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Loss of Serotonin Transporter Function Alters ADP-mediated Glycoprotein αIIbβ3 Activation through Dysregulation of the 5-HT2A Receptor*

  • Kendra H. Oliver
    Affiliations
    From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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  • Matthew T. Duvernay
    Affiliations
    From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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  • Heidi E. Hamm
    Correspondence
    To whom correspondence may be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 442 Robinson Research Bldg., 23rd Ave. South at Pierce, Nashville, TN 37232-6600.
    Affiliations
    From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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  • Ana M.D. Carneiro
    Correspondence
    To whom correspondence may be addressed: Dept. of Pharmacology, Vanderbilt University Medical Center, 461 Preston Research Bldg., 23rd Ave. South at Pierce, Nashville, TN 37232-6600.
    Affiliations
    From the Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grant MH090256 (to A. M. D. C.); Clinical and Translational Science Awards UL1TR000445, KL2TR000446, and TL1TR000447 (to Vanderbilt); and Molecular Libraries Probe Production Centers Network Grant U54MH084659 (to H. E. H.) and by American Heart Association Predoctoral Fellowship Award 14PRE19640007 (to K. H. O.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:July 15, 2016DOI:https://doi.org/10.1074/jbc.M116.736983
      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-([35S]thio)triphosphate and [3H]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.

      Introduction

      In the periphery, serotonin (5-HT)
      The abbreviations used are: 5-HT, 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid; SERT, serotonin transporter; SSRI, selective serotonin reuptake inhibitor; Cit, citalopram; 5-HT2AR, 5-HT2A receptor; SERT+/+, wild-type mice; SERT−/−, serotonin transporter knock-out mice; DOI, 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane; GTPγS, guanosine 5′-3-O-(thio)triphosphate; ANOVA, analysis of variance.
      is produced by enterochromaffin cells in the gastrointestinal tract, released into the plasma, and quickly taken up by platelets via the plasma membrane serotonin transporter (SERT). Following uptake, 5-HT is stored in dense granules by the actions of the vesicular monoamine transporter 2 (
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      Deletion of the serotonin transporter in rats disturbs serotonin homeostasis without impairing liver regeneration.
      ,
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      Serotonin uptake is largely mediated by platelets versus lymphocytes in peripheral blood cells.
      • Bismuth-Evenzal Y.
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      • Weizman A.
      • Rehavi M.
      Decreased serotonin content and reduced agonist-induced aggregation in platelets of patients chronically medicated with SSRI drugs.
      ). Chronic inhibition of SERT through selective serotonin reuptake inhibitors (SSRIs) (e.g. citalopram and paroxetine) leads to dramatically reduced platelet 5-HT granule content (
      • Carneiro A.M.
      • Cook E.H.
      • Murphy D.L.
      • Blakely R.D.
      Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans.
      ,
      • Abdelmalik N.
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      • Schene A.H.
      • Kamphuisen P.W.
      Effect of the selective serotonin reuptake inhibitor paroxetine on platelet function is modified by a SLC6A4 serotonin transporter polymorphism.
      ), altering peripheral 5-HT homeostasis and potentially modifying multiple physiological processes including hemostasis (
      • Namkung J.
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      Peripheral serotonin: a new player in systemic energy homeostasis.
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      The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats.
      ,
      • Blazevic S.
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      Molecular background and physiological consequences of altered peripheral serotonin homeostasis in adult rats perinatally treated with tranylcypromine.
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      • Côté F.
      Life without peripheral serotonin: insights from tryptophan hydroxylase 1 knockout mice reveal the existence of paracrine/autocrine serotonergic networks.
      ). Clinically, increased bleeding risk has been observed in patients taking SSRIs, and platelet aggregation is disrupted (
      • Carneiro A.M.
      • Cook E.H.
      • Murphy D.L.
      • Blakely R.D.
      Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans.
      ,
      • Tseng Y.-L.
      • Chiang M.-L.
      • Huang T.-F.
      • Su K.-P.
      • Lane H.-Y.
      • Lai Y.-C.
      A selective serotonin reuptake inhibitor, citalopram, inhibits collagen-induced platelet aggregation and activation.
      ). Here, we have characterized a similar effect in two distinct mouse models of lost SERT function, suggesting that sustained loss of SERT function influences hemostasis.
      Platelet dense granules contain 5-HT along with other platelet agonists including adenosine diphosphate (ADP), thromboxane (TXA2), and histamine. Appropriate platelet activation depends on the timely release of these factors (
      • Bismuth-Evenzal Y.
      • Gonopolsky Y.
      • Gurwitz D.
      • Iancu I.
      • Weizman A.
      • Rehavi M.
      Decreased serotonin content and reduced agonist-induced aggregation in platelets of patients chronically medicated with SSRI drugs.
      ,
      • Carneiro A.M.
      • Cook E.H.
      • Murphy D.L.
      • Blakely R.D.
      Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans.
      ,
      • Cerrito F.
      • Lazzaro M.P.
      • Gaudio E.
      • Arminio P.
      • Aloisi G.
      5HT2-receptors and serotonin release: their role in human platelet aggregation.
      ). Platelet aggregation is crucial early in thrombus formation (
      • Bismuth-Evenzal Y.
      • Gonopolsky Y.
      • Gurwitz D.
      • Iancu I.
      • Weizman A.
      • Rehavi M.
      Decreased serotonin content and reduced agonist-induced aggregation in platelets of patients chronically medicated with SSRI drugs.
      ,
      • Carneiro A.M.
      • Cook E.H.
      • Murphy D.L.
      • Blakely R.D.
      Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans.
      ,
      • Thompson N.T.
      • Scrutton M.C.
      • Wallis R.B.
      Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration.
      ). Aggregation, which is the bridging of platelet-platelet contacts, requires a conformational alteration in the glycoprotein αIIbβ3, leading to its activation and fibrinogen binding. 5-HT has been shown to enhance aggregation in a 5-HT2A receptor (5-HT2AR)-dependent manner (
      • Bismuth-Evenzal Y.
      • Gonopolsky Y.
      • Gurwitz D.
      • Iancu I.
      • Weizman A.
      • Rehavi M.
      Decreased serotonin content and reduced agonist-induced aggregation in platelets of patients chronically medicated with SSRI drugs.
      ,
      • Mercado C.P.
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      • Rusch N.J.
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      • Kilic F.
      A serotonin-induced N-glycan switch regulates platelet aggregation.
      • Lin O.A.
      • Karim Z.A.
      • Vemana H.P.
      • Espinosa E.V.
      • Khasawneh F.T.
      The antidepressant 5-HT2A receptor antagonists pizotifen and cyproheptadine inhibit serotonin-enhanced platelet function.
      ,
      • Adams J.W.
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      • Connolly D.T.
      APD791, 3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a novel 5-hydroxytryptamine 2A receptor antagonist: pharmacological profile, pharmacokinetics, platelet activity and vascular biology.
      • Przyklenk K.
      • Frelinger 3rd, A.L.
      • Linden M.D.
      • Whittaker P.
      • Li Y.
      • Barnard M.R.
      • Adams J.
      • Morgan M.
      • Al-Shamma H.
      • Michelson A.D.
      Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.
      ). The 5-HT2AR is the only serotonergic receptor found on platelets and potentiates platelet responses to weak agonists like ADP (
      • Li N.
      • Wallén N.H.
      • Ladjevardi M.
      • Hjemdahl P.
      Effects of serotonin on platelet activation in whole blood.
      ). Subthreshold concentrations of two different platelet agonists can exert a synergistic effect on platelet activation. One example includes dual ADP and 5-HT activation leading to increases in cytosolic [Ca2+] (
      • Thompson N.T.
      • Scrutton M.C.
      • Wallis R.B.
      Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration.
      ). However, the role of 5-HT during in vivo hemostasis remains unclear, particularly in the context of chronic SERT inhibition.
      To elucidate the underlying mechanisms of SSRI effects on platelet aggregation, a better understanding of acute versus chronic inhibition of SERT function during platelet activation is required. Acute and chronic blockage of SERT function results in distinct scenarios regarding the effects on 5-HT homeostasis. Acute inhibition of SERT blocks the amount of 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. Some have shown that acute SERT-mediated 5-HT uptake modulates platelet function (
      • Walther D.J.
      • Peter J.-U.
      • Winter S.
      • Höltje M.
      • Paulmann N.
      • Grohmann M.
      • Vowinckel J.
      • Alamo-Bethencourt V.
      • Wilhelm C.S.
      • Ahnert-Hilger G.
      • Bader M.
      Serotonylation of small GTPases is a signal transduction pathway that triggers platelet α-granule release.
      ,
      • Hummerich R.
      • Schloss P.
      Serotonin—more than a neurotransmitter: transglutaminase-mediated serotonylation of C6 glioma cells and fibronectin.
      ) in part through the transamination of small GTPase proteins important for platelet α-granule exocytosis. However, the effects of chronic loss of SERT function on platelet activation remain unresolved.
      In this study, we investigated the mechanisms by which chronic inhibition of SERT alters hemostatic function using two independent models (SERT−/− 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. Indeed, we found that mice lacking SERT function display a bleeding phenotype that can be rescued by the addition of wild-type platelets. Furthermore, we found that ADP-mediated αIIbβ3 activation was reduced in SERT−/− and citalopram-treated platelets due to loss of 5-HT2AR signaling and surface expression.

