SEROTONIN: A DIFFERENT PLAYER IN HYPERTENSION-ASSOCIATED THROMBOSIS

INTRODUCTION

Serotonin (5-hydroxytryptamine; 5-HT) was discovered in the lining of the gastrointestinal tract in 1935; a detailed history on its discovery and characterization was published several years later.¹ 5-HT is a derivative of the amino acid L-tryptophan, which is synthesized by tryptophan hydroxylase (TPH) with its two isoforms. TPH1 is responsible for the production of 5-HT in peripheral tissues² and TPH2 leads to the synthesis of 5-HT in the central nervous system.³ 5-HT is metabolized in liver and eventually excreted as 5-hydroxyindoleacetic acid. Platelets are likely the biological storage of circulating 5-HT. Platelets do not synthesize 5-HT but acquire it, mainly from the blood, after being secreted by the enterochromaffin cells of the intestine. The free 5-HT level in the blood is tightly regulated by a specific transporter, SERT (SLC6A4), which is expressed on the platelet surface.⁴ SERT removes 5-HT from the blood via a saturable reuptake mechanism.⁴ Once in the cytoplasm, 5-HT is sequestered by the vesicular monoamine transporter type 2 into intracellular dense granules. ^4–6 5-HT concentration in resting platelets is millimolar versus in the blood where it is in the low nanomolar range. ^{5, 6} Although, 5-HT is better known as a neurotransmitter with key roles in a variety of psychiatric diseases, it also has extra-cerebral roles: multifunctional signaling molecule, growth factor, endocrine hormone or paracrine messenger, from fetal to adult life.^{2, 7, 8}

5-HT in platelet aggregation

Platelets are derived from the fragmented cytoplasm of megakaryocytes and enter the circulation in an inactive form. The initial activation of platelets stabilizes them in hemostasis. Further platelet activation enlists more platelets at a fibrin-stabilized hemostatic area to form a thrombus after associating with the endothelium or each other. The role of circulating, free 5-HT in platelet adhesion, aggregation, and thrombus formation has not been resolved completely, but clinical and biochemical findings infer a complex process. An elevation in free 5-HT levels in plasma accelerates the exocytosis of dense and α-granules^{9, 10}; in turn, these secrete 5-HT and other procoagulant molecules that will mediate hemostasis. Supporting these hypotheses is the fact that platelets of 5-HT infused mice, in the absence of cardiovascular problem, show an enhanced aggregation profile; however, when the 5-HT-infused mice were injected with a selective 5-HT reuptake inhibitor (SSRI)^{11, 12} or a 5-HT_2A antagonist^{11, 13} the effect of elevated free 5-HT levels in plasma was reversed and the platelet aggregation profile normalized.^11–14 The importance of the plasma 5-HT level and platelet SERT in the platelet aggregation phenomenon is supported by findings in platelets of mice lacking the gene for TPH1^{11, 14} or the gene for SERT:¹¹ where granular secretion rates as well as the risk of thrombosis are significantly reduced.^{11, 12, 14} Following the interaction with von Willebrand factor and 5-HT_2A receptors, 5-HT-mediated platelet aggregation occurs via 5-HT_2A- or SERT-dependent pathways. ^{11, 12, 15–18}

