Dipyridamole Enhances NO/cGMP-Mediated Vasodilator-Stimulated Phosphoprotein Phosphorylation and Signaling in Human Platelets

Abstract

Background and Purpose-Dipyridamole and in particular dipyridamole in combination with low-dose aspirin are very effective in preventing recurrent stroke. However, the mechanism(s) underlying this dipyridamole effect have not been elucidated. Since dipyridamole inhibits the cGMP-specific phosphodiesterase type V in vitro, we hypothesized and tested whether therapeutically relevant dipyridamole concentrations enhance NO/cGMP-mediated effects in intact human platelets studied ex vivo. Methods-Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), an established marker of NO/cGMP effects in human platelets, was quantified by phosphorylation-specific antibodies and Western blots. Serotonin secretion and thromboxane synthase activity were determined by fluorometric quantification of derivatized serotonin and synthase products, respectively. Results-Endothelium-derived factors such as NO and prostaglandin I 2 are known to elevate both cGMP and cAMP levels with concomitant platelet inhibition and VASP phosphorylation. In our in vitro experiments, therapeutically relevant concentrations (3.5 mol/L) of dipyridamole amplified only cGMP-mediated VASP phosphorylation due to the NO donor sodium nitroprusside, but not cAMP-mediated effects. Furthermore, thromboxane synthase activity and serotonin secretion, events important for initial platelet activation, were inhibited by sodium nitroprusside, an effect also enhanced by dipyridamole, demonstrating the functional relevance of these observations. Finally, the ex vivo enhancement of NO/cGMP effects was also observed with platelets obtained from healthy volunteers treated with extended-release dipyridamole. Key Words: dipyridamole Ⅲ phosphodiesterase inhibitors Ⅲ platelet aggregation inhibitors Ⅲ stroke T he role of activated platelets in acute and chronic vascular diseases is well established. Accordingly, antiplatelet drugs significantly reduce the risk of severe events, such as ischemic stroke, myocardial infarction, and vascular death in atherosclerotic vascular disease. In numerous clinical studies, the efficacy of antiplatelet drugs in the treatment and secondary prevention of vascular diseases, particularly stroke, has been proven. [1][2][3] Platelet activation is inhibited by factors released from endothelial cells that constitute the inner cell layer of the vessel wall. The most important inhibitory endotheliumderived factors are NO and prostaglandin I 2 (PGI 2 ), which inhibit platelets by increasing the level of intracellular cyclic nucleotides. 4,5 Subsequent stimulation of cGMP-and cAMPdependent protein kinases leads to the phosphorylation of a variety of proteins. 6 In platelets, this inhibits agoniststimulated calcium signaling, 7,8 fibrinogen binding, 9 adhesion, 10 and aggregation 5 and stimulates phosphorylation of the cytoskeletal and focal adhesion protein vasodilatorstimulated phosphoprotein (VASP). 4,5,11 This phosphorylation correlates strongly with the inhibition of platelets. 9,11 Interestingly, antiplatelet treatment with clopidogrel or ticlo...

The neuron-astrocyte-microglia triad in CA3 after chronic cerebral hypoperfusion in the rat: Protective effect of dipyridamole

“…Experimentally we have demonstrated that dipyridamole significantly restores hippocampally based spatial memory (Melani et al 2010) and has a protective effect on hippocampal CA1pyramidal neurons (Lana et al 2014) 90 days after 2VO. The increases of extracellular adenosine (Figueredo et al 1999), together with increased VEGF production (Ernens et al 2010), and the potentiation of the NO system (Aktas et al 2003;Venkatesh et al 2010) help explaining the protecting effect of dipyridamole in this rat model of hypoperfusion. Dipyridamole has a positive effect on blood flow and angiogenesis through the nitrite/NO endocrine system (Venkatesh et al 2010), increases NO levels and decreases superoxide formation both in ischemic and non-ischemic animals (Pattillo et al 2011), and has pleiotropic pharmacological effects, such as antioxidant and antiinflammatory proprieties (Blake 2004;Eisert 2002;Hsieh et al 2010;Riksen et al 2005).…”

The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage

“…39 However, the mechanisms underlying this dipyridamole effect have not been fully elucidated. Dipyridamole has been shown to enhance nitric oxide (NO)/cGMP-mediated effects in intact human platelets 40 as well as to interfere with the ADPdependent platelet activation. 41 Both effects could be related to MRP4, assuming a possible effect of this transporter on the cytosolic cGMP concentration 28 as well as a role in adenosine nucleotide transport.…”

Additive Effects of Statin and Dipyridamole on Cerebral Blood Flow and Stroke Protection

“…By increasing cAMP and cGMP levels in platelets, DP reversibly inhibits platelet aggregation (Best et al, 1979). However, in addition to platelets, DP may also potentiate some of the vascular protective effects of endothelium-derived nitric oxide (NO), which increases cGMP by stimulating soluble guanylyl cyclase (Aktas et al, 2003). Endotheliumderived NO is an important regulator of cerebral blood flow (CBF), and endothelial NO synthasedeficient (eNOS À/À ) mice exhibit larger stroke size after focal cerebral ischemia (Huang et al, 1996).…”