      Results

      Decreased 5-HT Content in Platelets Isolated from 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 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 (
      • Malyszko J.
      • Urano T.
      • Knofler R.
      • Taminato A.
      • Yoshimi T.
      • Takada Y.
      • Takada A.
      Daily variations of platelet aggregation in relation to blood and plasma serotonin in diabetes.
      ). We used high performance liquid chromatography (HPLC) to measure whole blood levels of 5-HT and its major metabolite 5-hydroxyindoleacetic acid (5-HIAA). Whole blood from wild-type mice undergoing citalopram (Cit) treatment showed reduced 5-HT levels as compared with water-treated controls (Fig. 1A). No significant difference in 5-HIAA levels was found between water- and Cit-treated samples (Fig. 1B). To determine whether citalopram treatment alters hemostasis, we performed a tail bleed assay. We found that citalopram-treated mice exhibited a significantly longer tail bleed time as compared with water-treated controls (Fig. 1C). These findings indicate a phenotypic association between reduced 5-HT levels and increased bleeding time in mice treated with SSRIs.
      Figure thumbnail gr1
      FIGURE 1.Decreased 5-HT content in SERT−/− and citalopram-treated mice associated with bleeding phenotype. 5-HT and 5-HIAA were measured using HPLC from whole blood samples in both citalopram-treated platelets (water, n = 7; Cit, n = 8; A and B) and SERT−/− mice (SERT+/+, n = 11; SERT−/−, n = 14; D and E). Both citalopram-treated (p = 0.0003 (***), Mann-Whitney test) and SERT−/− mice (p < 0.0001 (****), Mann-Whitney test) have a significant reduction in 5-HT levels but no significant change in 5-HIAA. Tail bleed time (C and F) was also performed in citalopram-treated (water, n = 6; Cit, n = 6) and SERT−/− mice (SERT+/+, n = 11; SERT−/−, n = 8). Both citalopram-treated mice (p = 0.0087 (**), Mann-Whitney test) and SERT−/− mice (p = 0.0002 (***), Mann-Whitney test) show increased tail bleed time as compared with controls. Data are shown with median, range, and individual points. Error bars represent the range of the data set.