In a receptor–dependent pathway, 5-HT^{11, 13, 19} initiates G-protein-dependent signaling pathway to mobilize calcium from intracellular stores.^{6, 9, 15, 20–28} Free, calcium activates transglutaminase (TGase) which transamidates cytoplasmic 5-HT to the GTP-GDP hydrolysis domains of the small GTPases.²⁸ In the active, GTP-bound form, small GTPases promotes the exocytosis of the granule.^{12, 14, 28, 29} At this point the contribution of intracellular 5-HT to the SERT–dependent platelet aggregation pathway appears important. Once the free/unbound 5-HT is taken up by SERT in platelet cytosol, it is stored in dense granules. However, upon the saturation of dense granules, free/unbound 5-HT in the platelet cytosol is transamidated on the GTP-GDP hydrolysis domain of the small GTPases converting them to their active GTP-bound form, to enhance α-granule secretion.^{9, 11, 12, 14} Concurrently, the association between Rab4-GTP and SERT tethers the transporter to an intracellular compartment to prevent further rises in cytoplasmic 5-HT.^{12, 28, 29} Additionally, elevated plasma 5-HT activates p21 activating kinase (PAK) which phosphorylates the intermediate filament vimentin.^{30, 31} We reported that under physiological conditions SERT binds to vimentin during internalization from the plasma membrane.³² However, following the phosphorylation of vimentin, the level of SERT on phosphovimentin as well as the internalization rate of SERT from the plasma membrane to intracellular compartments are elevated.³² Based on these findings we propose that plasma 5-HT at high levels leads to abnormalities in the platelet trafficking of SERT, which reduces the density of SERT molecules on the plasma membrane. These events are correlated with the platelet aggregation process. Mechanistically, these finding investigate the link between elevated plasma 5-HT and loss of surface SERT by focusing on the intracellular tethering of SERT by Rab4²⁸ and the 5-HT-mediated phosphorylation of vimentin that promotes SERT internalization³² in platelets (Fig. 1). In supporting our findings on the importance of SERT and SSRI in platelet aggregation process, studies with a different approach, showed that stimulation of 5-HT_2A decreases the adhesive property of platelet by shedding of GPIb from the platelet surface.³³ However, platelet SERT clears 5-HT from plasma which becomes no longer available to stimulate 5-HT_2A stimulation, in the presence of SSRI platelet loses adhesive properties.³³

Figure 1.

(A) High 5-HT leads to abnormalities in the platelet trafficking of SERT, which reduces the density of SERT molecules on the plasma membrane. (B) Based on our findings for the known actions of 5-HT in platelets, we propose that high levels of uptake leads to saturation of dense granules and 5-HT appears in the platelet cytoplasm. VMAT is disabled and cannot remove 5-HT in dense granules anymore. This leads to the serotonylation of small GTPases such as Rab4, Rho and Rac via Ca²⁺-activated TGase. In GTP form, Rab4 binds and prevents the translocation of SERT to the plasma membrane. (C) These processes are involved in platelet activation and aggregation in a two-step process: in the first step elevated 5-HT controls the platelet 5-HT uptake rates via altering the membrane trafficking of plasma membrane SERT which in turn elevates the plasma 5-HT level further; then in the second step the elevated plasma 5-HT level activates 5-HT_2A which accelerates the exocytosis of α-granules. Secretion of prothrombotic molecules from α-granules to the plasma with high levels of 5-HT propagates the thrombus formation. However, even at the highest levels of plasma 5-HT, there are always a number of SERT molecules on the plasma membrane that still continue to clear plasma 5-HT, but with a lower rate, most probably until the plasma 5-HT level returns to the physiological level.

In a recent study, we reported that at elevated level plasma 5-HT was associated with a change in the density and/or composition of N-glycans on the platelet surface and this abnormality was allied with an enhanced rate of platelet aggregation.¹¹ Earlier studies with the platelets of fawn-hooded rats showed a connection between plasma 5-HT level and platelet functions.^34–37 The genetic disorder in the platelet function of fawn-hooded rat appears similar to the storage pool disease. The reduced level of ATP, ADP and 5-HT in the platelets and the glycoprotein structure on the surface of platelets of fawn-hooded rates reduce their aggregation rates and contribute to the platelet disorder.^34–37

