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",isbn:"978-1-83968-681-8",printIsbn:"978-1-83968-680-1",pdfIsbn:"978-1-83968-682-5",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"ddc0dea7e5b98335c187688d9c0c5b42",bookSignature:"Dr. Urvashi Sharma",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/10298.jpg",keywords:"Internet of Things, Smart Biosensor and Hardware, Reliability, Patients Data, Context-Specific and Aware, Integrated and Connected, Funding Structures, Policy and Its Implications, Electronic Medical Records, Electronic Health Records, Design, Implementation and Evaluation",numberOfDownloads:60,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 3rd 2020",dateEndSecondStepPublish:"October 1st 2020",dateEndThirdStepPublish:"November 30th 2020",dateEndFourthStepPublish:"February 18th 2021",dateEndFifthStepPublish:"April 19th 2021",remainingDaysToSecondStep:"5 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Sharma obtained her Ph.D. from Brunel University London, U.K. Her work has contributed to understanding the role of a user and the context in relation to the successful application of e-health modalities in primary care settings in the U.K.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"105398",title:"Dr.",name:"Urvashi",middleName:null,surname:"Sharma",slug:"urvashi-sharma",fullName:"Urvashi Sharma",profilePictureURL:"https://mts.intechopen.com/storage/users/105398/images/system/105398.jpg",biography:"Dr Urvashi Sharma started her research career as a biomedical engineer exploring barriers and facilitators to remote patient monitoring and use of electronic health records. Her work has contributed to understanding the role of a user and the context in relation to successful application of e-health modalities in primary care settings in the U.K. Through her work, she also explored whether employing randomised controlled trials to ascertain the effectiveness of technological interventions is viable. 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Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"878",title:"Phytochemicals",subtitle:"A Global Perspective of Their Role in Nutrition and Health",isOpenForSubmission:!1,hash:"ec77671f63975ef2d16192897deb6835",slug:"phytochemicals-a-global-perspective-of-their-role-in-nutrition-and-health",bookSignature:"Venketeshwer Rao",coverURL:"https://cdn.intechopen.com/books/images_new/878.jpg",editedByType:"Edited by",editors:[{id:"82663",title:"Dr.",name:"Venketeshwer",surname:"Rao",slug:"venketeshwer-rao",fullName:"Venketeshwer Rao"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"4816",title:"Face Recognition",subtitle:null,isOpenForSubmission:!1,hash:"146063b5359146b7718ea86bad47c8eb",slug:"face_recognition",bookSignature:"Kresimir Delac and Mislav Grgic",coverURL:"https://cdn.intechopen.com/books/images_new/4816.jpg",editedByType:"Edited by",editors:[{id:"528",title:"Dr.",name:"Kresimir",surname:"Delac",slug:"kresimir-delac",fullName:"Kresimir Delac"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"55699",title:"Immuno-Thrombotic Effects of Platelet Serotonin",doi:"10.5772/intechopen.69349",slug:"immuno-thrombotic-effects-of-platelet-serotonin",body:'Serotonin (5-HT) is a well-known neurotransmitter, which regulates neural activity and a variety of neuropsychological processes [1]. As it has been shown to be involved in the regulation of systemic and cellular functions, alterations in serotonin concentration in the body are associated with many different diseases, such as irritable bowel syndrome, restless legs syndrome, sudden infant death syndrome, autism, headache, insomnia, anxiety, depression, anorexia, schizophrenia, Parkinson’s and Alzheimer’s disease, pulmonary hypertension, and myocardial infarction. 5-HT was first described in 1930 by Vittrorio Erspamer who isolated it from enterochromafin cells of the gut [1]. Only a small amount of 5-HT is synthesized in brain (5%), whereas 95% is produced by the enterochromafin cells of the gastrointestinal (GI) tract. 5-HT is synthesized from the essential amino acid
5-HT biosynthesis and receptor distribution in brain (A) and periphery (B). Serotonin (5-HT), serotonin transporter (SERT), monoamine oxidase (MAO), 5-hydroxyindole acetic acid (5-HIAA), 5-hydroxytryptophan (5-HTP), tryptophan (TRP), and vesicular monoamine transporter (VMAT). For the details, see the text.