      Reduced 5-HT Whole Blood Levels and Increased Tail Bleed Times in SERT−/−

      We observed very little measurable 5-HT in SERT−/− platelets (Fig. 1D). Similar to whole blood samples isolated from citalopram-treated mice, there was no significant difference in 5-HIAA between SERT+/+ and SERT−/− mice (Fig. 1E). We found that bleeding time was significantly increased in SERT−/− mice as compared with SERT+/+ (Fig. 1F). These data show that SERT function modulates whole blood 5-HT levels and that SERT inhibition is associated with increased bleeding time.

      Bleeding in SERT−/− Mice Is Rescued by Transfusion of SERT+/+ Platelets

      As 5-HT potentiates platelet activation and previous studies have shown that SERT function alters platelet aggregation (
      • Carneiro A.M.
      • Cook E.H.
      • Murphy D.L.
      • Blakely R.D.
      Interactions between integrin αIIbβ3 and the serotonin transporter regulate serotonin transport and platelet aggregation in mice and humans.
      ), we investigated whether the SERT−/− bleeding phenotype resulted from platelet functional defects. We first measured the number of platelets in SERT−/− and SERT+/+ whole blood samples. No significant difference in the number of platelets was observed between genotypes (Fig. 2A). To determine whether addition of SERT+/+ platelets rescued the SERT−/− bleeding phenotype, mice were transfused with gel-filtered platelets resuspended in saline via jugular vein injection. A tail bleed was performed 3 min after transfusion of SERT+/+ platelets to SERT+/+ and SERT−/− mice (Fig. 2B). In control experiments, saline-transfused SERT−/− mice showed a significantly longer tail bleed time as compared with SERT+/+ mice. We found that addition of gel-purified platelets (2 × 107 in 200 μl of saline) to SERT+/+ mice did not significantly alter tail bleed time. The difference in SERT+/+ and SERT−/− bleeding time was abolished following platelet transfusion, indicating that hemostasis was rescued in SERT−/− mice. These findings support the conclusion that the bleeding phenotype of the SERT−/− mice results from a defect in platelet function.
      Figure thumbnail gr2
      FIGURE 2.Bleeding phenotype can be rescued with transfusion of wild-type platelets. A, platelet counts were determined in SERT+/+ (n = 14) and SERT−/− mice (n = 6). There was no significant difference in the number of platelets (p = 0.8890, Mann-Whitney test). B, tail bleed time was determined 3 min after transfusion of wild-type mouse platelets and was measured in SERT+/+ and SERT−/− mice with the addition of either saline (SERT+/+, n = 5; SERT−/−, n = 6) or platelets (SERT+/+, n = 6; SERT−/−, n = 6). Tail bleed time in saline-transfused SERT−/− was significantly increased compared with SERT+/+ (p = 0.0039 (***)) and was rescued to SERT+/+ bleeding time following platelet transfusion (saline-infused SERT−/− versus platelet-infused SERT−/−, p = 0.002 (##); saline-infused SERT+/+ versus platelet-infused SERT−/−, p = 0.9893; saline-infused SERT+/+ versus platelet-infused SERT+/+, p = 0.9837; Tukey's multiple comparison test following two-way ANOVA). Data are shown with median, range, and individual points. Error bars represent the range of the data set.

      ADP-mediated αIIbβ3 Activation Is Reduced in SERT−/− Platelets

      SERT−/− Platelets Respond Normally to Thrombin

      Gel-filtered platelets from SERT+/+ and SERT−/− mice exposed to 10 and 200 nm thrombin had similar αIIbβ3 activation as measured by JON/A binding (Fig. 3A). Following thrombin stimulation, there were no significant differences between the SERT−/− and SERT+/+ platelet P-selectin expression (CD62p binding) following post-test analysis (Fig. 3B). These findings reveal that platelet activation with thrombin is not altered in SERT−/− platelets.
      Figure thumbnail gr3
      FIGURE 3.Platelet ADP-mediated αIIbβ3 activation is reduced in SERT−/− mice. A, JON/A binding in SERT−/− (n = 6) and SERT+/+ (n = 6) mice with 10 and 200 nm thrombin shows no difference based on genotype (Tukey's post-test following two-way ANOVA: thrombin, p < 0.0001). B, P-selectin binding did account for some variation (two-way ANOVA: thrombin, p < 0.0001; genotype, p = 0.032) but did not show any difference following post-test (Tukey's multiple comparison test). C, ADP-mediated JON/A binding is significantly reduced in the SERT−/− platelets at 10 μm ADP (10 μm ADP-treated SERT+/+ versus 10 μm ADP-treated SERT−/−, p = 0.0354 (*)) but not at 1 μm ADP (two-way ANOVA revealed an overall effect of ADP (p = 0.0001) and an interaction effect (p = 0.026); additionally, 1 μm ADP-treated SERT+/+ versus 1 μm ADP-treated SERT−/− showed a significant effect (p = 0.999) using Tukey's multiple comparison test). D, there is no difference in P-selectin expression in SERT−/− and SERT+/+ mice (two-way ANOVA: ADP, p = 0.0473; Tukey's multiple comparison test, not significant). Data are shown with median, range, and individual points. Error bars represent the range of the data set. gMFI, geometric mean fluorescence intensity; PE, phycoerythrin.

      SERT−/− Platelets Have Reduced ADP-mediated αIIbβ3 Activation

      We next tested ADP platelet activation in SERT−/− platelets. We found that 10 μm, but not 1 μm, ADP-mediated αIIbβ3 activation is significantly reduced in the SERT−/− platelets (Fig. 3C). No difference in P-selectin expression was observed in SERT−/− or SERT+/+ platelets (Fig. 3D). These data demonstrate that loss of SERT function leads to a deficiency in the ADP-mediated inside-out signaling that triggers activation of αIIbβ3.