5-HT infusion in mice elevates plasma 5-HT levels and alters the terminal N-glycan content of platelet surface proteins; this enhances platelet aggregation, establishing the surface glycan as a key mediator in platelet activation. Others reported that sialic acid at the terminal position of N-glycans promoted the cell-cell adhesion by acting as a ligand for receptors such as P- and E-selectins.³⁸ Therefore this feature of sialic acids could also be expected for the aggregation characteristics of platelets. However, before our studies, neither the involvement of 5-HT in N-glycan switching nor the role of N-glycans on platelet aggregation was reported.¹¹ Comparing the 5-HT infused TPH1-KO and SERT-KO mice models with wild type counterparts, we found that the plasma membrane of platelets isolated from 5-HT-infused mice had predominantly N-glycolyl-neuraminic acid (Neu5Gc) containing N-glycans.¹¹ Plasma 5-HT at a high level elevates the Neu5Gc level on the platelet surface, not only promoting its biosynthesis through the catalytic activity of CMP-N-acetyl-neuraminic acid hydroxylase (CMAH) but also promoting the translocation rates of the vesicles carrying Neu5Gc containing N-glycans to the plasma membrane (Fig. 2). 5-HT-infused SERT KO mice (deficient in intracellular 5-HT) showed nearly 2-fold higher CMAH activity than the control mice. Since Neu5Gc is formed from Neu5Ac via the catalytic action of CMAH, the predominance of Neu5Gc on platelets of 5-HT infused mice suggests that 5-HT signaling enhances the catalytic function of CMAH in a 5-HT_2A receptor –dependent pathway and elevates the density of Neu5Gc on the platelet surface.¹¹ These findings concurred with FACS analysis of platelets stained with Neu5Gc antibodies.

Figure 2.

The stimulation of cells with 5-HT activates, via 5-HT_2A signaling, the production of Neu5Gc via the catalytic function of CMAH. 5-HT signaling is mediated by the G protein-coupled 5-HT_2A which facilitates the formation of Inositol 1,4,5-triphosphate (IP₃) resulting in a rise of cytoplasmic Ca²⁺ in platelets. Based on our reported and unpublished data, we propose that elevated intracellular Ca²⁺ activates CMAH, which elevates the number of Neu5Gc containing N-glycans on the plasma membrane of platelets. Our studies showed that 5-HT signaling is important either on trafficking of Neu5Gc (presented in the diagram in blue) containing vesicles to the plasma membrane or enhancing the catalytic ability of CMAH or both in inducing platelet activation and aggregation.

In summary, the involvement of 5-HT in platelet aggregation mechanism appears to be through multiple pathways.

5-HT in hypertension-associated platelet aggregation

The plasma 5-HT level is elevated in various conditions, including hypertension^39–43 and thrombosis.^{6, 9–11, 12, 42} However, in the absence of cardiovascular disease, in vivo administration of 5-HT does not increase systolic blood pressure¹² suggesting that the elevation in plasma 5-HT level could be a consequence rather than a cause of some forms of hypertension. The action of plasma 5-HT on blood pressure can be varied with its acute or chronic elevation and the location in the circulation system. For example, the blood pressure of deoxycorticosterone acetate (DOCA)-salt hypertensive rats was decreased over the course of one month of 5-HT infusion.⁴³ If the prothrombotic action of elevated 5-HT level in plasma were ignored, its blood pressure lowering capacity would be a novel approach to the hypertension studies.

Hypertension is characterized by an increased vascular resistance that can be caused by vasoconstriction.⁴⁴ Several studies report an elevation in plasma 5-HT concentration in hypertensive subjects that concurrently have diabetes and coronary artery disease^{8, 20, 21, 39, 40, 42, 43} as well as in a variety of other cardiovascular pathologies including cerebrovascular disease and arterial thrombosis.^{6, 11–14} Since these conditions share platelet activation as a disease mechanism, attention has focused on identifying the complex mechanisms by which 5-HT may promote platelet activation. The cause of elevated 5-HT could be due to an increase in the exocytosis rates of dense granules and/or 5-HT secretion from the enterochromaffin cells of the intestine. 5-HT is also a well-known arterial vasoconstrictor, as described by Rapport, as early as 1945.⁴⁴

In many forms of experimental and human hypertension^{2, 8, 16, 39–43} plasma 5-HT levels are reported as elevated but thrombosis is not commonly encountered in hypertension. Biochemical and functional analyses of lungs, platelets, and pulmonary artery smooth muscle cells showed a direct involvement of the SERT/RhoA/Rho kinase signaling pathway in 5-HT–mediated platelet activation during pulmonary hypertension.⁴⁵

