kinase A (PKA) activity. 20 This effect is mediated through the ␤ 2 -subtype of ␤AR and is not associated with any change in intraplatelet Ca 2ϩ . ␤ 2 AR stimulation inhibits platelet adhesion to human umbilical vein endothelial cell monolayers, an effect abolished by NOS inhibition.…”

“…19 Additionally, platelets have been found to have a measurable basal level of NOS activity, as determined from the conversion of L-arginine to L-citrulline. 12,16,20 Regulation of Platelet NOS3 Activity and NO Production: Differences From Endothelial Cells NOS3 in platelets was initially considered similar to that found in endothelial cells, in terms of its dependence on intracellular Ca 2ϩ for activation. Although platelet NOS3 was initially characterized as a Ca 2ϩ -calmodulin sensitive enzyme, 10 recent evidence suggests that activation of this enzyme more usually occurs independently of intracellular Ca 2ϩ .…”

β-Actin regulates platelet nitric oxide synthase 3 activity through interaction with heat shock protein 90

“…NOS activity was assessed by measuring the conversion of L-[ 3 H]arginine to L-[ 3 H]citrulline, and cGMP was determined by RIA, both in GFP in suspension (unstirred) at 37°C, as described (6,17,35). 2؉ .…”

“…Its actions in target cells are largely mediated through the activation of soluble guanylyl cyclase and, hence, increased formation of cyclic guanosine-3Ј,5Ј-monophosphate (cGMP). Inhibition of NO production by platelets gives rise to their activation as well as increased adhesion to vascular endothelium (5,6), which is a precursor to intravascular thrombosis. Thus, platelet NO biosynthesis is crucial for the maintenance of blood fluidity and the prevention of thrombus formation in uninjured healthy vessels physiologically.…”