      5-HT2AR Stimulation Enhances ADP-mediated αIIbβ3 Activation

      To examine the role of 5-HT in ADP-mediated platelet activation, we distinguished the role of two plasma membrane proteins that are responsive to 5-HT in platelets: SERT and the 5-HT2AR. We performed a dose-response curve with citalopram and paroxetine, two structurally distinct SSRIs, and found that they block intact 5-[3H]HT platelet uptake with an IC50 of 1.5 × 10−11 and 1.3 × 10−11, respectively (Fig. 4A). Platelet activation with 1 and 10 μm ADP was examined with the addition of either 5-HT2AR antagonists (ketanserin and methysergide) or SERT inhibitors (citalopram and paroxetine) 5 min before ADP activation. Both ketanserin and methysergide treatment significantly reduced ADP-mediated activation at 1 μm (Fig. 4B). Acute inhibition of 5-HT uptake by SERT with citalopram (10 nm) or paroxetine (10 nm) had no effect on ADP-mediated αIIbβ3 activation despite effectively blocking SERT-mediated 5-HT uptake. These data reveal a role for 5-HT2AR activation, but not 5-HT uptake, in ADP-mediated αIIbβ3 inside-out activation.
      Figure thumbnail gr4
      FIGURE 4.ADP requires 5-HT2A receptor activation for ADP/5-HT-mediated αIIbβ3 synergy, which is disrupted in SERT−/− platelets. A, isolated platelet uptake of 5-[3H]HT blocked with either citalopram or paroxetine (log(inhibition) versus normalized response-variable slope: citalopram robust sum of squares, 20.58; paroxetine robust sum of squares, 22.92). B, JON/A binding following 15-min ADP activation was measured using geometric mean fluorescence intensity (gMFI) following a 5-min preincubation with either 5-HT2A receptor antagonist ketanserin or methysergide or selective serotonin reuptake inhibitor citalopram or paroxetine on the same platelet preparation (n = 6). Both ketanserin and methysergide treatment significantly reduced ADP αIIbβ3 activation at both 1 (ketanserin, p = 0.0409 (*); methysergide, p = 0.0041 (**)) and 10 μm (ketanserin, p = 0.0187 (*); methysergide, p < 0.0001 (****); Dunnett's test with two-way repeated measures ANOVA: interaction, p = 0.0103; ADP, p < 0.0001; inhibitors, p < 0.0001; subjects (matching), p < 0.0001). Non-parallel experiments examined JON/A binding following incubation with 5-HT alone (C), ADP 1 μm + 5-HT (p < 0.0001 (***)) (D), or ADP/5-HT following a 5-min incubation with ketanserin (Sidak's test following two-way ANOVA) (E). Data are shown with median, range, and individual points. Error bars represent the range of the data set.

      SERT−/− Platelets Have Reduced ADP/5-HT Synergy

      To explore the role of 5-HT2AR signaling in the context of the SERT−/− model, we examined JON/A binding to activated αIIbβ3 in the context of simultaneous 5-HT and ADP receptor stimulation. 5-HT alone was unable to elicit activation of the αIIbβ3 integrin at either 1 or 10 μm concentration in either SERT+/+ or SERT−/− platelets (Fig. 4C). However, using a submaximal concentration of ADP (1 μm), we observed that 10 μm 5-HT potentiated the ADP-mediated activation of αIIbβ3 in SERT+/+ platelets. 5-HT was unable to potentiate ADP-mediated platelet activation in SERT−/− platelets (Fig. 4D). In a separate experiment, we found that incubation with ketanserin prevented the 5-HT-dependent potentiation of αIIbβ3 activation in SERT+/+ platelets (Fig. 4E). These results demonstrate that submaximal stimulation of ADP receptors is potentiated by co-stimulation of 5-HT2AR, which synergizes to mediate αIIbβ3 inside-out activation. Additionally, these data indicate a defect in 5-HT2AR signaling within SERT−/− platelets.

      SERT−/− Platelets Have Reduced 5-HT2AR Surface Expression

      ADP Levels and ADP Receptor Expression Are Retained in SERT−/− Platelets

      We first explored whether the loss of ADP-mediated αIIbβ3 activation was due to a deficit in ADP-dependent signaling. We examined ADP receptor (P2Y1 and P2Y12) expression levels via [3H]ADP binding that was competitively blocked with ADP receptor-specific antagonists. This assay was optimized in SERT+/+ platelet membrane preparations and shown as percentage of total [3H]ADP binding blocked in the presence of 2,2′-pyridylisatogen tosylate, a P2Y antagonist (Fig. 5A). Competitive inhibition with MRS2179 (P2Y1-specific antagonist) and ticagrelor (P2Y12-specific antagonist) was used to determine relative receptor binding in SERT+/+ and SERT−/− platelet membrane preparations (Fig. 5, C and D). P2Y1 accounted for ∼46.42% of [3H]ADP binding, whereas P2Y12 accounted for ∼55.89% of total [3H]ADP binding. Inhibition at 1 μm for both MRS2179 and ticagrelor was used to determine percentage of [3H]ADP binding to P2Y1 in platelet membranes. We found no statistically significant difference in either P2Y12 or P2Y1 binding between SERT+/+ and SERT−/− membrane preparations. Additionally, we measured whole blood ADP levels using an ELISA and observed no difference between SERT+/+ and SERT−/− preparations (Fig. 5E). These data demonstrate that the ADP whole blood levels and receptor numbers are not different in SERT−/− platelets.
      Figure thumbnail gr5
      FIGURE 5.SERT−/− platelets have reduced 5-HT2A receptor surface expression but no change in P2Y1 and P2Y12. A and B, competitive inhibition with 2,2′-pyridylisatogen tosylate (PIT) (dual P2Y1 and P2Y12 antagonist; IC50 = 7.80 × 10−5, 83.71%), MRS2179 (P2Y1-specific antagonist; IC50 = 2.08 × 10−5, 46.42%), and ticagrelor (Ticag.) (P2Y12-specific antagonist; IC50 = 1.25 × 10−7, 55.89%) was used with [3H]ADP to determine relative binding of receptors. Single point inhibition was used to determine percentage [3H]ADP binding for P2Y12 (n = 9 SERT+/+; n = 7 SERT−/−; p = 0.4592, Mann-Whitney test) (C) and P2Y1 receptors (n = 9 SERT+/+; n = 7 SERT−/−; p = 0.9646, Mann-Whitney test) (D) in SERT+/+ and SERT−/− platelets. Data are shown with median, range, and individual points. E, ADP whole blood levels were determined by ELISA (n = 6; p = 0.675, Mann-Whitney test). F, [35S]GTPγS incorporation was used to measure 5-HT2A receptor-specific G-protein activation in purified platelet membranes (n = 8; p = 0.0002 (***), Mann-Whitney test). G, 5-HT2AR specific saturation curve with [3H]ketanserin is shown for both SERT+/+ and SERT−/− platelets. H, calculated Bmax values are significantly reduced in SERT−/− platelets (n = 5 SERT+/+; n = 6 SERT−/−; p = 0.0173 (*), Mann-Whitney test). There was no significant difference in Kd between SERT+/+ and SERT−/− [3H]ketanserin binding (SERT+/+ median, 7.32 nm; SERT−/− median, 6.71 nm; p = 0.134, Mann-Whitney test). Error bars represent the range of the data set.