When the plasma 5-HT level, systolic blood pressure (SBP) and the rate of platelet aggregation are compared in two preclinical models of hypertension (angiotensin II-infused for 7 days or cocaine-injected for 30 min),^{16, 46} the blood plasma 5-HT levels as well as the SBP are found as elevated in both hypertensive models, but only cocaine and not Ang II, exerted a pro-thrombotic effect.^{16, 46}

The platelets in hypertensive subjects have an abnormal biological profile compared to platelets of normal subjects.⁴⁷ The abnormal endothelium in hypertension promotes platelet activation, aggregation, and thrombosis and these maintain further elevation of SBP. Whether different mechanisms of hypertension differentially predispose platelets to thrombosis is not clear yet. To address this issue, the effect of elevated plasma 5-HT on platelets was compared in angiotensin II-infused mice (mimics the activated renin-angiotensin system)¹⁶ with cocaine-injected (sympathetic nervous system is activated) mice.⁴⁶

Cocaine-induced mouse model of hypertension

Cocaine is a powerful sympathomimetic agent that causes acute elevations in arterial pressures; the net effect of cocaine on SBP and diastolic blood pressures in humans is an increase of 20 mm Hg and 10 mm Hg respectively.^48–50 In peripheral tissues, cocaine produces a sympathomimetic response by inhibiting the reuptake of 5-HT and catecholamines leading to a transient bradycardia followed by tachycardia, hypertension, and acute thrombosis in coronary arteries.^{24, 51–53} Studies agree that cocaine-related cardiovascular effects do not require preexisting vascular disease.⁵¹

Through a variety of mechanisms, cocaine increases the risk of thrombosis.⁵¹ Even in the absence of systemic platelet activation, endothelial dysfunction, or cardiovascular complications, cocaine is associated with acute thrombosis of coronary arteries.^51–55 Autopsy studies have demonstrated the presence of coronary atherosclerosis in young cocaine users along with associated thrombus formation;⁵² thus, cocaine use is associated with premature coronary atherosclerosis and thrombosis. Platelets isolated from cocaine-injected mice appear hyper-reactive and form thrombi as a result of elevated exocytosis of α-granules and of plasminogen-activator inhibitor level but a decreased antithrombin-III level.^{51, 53, 55}

The role of 5-HT in cocaine-mediated thrombus formation is poorly understood. Cocaine acts as a ligand on SERT and reduces the 5-HT reuptake rates of the cells. However, when mice were injected with cocaine, their plasma 5-HT was elevated to the level found in SSRI-injected mice;^{12, 46} however, contrary to the effects of SSRI, platelets became hyper-reactive.⁵⁴ Platelets of cocaine-injected TPH1-KO mice compared with platelets isolated from TPH1-KO mice had higher aggregation rates by 10%; surprisingly, in cocaine-injected DAT-KI mice (Cocaine-insensitive dopamine transporter-knocked) platelets were already prothrombotic with aggregation rates 20% higher than the rates observed in the control group.⁴⁶ DAT-KI mice are introduced genetically with cocaine-insensitive DAT which made them 70-fold less sensitive to cocaine but fully functional for dopamine uptake.⁵⁶ Our findings suggest participation of the sympathetic nervous system in cocaine-mediated platelet aggregation via the synergistic actions of 5-HT and nonserotonergic amines such as catecholamines.⁴⁶

Ang II-induced mouse model of hypertension

Ang II, part of the renin-angiotensin-aldosterone system contributes to hypertension.⁵⁷ Whether Ang II-mediated hypertension is associated with a prothrombotic state remains controversial. Whereas some studies report a prothrombotic effect of Ang II^57–59, others report a protective effect of Ang II that is independent of its pressor effects^60–62 and may rely on elevated prostacyclin or nitric oxide to inhibit platelet aggregation.^{62, 63} Thus, we studied the correlation between elevated serum Ang II and the adhesive properties of platelets.¹⁶ The hypertensive model was established on adult C57BL/6J male mice infused isotonic saline or Ang II via osmotic mini-pumps.¹⁶ Baseline SBP was measured on 6 consecutive days prior to insertion of the mini-pumps. Then, SBP in each mouse was averaged from ≥6 trials each day for one week after saline- or Ang- II infusion. Ang II infused for one week increased SBP from 101±8.6 mm Hg to 180±12 mm Hg (average increase of 78%).

