Abstract
Reduced platelet aggregation and a mild bleeding phenotype have been observed in patients chronically taking selective serotonin reuptake inhibitors (SSRIs). However, it remains unclear how SSRIs, which inhibit the plasma membrane serotonin transporter (SERT), modulate hemostasis. Here, we examine how sustained inhibition of SERT activity alters serotonergic signaling and influences platelet activation and hemostasis. Pharmaceutical blockade (citalopram dosing) or genetic ablation (SERT(-/-)) of SERT function in vivo led to reduced serotonin (5-hydroxytryptamine (5-HT)) blood levels that paralleled a mild bleeding phenotype in mice. Transfusion of wild-type platelets to SERT(-/-) mice normalized bleeding times to wild-type levels, suggesting that loss of SERTs causes a deficiency in platelet activation. Although SERT(-/-) platelets displayed no difference in P-selectin or αIIbβ3 activation upon stimulation with thrombin, ADP-mediated αIIbβ3 activation is reduced in SERT(-/-) platelets. Additionally, synergistic potentiation of αIIbβ3 activation by ADP and 5-HT is lost in SERT(-/-) platelets. Acute treatment of wild-type platelets with 5-HT2A receptor (5-HT2AR) antagonists or SSRIs revealed that functional 5-HT2ARs, not SERTs, are necessary for the synergistic activation of αIIbβ3 by dual 5-HT/ADP stimulation. Pharmacological studies using radiolabeled guanosine 5'-3-O-([(35)S]thio)triphosphate and [(3)H]ketanserin revealed that platelets isolated from SERT(-/-) or citalopram-treated mice have reduced activation of G-proteins coupled to 5-HT2ARs and receptor surface expression. Taken together, these data demonstrate that sustained SERT loss of function reduces 5-HT2AR surface expression that is critical for the synergistic activation of αIIbβ3 by 5-HT and ADP. These results highlight an antiplatelet strategy centered on blocking or desensitizing 5-HT2AR to attenuate ADP-mediated αIIbβ3 activation.
Highlights
Platelet dense granules contain 5-HT along with other platelet agonists including adenosine diphosphate (ADP), thromboxane (TXA2), and histamine
Decreased 5-HT Content in Platelets Isolated from serotonin transporter (SERT)؊/؊ and Citalopram-treated Mice Parallels Bleeding Phenotype
Mice Treated with Citalopram for 6 Days Have Reduced Whole Blood 5-HT Levels and Increased Tail Bleed Time—To determine whether selective serotonin reuptake inhibitors (SSRIs) alter blood 5-HT homeostasis, mice were exposed to citalopram-treated water for 6 days, 1 day beyond the lifetime of a circulating platelet in a mouse [22]
Summary
5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; SERT, serotonin transporter; SSRI, selective serotonin reuptake inhibitor; Cit, citalopram; 5-HT2AR, 5-HT2A receptor; SERTϩ/ϩ, wild-type mice; SERTϪ/Ϫ, serotonin transporter knock-out mice; DOI, 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane; GTP␥S, guanosine 5Ј-3-O-(thio)triphosphate; ANOVA, analysis of variance. 5-HT carried into the cytosol during platelet activation, whereas chronic blockage of SERT slowly depletes granule 5-HT, resulting in loss of 5-HT secretion and 5-HT signaling during platelet activation. The effects of chronic loss of SERT function on platelet activation remain unresolved. We investigated the mechanisms by which chronic inhibition of SERT alters hemostatic function using two independent models (SERTϪ/Ϫ and 6-day citalopram treatment). We hypothesized that the bleeding effects noted with the disruption of SERT function are due to altered 5-HT2AR signaling during platelet activation. We found that mice lacking SERT function display a bleeding phenotype that can be rescued by the addition of wild-type platelets. We found that ADP-mediated ␣IIb3 activation was reduced in SERTϪ/Ϫ and citalopram-treated platelets due to loss of 5-HT2AR signaling and surface expression
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