      5-HT2AR G-protein Activation and Surface Expression Levels Are Reduced in SERT−/− Platelets

      Based on our findings, the 5-HT2AR plays an important role in ADP-mediated αIIbβ3 activation, and this effect is reduced in SERT−/− platelets. Therefore, we purified platelet membranes and measured [35S]GTPγS incorporation to 5-HT2AR following activation with 1-[2,5-dimethoxy-4-iodophenyl]-2-aminopropane (DOI), a 5-HT2AR-specific agonist. We observed a significant reduction in [35S]GTPγS incorporation to SERT−/− membrane preparations as compared with SERT+/+ (Fig. 5F; n = 8; p = 0.0002, Mann-Whitney test). To determine whether this reduction in G-protein activation was caused by reduced 5-HT2AR surface expression, we used radiolabeled [3H]ketanserin to measure surface levels of the 5-HT2AR on intact platelets. A saturation curve was performed to examine the number of surface receptors (Bmax) of 5-HT2AR (Fig. 5G). SERT−/− platelets showed significantly reduced Bmax for 5-HT2AR surface levels as compared with those observed in SERT+/+ platelets (Fig. 5H). There was no significant difference in Kd between SERT+/+ and SERT−/− [3H]ketanserin binding (SERT+/+ median, 7.32 nm; SERT−/− median, 6.71 nm; p = 0.134, Mann-Whitney test). These results show reduced 5-HT2AR signaling in SERT−/− platelets, likely resulting from reduced 5-HT2AR surface expression.

      Citalopram-treated Platelets Recapitulate the SERT−/− Platelet Phenotype: Citalopram-treated Platelets Have Reduced ADP-mediated αIIbβ3 Activation and Reduced 5-HT2AR Surface Expression

      To determine whether we could recapitulate the loss of 5-HT2AR surface expression using an alternative model of lost SERT function, we treated mice chronically with citalopram for 6 days. Similar to SERT−/− platelets, citalopram-treated platelets showed reduced ADP-dependent αIIbβ3 activation compared with water-treated controls (Fig. 6A). In control water-treated mouse platelets, αIIbβ3 activation by ADP was enhanced by 5-HT, whereas 5-HT potentiation of ADP signaling was absent in citalopram-treated mouse platelets (Fig. 6B). Furthermore, 5-HT2AR levels were reduced in platelets isolated from citalopram-treated mice (Fig. 6C). These findings establish that 6-day treatment with SSRI is sufficient to reduce platelet expression of 5-HT2AR and decrease the capacity of platelets to be synergistically activated by ADP and 5-HT. Additionally, these findings demonstrate that the loss of 5-HT2AR surface expression and platelet function in SERT−/− mice is not due to loss of the SERT protein but rather results from sustained loss of SERT function. Finally, to test whether premature exposure to 5-HT could alter ADP-mediated αIIbβ3 activation, wild-type platelets were incubated with serotonin (10 μm) for 15 min, washed, and resuspended. They were then activated with 10 μm ADP, and JON/A binding was measured (Fig. 6D). We found that platelets pretreated with 5-HT for 15 min lost ADP-mediated αIIbβ3 activation similarly to both SERT−/− and citalopram-treated platelets. These results suggest that the preactivation or possible desensitization of the 5-HT2AR reduces platelet ADP-mediated αIIbβ3 activation.
      Figure thumbnail gr6
      FIGURE 6.Citalopram-treated mice have reduced ADP/5-HT synergy and reduced 5-HT2A surface expression. A, citalopram ADP-mediated JON/A binding was probed (n = 6; two-way ANOVA followed by Tukey's post-test: PBS-stimulated, water-treated mouse platelets versus 10 μm ADP-stimulated, water-treated mouse platelets, p < 0.0001; PBS-stimulated, citalopram-treated mouse platelets versus 10 μM ADP-stimulated, citalopram-treated mouse platelets, p = 0.025; 10 μm ADP-stimulated, water-treated mouse platelets versus 10 μm ADP-stimulated, citalopram-treated mouse platelets, p = 0.0056 (**)). B, ADP/5-HT synergy toward JON/A binding was also probed in citalopram-treated mice (n = 6; two-way ANOVA followed by Tukey's post-test: PBS-stimulated, water-treated mouse platelets versus PBS-stimulated, citalopram-treated mouse platelets, p = 0.5501; 1 μm ADP- and 1 μM 5-HT-stimulated, water-treated mouse platelets versus 1 μm ADP- and 1 μm 5-HT-stimulated, citalopram-treated mouse platelets, p = 0.0452 (*); 1 μm ADP- and 10 μm 5-HT-stimulated, water-treated mouse platelets versus 1 μm ADP- and 10μm 5-HT-stimulated, citalopram-treated mouse platelets, p = 0.009 (**)). C, surface 5-HT2A receptor levels were also determined using 20 nm [3H]ketanserin binding on intact platelets (water, n = 4; Cit treatment, n = 6; p = 0.0095 (**), Mann-Whitney test). D, wild-type platelets that were pretreated (Pre-t) with 5-HT for 15 min, washed, and activated with 10 μm ADP show decreased JON/A binding. Data are shown with median, range, and individual points.