5-HT can also be taken up from plasma into several cells—such as platelets—via the 5-HT transporter (5-HTT, SERT). After uptake, 5-HT can be then stored in vesicles and granules through the action of vesicular monoamine transporter (VMAT)-1/2 which is expressed in neurons, neuroendocrine cells, and platelets. The largest quantity of serotonin is believed to be stored in platelets, from where it can be released upon platelet activation, for example, during thrombus formation or inflammatory reactions. Interestingly, chemical precursors of 5-HT can pass across the blood-brain barrier, but 5-HT cannot, thereby effectively isolating the brain 5-HT pool from the periphery and vice versa. In the brain, 5-HT regulates several complex networks, such as mood, perception, reward, anger, memory, appetite, attention, and sexuality. There are two major routes of 5-HT metabolism, which convert 5-HT to melatonin and 5-HIAA. 5-HT is metabolized by neurons and endothelial cells by monoamine oxidases (MAOs) and the products of this breakdown are then excreted by the kidney [3, 5–7].
Peripheral 5-HT regulates heart development and rate, valvulopathy, pain, nociception, embryonic development, vasoconstriction/vasodilatation, blood flow, hemostasis, and many other important processes. Platelets are not able to synthesize serotonin, but take it up from plasma via 5-HTT, store it in dense granules (via VMAT-1), and release it into the blood during their activation. Platelet serotonin has not only well-established autocrine functions during platelet activation and thrombus growth but also paracrine functions in the vasculature including modulation of endothelial, smooth muscle, and immune cell function.
Platelets store 5-HT in their dense granules at millimolar range and secrete it after activation [8]. Dense granule and 5-HT release support the recruitment of circulating platelets to preformed thrombi, thereby leading to thrombus growth. This process is mediated through the interaction between 5-HT and its receptor 5HT2A expressed on circulating platelets. Activated 5-HT2A receptor transduces the signal to Gq-phospholipase C (PLC) β-signaling cascade. Enhanced PLCβ activity results in intracellular Ca2+mobilization from the store through inositol 3-phosphate (IP3) receptor and mediates 1,2-diacylglycerol (DAG)-dependent protein kinase C (PKC) activation, thereby amplifying platelet reactivity (Figure 2).
Autocrine effects of platelet 5-HT. Activated platelets release 5-HT, thereby amplifying platelet activation and the recruitment of circulating platelets. Binding of platelet 5-HT to the 5-HT2A receptor induces activation of PLCβ-signaling cascade and upstream effectors which support platelet reactivity. Receptor-ligand interactions also regulate 5-HTT uptake kinetics by interconnecting several signaling pathways. For the details, see the text.
In addition to the mobilization of cytosolic Ca2+ [9, 10], receptor-ligand interactions are also known to regulate 5-HT uptake kinetics. In human platelets, the rise of cytoplasmic Ca2+ in the absence of exocytosis reduces 5-HT transport into the cytoplasm, thereby decreasing the release of 5-HT [9]. Interestingly, rabbit platelets activated in the presence of the extracellular Ca2+ chelator ethylene tetraacetic acid also displayed a decrease in 5-HT transport activity [11, 12]. Consistently, human platelets treated with the membrane permeant Ca2+ chelator BAPTA-AM also had reduced 5-HT transport in the presence of extracellular Ca2+ [9]. Activation of the Orai1 Ca2+ channel induces a robust Ca2+ influx called store-operated Ca2+ entry (SOCE), which is triggered through the release of Ca2+ from intracellular stores. This process is controlled by functional coupling of activated stromal interaction molecule 1 (STIM1) to Orai1 [13]. Interestingly, strongly reduced SOCE was found in 5Htt−/− platelets [14]. This suggests that secreted platelet 5-HT contributes to the regulation of SOCE through binding to 5-HT2A which activates Gq-PLCβ-mediated Ca2+ store release, thereby further activating STIM1/Orai1 complex. Interestingly, SOCE-induced signal can strongly inhibit 5-HT uptake in human platelets via 5-HTT [9, 11]. This could be an important step to keep 5-HT outside of platelets, thereby increasing extracellular 5-HT concentration and permanently activating 5-HT2A on the platelet surface. Therefore, 5-HT cannot enter the platelet cytosol during SOCE. Interestingly, 5-HTT contains several consensus sites for PKC. It has been shown that PKC activity is required for the internalization of the transporter suggesting a link between 5-HT uptake and intracellular Ca2+ level [15–18]. Altogether, Ca2+ signaling, Ca2+ store release, and Ca2+ influx through SOCE play an important regulatory role for 5-HT cycling in human and mouse platelets.