5-HT and platelet aggregation in Ang II and cocaine models

After confirmation of a SBP increase following cocaine-injection or Ang II-infusion, plasma 5-HT concentration in saline, Ang- infused, and cocaine-injected mice was determined.^{16, 46} Neither 30 min cocaine-injection nor 7 days Ang II infusion caused a change in circulating platelet counts (not shown). However, accentuated collagen-induced aggregation was observed in platelets from cocaine-injected mice; this was on average 50% higher compared to that observed in platelets from saline- or Ang II-infused mice.^{16, 46} These findings were confirmed by showing a significant elevation in P-selectin (marker of platelet activation) by flow cytometry. Thus, exposure to cocaine coincides with a heightened platelet response to collagen and enhanced platelet activation (Table 1). The 5-HT level was 2.8-fold higher in cocaine-injected mice than Ang II-infused mice but in both groups, Ang II and cocaine, the levels of SBP and 5-HT levels were elevated. Hypertension was associated with one week Ang II-infusion or 30 min cocaine-injection, plasma 5-HT levels were relatively elevated, but only the platelets of cocaine-injected mice were hyperreactive.

TABLE 1.

The summary of our findings in 4 groups of mice model system

Mice-Infused	Saline	5-HT	Ang II	Cocaine
SBP (mm Hg)	101 ± 8.6	106 ± 14	180 ± 19	146 ± 15
[5-HT] in plasma (ng/ml blood)	0.85 ± 0.04	2.74 ± 0.37	1.24 ± 0.27	3.50 ± 0.18
Platelet aggregation	35.8 ± 12	70.7 ± 7.63	32 ± 2.06	64.33 ± 4.04
FACS analysis of P-selectin	181.7 ± 8.00	316.05 ± 2.61	145 ± 7.35	325 ± 11.2

Adult C57BL/6J male mice which were inserted osmotic pumps filled with 5-HT (0.05 mg/ml, with an infusion rate of 1.66 µg/kg/hr)¹² or Ang II (24 µg Ang II/gram body weight with infusion rate of 2 ng/g/min)¹⁶ for 24 hrs or 7 days, respectively. Also, a set of C57BL/6J mice were injected with cocaine at 30 mg/kg for 30 min.⁴⁶ The baseline SBP was measured on 6 consecutive days prior to insertion of mini-pumps. All assays were performed in triplicate. mean ± SEM; n=15 for all groups.

The known effects of drugs interacting with SERT activity on platelet function in the setting of systemic hypertension

Plasma 5-HT levels are elevated in a variety of pathologies including hypertension, thrombotic disease and carcinoid syndrome. The direct contribution of 5-HT to thrombosis is difficult to assess in complex diseases such as hypertension. However, in carcinoid syndrome, carcinoid tumors overproduce 5-HT and the increased circulating level of 5-HT (and other hormones) is correlated with the formation of disseminated intravascular coagulation. Although the evidence linking 5-HT to hypercoagulable states is mainly correlative, Sarpogrelate-ANPLAG® is marketed in Japan, China and Korea as an anti-platelet agent.^64–66

These implicate that 5-HT in human diseases associated with abnormal coagulation. One of the best examples in connecting 5-HT and hypertension is “serotonin syndrome” which generally occurs if the physiological 5-HT level is elevated by using two of the following drugs at the same time, triptans, SSRI, selective serotonin/norepinephrine reuptake, ecstasy, LSD, monoamine oxidase inhibitors, meperidine, dextromethorphan. The combination of these drugs causes the elevation of 5-HT level significantly higher than the physiological level which creates a prethrombotic environment for platelet. Despite the availability of a wide range of effective BP-lowering agents, a substantial proportion of patients with hypertension fail to achieve target BP levels. The majority of patients with hypertension need at least two drugs to achieve BP control so it is important to identify new medication targets.