      Discussion

      Our research has demonstrated how loss of SERT function leads to platelet dysfunction, providing mechanistic insight into the increased bleeding times previously observed in patients taking SSRIs (
      • Bismuth-Evenzal Y.
      • Gonopolsky Y.
      • Gurwitz D.
      • Iancu I.
      • Weizman A.
      • Rehavi M.
      Decreased serotonin content and reduced agonist-induced aggregation in platelets of patients chronically medicated with SSRI drugs.
      ,
      • Andrade C.
      • Sandarsh S.
      • Chethan K.B.
      • Nagesh K.S.
      Serotonin reuptake inhibitor antidepressants and abnormal bleeding: a review for clinicians and a reconsideration of mechanisms.
      ). We characterized a bleeding phenotype in two independent models of lost SERT function, genetic and pharmacologic. This effect was rescued by addition of purified platelets with intact SERT function, indicating that the actions of SSRIs are mediated primarily via alteration of platelet function. Sustained loss of SERT function reduces 5-HT2AR surface expression, leading to reduced ADP-mediated αIIbβ3 activation. Therefore, our findings support a novel mechanism for the reduced aggregation in SSRI-treated patients: reduced 5-HT2AR surface expression and defective ADP-mediated αIIbβ3 activation.
      There are three possibilities by which SSRIs may mediate their effects on platelets: 1) acute blockage of SERT function and 5-HT uptake, 2) depletion of granule 5-HT and loss of 5-HT secretion during activation, and 3) increased extracellular 5-HT levels. Many studies concluding that acute uptake of 5-HT contributes to platelet function used SSRIs as tool compounds to acutely inhibit 5-HT uptake. However, these studies utilized SSRIs at high concentrations (>10 μm) that are known to exert off-target effects (e.g. the σ1 receptor) (
      • Tseng Y.-L.
      • Chiang M.-L.
      • Huang T.-F.
      • Su K.-P.
      • Lane H.-Y.
      • Lai Y.-C.
      A selective serotonin reuptake inhibitor, citalopram, inhibits collagen-induced platelet aggregation and activation.
      ,
      • Maurice T.
      • Su T.-P.
      The pharmacology of σ-1 receptors.
      ). Comparatively, our study uses concentrations of SSRIs that block platelet-mediated SERT uptake but are far below the reported concentrations resulting in off-target effects (10 nm; Fig. 4). Here, we observed no change in ADP-mediated αIIbβ3 activation following acute SSRI treatment, suggesting that acute 5-HT uptake is not required for inside-out activation of αIIbβ3 by weak agonists. However, we observed reduced ADP-mediated αIIbβ3 activation following chronic SSRI treatment.
      Others have suggested that acute uptake of 5-HT by SERT leads to a receptor-independent signaling pathway via the post-translational modification of intracellular proteins (
      • Walther D.J.
      • Peter J.-U.
      • Winter S.
      • Höltje M.
      • Paulmann N.
      • Grohmann M.
      • Vowinckel J.
      • Alamo-Bethencourt V.
      • Wilhelm C.S.
      • Ahnert-Hilger G.
      • Bader M.
      Serotonylation of small GTPases is a signal transduction pathway that triggers platelet α-granule release.
      ). This process, known as serotonylation, involves the covalent attachment of 5-HT molecules to proteins mediated by the enzyme transglutaminase, resulting in transamination of small GTPases important for platelet α-granule secretion (
      • Walther D.J.
      • Peter J.-U.
      • Winter S.
      • Höltje M.
      • Paulmann N.
      • Grohmann M.
      • Vowinckel J.
      • Alamo-Bethencourt V.
      • Wilhelm C.S.
      • Ahnert-Hilger G.
      • Bader M.
      Serotonylation of small GTPases is a signal transduction pathway that triggers platelet α-granule release.
      ,
      • Ahmed B.A.
      • Jeffus B.C.
      • Bukhari S.I.
      • Harney J.T.
      • Unal R.
      • Lupashin V.V.
      • van der Sluijs P.
      • Kilic F.
      Serotonin transamidates Rab4 and facilitates its binding to the C terminus of serotonin transporter.
      ). In our experiments, we demonstrated no difference in P-selectin antibody as a measure for α-granule exocytosis in SERT−/− platelet following thrombin activation (Fig. 3B). These data would suggest that α-granule exocytosis is intact following the chronic loss of SERT function. Instead, we observed that chronic loss of SERT function led to reduced 5-HT2AR signaling and reduced ADP-mediated αIIbβ3 activation, which is necessary for proper aggregation in vivo.
      In conjunction with depleting platelet granule 5-HT levels, one would expect that chronic SERT inhibition would increase plasma concentrations of 5-HT as suggested by the effects of SSRIs in the CNS (
      • Gartside S.E.
      • Umbers V.
      • Hajós M.
      • Sharp T.
      Interaction between a selective 5-HT1A receptor antagonist and an SSRI in vivo: effects on 5-HT cell firing and extracellular 5-HT.
      ,
      • Artigas F.
      • Romero L.
      • de Montigny C.
      • Blier P.
      Acceleration of the effect of selected antidepressant drugs in major depression by 5-HT1A antagonists.
      ). However, because 5-HT2AR stimulation by 5-HT alone does not lead to platelet activation, loss of SERT function likely leads to local increases in plasma 5-HT levels within the portal vein and indirectly triggers internalization of the 5-HT2AR. It has been demonstrated that 5-HT induces internalization of 5-HT2AR in a β-arrestin-dependent manner within 30 min of initial exposure (
      • Schmid C.L.
      • Raehal K.M.
      • Bohn L.M.
      Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo.
      ,
      • Bohn L.M.
      • Schmid C.L.