After Ca2+ store release and PKC activation, integrins exposed and activated on the platelet surface support aggregation and thrombus formation. In β3 integrin-deficient platelets, 5-HT uptake was strongly reduced, indicating a functional crosstalk between 5-HTT and β3 integrin [19]. Integrin activation defect in response to glycoprotein VI (GPVI) or C-type lectin-like receptor 2 (CLEC-2) stimulation was found in 5Htt−/− mouse platelets, which was fully rescued in the presence of extracellular 5-HT [14]. The physical interaction between 5-HTT and β3 seems to be dispensable for β3 integrin activation. The observed integrin activation defect is due to the lack of the secreted platelet 5-HT which further amplifies “inside-out” activation of integrins through Ca2+-dependent and independent pathways mediated by Ca2+- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI) and PKC, respectively.
Although 5-HT is mainly stored in dense granules, intracellular-free 5-HT in the cytoplasm has been proposed to activate diverse biological processes called serotonylation. It has been shown that small-guanosine triphosphate-binding protein (GTP)-ases covalently bind 5-HT, thereby changing the structure and activity of GTPase, leading to α-granule exocytosis from platelets. This process requires tissue transglutaminase and factor XIIIa, both activated by mobilized Ca2+. Transglutaminase may mediate the transamidation of small GTPases, like cytoplasmic Ras homolog gene family member A (RhoA) and a small GTP-binding protein Rab4. Serotonylation in turn blocks the inactivation of both molecules. A complex composed of Ca2+ and calmodulin (CaM) may also activate guanine exchange factors (GEFs), which induce the exchange of guanosine di- (GDP) to triphosphate (GTP) on RhoA and Rab4 and thus stimulates activation of the respective protein. These two active molecules play an important role in cytoskeleton rearrangement, exocytosis of α-, and dense granule contents. Some bioactive molecules stored in platelet granules, such as fibrinogen and factor V, are also known to be serotonylated [8]. Upon platelet activation, these proteins are exposed at the platelet surface and are used to mark a subpopulation of highly activated, pro-coagulant platelets, the so-called collagen and thrombin-activated (COAT) platelets. Coated platelets express high levels of phosphatidylserine and strongly support prothrombinase activity [8, 20].
Besides the dopamine transporter (DAT), the noradrenaline transporter (NET), and the organic transporter (OCT), 5-HTT is an important 5-HT transporter to regulate 5-HT uptake from the blood plasma and reuptake of the released platelet 5-HT in certain physiological conditions. 5-HTT is encoded by the SLC6A4 gene containing 14 exons. The protein structure of 5-HTT contains 12 transmembrane domains. In humans, the splice variants of 5-HTT and their mutations are associated with several pathologies, such as anxiety, suicide, depression, substance abuse, autism, and neurogenic disorders [21–24]. 5-HTT is abundantly expressed not only on neurons, endothelial cells, mast cells, immune cells, in intestine, and vasculature, but also in platelets [25, 26]. It is well established that in platelets 5-HTT plays an important role in the uptake of 5-HT from the circulation. Monoamine transporters are thought to be able to compensate for one another where they are co-expressed. For example, 5-HT may be taken up in venous vessels independently of 5-HTT expression [25, 27]. Interestingly, and in sharp contrast to venous vessels, genetic ablation of 5Htt in mice completely abolished 5-HT uptake in platelets, since no detectable secreted 5-HT was observed upon platelet activation, indicating an essential role of 5-HTT for 5-HT uptake into platelets, which cannot be compensated by other transporters [14]. Altogether, these results highlight the cell-type-specific regulation of 5-HT uptake in mammalian cells.