The cardiovascular effects of 5-HT are not uniform: bradycardia or tachycardia, hypotension or hypertension, vasodilatation or vasoconstriction. These responses are mediated by 5-HT₁, 5-HT₂, 5-HT₃, 5-HT₄, 5-HT_5A/5B, and 5-HT₇ receptors as well as by a tyramine-like action in the central nervous system, autonomic ganglia, postganglionic nerve endings, vascular smooth muscle, endothelium and cardiac tissue. For example, BP response to administration of 5-HT is triphasic: initial short-lasting vasodepressor phase (reflex bradycardia due to 5-HT3 receptors on vagal afferents), a middle vasopressor phase (5-HT_2A receptors) and a late, longer-lasting, vasodepressor phase (direct vasorelaxation by activation of 5-HT7 receptors located on vascular smooth muscle, inhibition of the vasopressor sympathetic outflow by sympatho-inhibitory 5-HT_1A/1B/1D receptors and release of endothelial nitric oxide by 5-HT_2B and/or 5-HT_1B/1D receptors). Furthermore, central administration of 5-HT can cause both hypotension (5-HT_1A receptors) and hypertension (5-HT₂ receptors).

Because of this complexity, until now, only 2 drugs have been found to have blood pressure lowering effect. Both work on alpha1-adrenoceptors as well as 5-HT receptors. Ketanserin affects baroreflex function by blocking 5-HT_2A receptors and/or alpha1-adrenoceptors through central and/or peripheral mechanisms.^{66, 67} On the other side, Urapidil has an alpha-blocking effect and also a central sympatholytic effect mediated via stimulation of 5HT_1A receptors in the central nervous system.^{68, 69}

Further studies on the role of 5-HT in hypertension, new compounds with high affinity and selectivity for the different 5-HT receptor subtypes together with SERT may be used as a therapy in the hypertension armamentarium.

CONCLUDING REMARKS

5-HT in peripheral tissues as well as the central nervous system has a rich history in pharmacology and this has translated into a widely exploited therapeutic target. New insights into the regulation of SERT and 5-HT_2A functions in platelet biology may open new dimensions in the targeting of the additive effect of plasma 5-HT on platelet aggregation. We have focused here on the known mechanisms in which 5-HT impacts platelet biology in cardiovascular diseases.

Elevations of plasma 5-HT levels have been reported in a variety of cardiovascular pathologies including hypertension, atherosclerosis, coronary artery disease, angina, and arterial thrombosis.^{6, 14, 11, 12, 13, 39, 40} Since these conditions share platelet activation as a disease component, attention has focused on identifying the complex mechanisms by which 5-HT may promote platelet activation.

Considering the role of 5-HT in platelet aggregation, a loss of platelet SERT coupled with elevated plasma 5-HT may play a significant role in cardiovascular diseases, diabetes mellitus, metabolic syndrome, atherosclerosis, and peripheral arterial disease; this is thought to reflect a common prothrombotic state. Numerous factors have been identified that confer this increased susceptibility to thrombosis, including a loss of endothelial-derived nitric oxide, vascular smooth muscle cell hypertrophy, hyperinsulinemia, and other metabolic abnormalities, obesity, and inflammation.^{1, 37, 20, 21} The development of possible antithrombotic therapies for patients with cardiovascular disease has focused on reducing these risk factors rather than on targeting endogenous mechanisms responsible for the prothrombosic state. In this regard, therapies designed to promote the expression of SERT on the platelet surface and thereby reduce plasma levels of 5-HT may represent a novel approach to alleviating thrombotic events as well as controlling blood pressure.