      Serotonin receptor signaling and regulation via β-arrestins.
      ). 5-HT2AR desensitization has been shown in the brain following prolonged exposure to elevated levels of serotonin in citalopram-treated mice (
      • Schaff M.
      • Receveur N.
      • Bourdon C.
      • Ohlmann P.
      • Lanza F.
      • Gachet C.
      • Mangin P.H.
      β-Arrestin-1 participates in thrombosis and regulates integrin αIIbβ3 signalling without affecting P2Y receptors desensitisation and function.
      ,
      • Yamauchi M.
      • Miyara T.
      • Matsushima T.
      • Imanishi T.
      Desensitization of 5-HT2A receptor function by chronic administration of selective serotonin reuptake inhibitors.
      ). However, it has yet to be validated whether extracellular plasma 5-HT levels increase following SSRI treatment. Evaluating changes in plasma (i.e. extracellular) 5-HT levels as compared with platelet (i.e. intracellular) 5-HT levels following SSRI treatment has been challenging. Measuring plasma 5-HT levels has proven difficult due to very low concentration by extraordinarily efficient liver metabolism as well as the free diffusion of 5-HT out of circulation (
      • Moore T.C.
      • Eiseman B.
      Serotonin metabolism in the isolated perfused canine liver.
      ,
      • Anderson G.M.
      • Barr C.S.
      • Lindell S.
      • Durham A.C.
      • Shifrovich I.
      • Higley J.D.
      Time course of the effects of the serotonin-selective reuptake inhibitor sertraline on central and peripheral serotonin neurochemistry in the rhesus monkey.
      • Anderson G.M.
      • Cook E.H.
      Commentary on “platelet studies in autism spectrum disorder patients and first-degree relatives.”.
      ). Furthermore, levels of plasma 5-HT can vary greatly, possibly due to contamination by platelet granule release during plasma purification (
      • Brenner B.
      • Harney J.T.
      • Ahmed B.A.
      • Jeffus B.C.
      • Unal R.
      • Mehta J.L.
      • Kilic F.
      Plasma serotonin levels and the platelet serotonin transporter.
      ,
      • Matondo R.B.
      • Punt C.
      • Homberg J.
      • Toussaint M.J.
      • Kisjes R.
      • Korporaal S.J.
      • Akkerman J.W.
      • Cuppen E.
      • de Bruin A.
      Deletion of the serotonin transporter in rats disturbs serotonin homeostasis without impairing liver regeneration.
      ,
      • Anderson G.M.
      • Cook E.H.
      Commentary on “platelet studies in autism spectrum disorder patients and first-degree relatives.”.
      ,
      • Lau W.K.
      • Chan-Yeung M.M.
      • Yip B.H.
      • Cheung A.H.
      • Ip M.S.
      • Mak J.C.
      • COPD Study Group of the Hong Kong Thoracic Society
      The role of circulating serotonin in the development of chronic obstructive pulmonary disease.
      ). Reported plasma 5-HT levels fluctuate between investigators and preparations, but our data clearly demonstrate that 5-HT homeostasis is altered by loss of SERT function (
      • Jernej B.
      • Vladić A.
      • Cicin-Sain L.
      • Hranilović D.
      • Banović M.
      • Balija M.
      • Bilić E.
      • Sucić Z.
      • Vukadin S.
      • Grgicević D.
      Platelet serotonin measures in migraine.
      ,
      • Singh P.
      • Fletcher T.W.
      • Li Y.
      • Rusch N.J.
      • Kilic F.
      Serotonin uptake rates in platelets from angiotensin II-induced hypertensive mice.
      • Walther D.J.
      • Peter J.U.
      • Bashammakh S.
      • Hörtnagl H.
      • Voits M.
      • Fink H.
      • Bader M.
      Synthesis of serotonin by a second tryptophan hydroxylase isoform.
      ). Furthermore, our data demonstrate that improper exposure of platelets to 5-HT likely desensitizes the receptor, leading to reduced ADP-mediated αIIbβ3 activation (Fig. 6D). Our findings indicate that chronic loss of SERT function reduces 5-HT2AR surface expression and signaling in each of two models of altered 5-HT homeostasis.
      It has previously been established that 5-HT plays a synergistic role with ADP activation during platelet aggregation (
      • Cerrito F.
      • Lazzaro M.P.
      • Gaudio E.
      • Arminio P.
      • Aloisi G.
      5HT2-receptors and serotonin release: their role in human platelet aggregation.
      ,
      • Thompson N.T.
      • Scrutton M.C.
      • Wallis R.B.
      Synergistic responses in human platelets. Comparison between aggregation, secretion and cytosolic Ca2+ concentration.
      ,
      • Berger J.S.
      • Becker R.C.
      • Kuhn C.
      • Helms M.J.
      • Ortel T.L.
      • Williams R.
      Hyperreactive platelet phenotypes: relationship to altered serotonin transporter number, transport kinetics and intrinsic response to adrenergic co-stimulation.
      ,
      • Hirsh-Rokach B.
      • Spectre G.
      • Shai E.
      • Lotan A.
      • Ritter A.
      • Al-Aieshy F.
      • Malmström R.E.
      • Varon D.
      • Alcalai R.
      Differential impact of selective serotonin reuptake inhibitors on platelet response to clopidogrel: a randomized, double-blind, crossover trial.
      ). Here, we demonstrate that 5-HT2AR stimulation potentiates ADP-dependent signaling to activate αIIbβ3 (i.e. JON/A binding), leading to aggregation. Although 5-HT2AR stimulation alone did not influence αIIbβ3-activation, dual stimulation of 5-HT2AR with a submaximal concentration of ADP enhanced JON/A binding. 5-HT2AR stimulation may be required to enhance P2Y1 Gq-mediated signaling and Gi-mediated P2Y12 activation converging on αIIbβ3 inside-out activation. It is well known that the 5-HT2AR can signal through multiple non-G-protein pathways including β-arrestins (
      • Schmid C.L.
      • Raehal K.M.
      • Bohn L.M.
      Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo.
      ,
      • Stahl E.L.
      • Zhou L.
      • Ehlert F.J.
      • Bohn L.M.
      A novel method for analyzing extremely biased agonism at G protein-coupled receptors.
      ,
      • Schmid C.L.
      • Bohn L.M.
      Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a β-arrestin2/Src/Akt signaling complex in vivo.
      ). Therefore, although canonical 5-HT2AR signaling would be redundant relative to ADP-mediated P2Y1 activation, alternative signaling pathways downstream of β-arrestins, such as ERK1/2 and c-Src, could culminate in αIIbβ3 activation (
      • Schmid C.L.
      • Raehal K.M.
      • Bohn L.M.
      Agonist-directed signaling of the serotonin 2A receptor depends on β-arrestin-2 interactions in vivo.
      ,
      • Stahl E.L.
      • Zhou L.
      • Ehlert F.J.
      • Bohn L.M.
      A novel method for analyzing extremely biased agonism at G protein-coupled receptors.
      ,
      • Schmid C.L.
      • Bohn L.M.
      Serotonin, but not N-methyltryptamines, activates the serotonin 2A receptor via a β-arrestin2/Src/Akt signaling complex in vivo.
      • Li Z.
      • Delaney M.K.
      • O'Brien K.A.
      • Du X.
      Signaling during platelet adhesion and activation.
      ). Additionally, 5-HT2AR signaling through arachidonic acid, 2-arachidonylglycerol, calmodulin, or AKT could also synergize with ADP signaling (
      • Quinn J.C.
      • Johnson-Farley N.N.
      • Yoon J.
      • Cowen D.S.
      Activation of extracellular-regulated kinase by 5-hydroxytryptamine2A receptors in PC12 cells is protein kinase C-independent and requires calmodulin and tyrosine kinases.
      • Johnson-Farley N.N.
      • Kertesy S.B.
      • Dubyak G.R.
      • Cowen D.S.
      Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells.
      ,
      • Göoz M.
      • Göoz P.
      • Luttrell L.M.
      • Raymond J.R.
      5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-α-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells.
      • Miller K.J.
      • Mariano C.L.
      • Cruz W.R.
      Serotonin 5HT2A receptor activation inhibits inducible nitric oxide synthase activity in C6 glioma cells.
      ). Further experiments are required to determine what signaling pathways are necessary and sufficient for maximal αIIbβ3 activation via ADP/5-HT synergy.
      Clinically, targeted inhibition of the 5-HT2AR has been efficacious (
      • Przyklenk K.
      • Frelinger 3rd, A.L.
      • Linden M.D.
      • Whittaker P.
      • Li Y.
      • Barnard M.R.
      • Adams J.
      • Morgan M.
      • Al-Shamma H.
      • Michelson A.D.
      Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.
      ). APD791, an inverse agonist of 5-HT2AR, is currently in clinical trials and attenuated recurrent thrombosis irrespective of the time of treatment (
      • Adams J.W.
      • Ramirez J.
      • Shi Y.
      • Thomsen W.
      • Frazer J.
      • Morgan M.
      • Edwards J.E.
      • Chen W.
      • Teegarden B.R.
      • Xiong Y.
      • Al-Shamma H.
      • Behan D.P.
      • Connolly D.T.
      APD791, 3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a novel 5-hydroxytryptamine 2A receptor antagonist: pharmacological profile, pharmacokinetics, platelet activity and vascular biology.
      ,
      • Przyklenk K.
      • Frelinger 3rd, A.L.
      • Linden M.D.
      • Whittaker P.
      • Li Y.
      • Barnard M.R.
      • Adams J.
      • Morgan M.
      • Al-Shamma H.
      • Michelson A.D.
      Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.
      ). No increase in bleeding time in the presence of APD791 was observed as compared with other antiplatelet therapies. Additionally, APD791 was able to block 5-HT-dependent platelet activation over a short time scale (2 h) (
      • Adams J.W.
      • Ramirez J.
      • Shi Y.
      • Thomsen W.
      • Frazer J.
      • Morgan M.
      • Edwards J.E.
      • Chen W.
      • Teegarden B.R.
      • Xiong Y.
      • Al-Shamma H.
      • Behan D.P.
      • Connolly D.T.
      APD791, 3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide, a novel 5-hydroxytryptamine 2A receptor antagonist: pharmacological profile, pharmacokinetics, platelet activity and vascular biology.
      ,
      • Przyklenk K.
      • Frelinger 3rd, A.L.
      • Linden M.D.
      • Whittaker P.
      • Li Y.
      • Barnard M.R.
      • Adams J.
      • Morgan M.
      • Al-Shamma H.
      • Michelson A.D.
      Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis.
      ). Our data provide mechanistic insight into the reduced aggregation previously observed in SSRI-treated patients and support explorations of peripherally restricted, well tolerated SSRIs or 5-HT2AR antagonists as an antiplatelet therapeutic approach. The therapeutic implications of this work suggest a novel approach targeting the 5-HT signaling systems via SERT inhibition, leading to reduced 5-HT2AR-mediated platelet aggregation.

      Author Contributions

      K. H. O. designed, performed, analyzed, and interpreted results and wrote the manuscript. H. E. H. and A. M. D. C. both equally designed, interpreted results, and wrote the manuscript. Scientific interpretation, experiment guidance, and manuscript editing was performed by M. T. D.

      Acknowledgments

      We acknowledge Drs. Matthew Mazalouskas and Michael Dohn for assistance with editing of the manuscript. We also thank Jae Maeng for animal husbandry. Additionally, we thank the undergraduate researchers who participated, including Cecilia Li and Cynthia Mairink.

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