5-HTT can be targeted by several antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) (cf., Section 5), which are widely used in the treatment of psychiatric diseases to increase 5-HTT concentrations in the synaptic space. The blockade of 5-HTT with the SSRI citalopram reduces the aggregation response to collagen in human platelets [28] due to reduced phosphorylation of a tyrosine-protein kinase Syk in the GPVI signalosome. Syk can also bind and phosphorylate 5-HTT suggesting an Syk-mediated functional crosstalk between 5-HTT and GPVI complex. Interestingly, 5Htt−/− mouse platelets could not show any abnormalities in the tyrosine phosphorylation cascade of the GPVI signalosome, as Syk phosphorylation was normal after GPVI stimuli. Consequently, Syk and 5-HTT interaction seems to be dispensable for the initial activation of GPVI complex, but enhanced Syk activity may regulate the 5-HT uptake in platelets [29].
During degranulation, activated platelets secrete a significant amount of 5-HT from dense granules which is clinically relevant to induce acute thrombotic events [30, 31] by promoting vasoconstriction and cellular activation of neighboring platelets and lymphocytes through their 5-HT receptors.
5-HT receptors expressed on endothelial, smooth muscle, and immune cells respond to platelet-derived 5-HT (Figure 3). 5-HT has growth-promoting effects on endothelial cells, which may facilitate tissue healing after vascular damages [32]. However, 5-HT may also exert dual effects either stimulating constriction or dilatation of microvasculature. In the liver, 5-HT appears to mainly promote constriction of hepatic sinusoid vessels, since mice lacking peripheral 5-HT display elevated sinusoidal perfusion under physiological and pathological conditions [33]. By contrast, platelet-derived 5-HT coordinates the formation of gaps between endothelial cells in the joint microvasculature, which in arthritic conditions may contribute to inflammation [34]. How these processes are regulated is still not clear but presumably may involve differential signaling pathways through specific 5-HT receptors expressed on vascular endothelial and smooth muscle cells.
Paracrine effects of platelet 5-HT. Secretion of platelet 5-HT modulates the function of endothelial and smooth muscle cells either promoting vessel constriction or dilatation. Platelet 5-HT influences several functions of immune cells, indicating their importance in the regulation of immune cell response and activities under pathophysiological conditions. For the details, see the text.
Platelet-derived 5-HT can regulate the function of T- and B-cells, natural killer cells, monocytes, and neutrophils under certain conditions [35–38]. In the spleen, 5-HT increases monocyte differentiation into dendritic cells and early naive T-cell activation via the 5-HT2A receptor [38, 39]. Furthermore, it also has been shown that lymphocytic cytokine levels in mice are reduced after treatment with SSRI [40]. In a mouse model of viral hepatitis, the release of 5-HT by platelets was responsible for tissues damage caused by CD8 (+) T-cells, microcirculatory events, and reduced clearance of infiltrated viruses [33]. Moreover, specific antagonism of 5-HT receptors in mice attenuated asthmatic attacks and sepsis [37, 41].
5-HT released from dense granules upon activation by the inflamed endothelium also contributes to the recruitment of immune cells to the vascular wall [37]. Indeed, platelet-derived 5-HT promotes leukocyte migration, possibly via activation of endothelial cells, thereby enhancing P-selectin exposure and IL-8 release [37], which trigger neutrophil rolling, adhesion, and extravasation. Moreover, locally increased levels of platelet-released 5-HT had paracrine effects on endothelial cells, thereby inducing microvasculature leakage through the activation of transglutaminase and the phosphorylation of vimentin [42]. By contrast, in solid tumors platelet-released 5-HT has been described as a major regulator of the tumor vascular homeostasis that continuously prevent bleeding. Interestingly, tumor-infiltrating leukocytes have been identified as the cause of tumor bleeding [43, 44]. Altogether, these studies suggest that under specific conditions, platelet-released 5-HT promotes clot formation and modulates immune cell functions.
In humans, 5-HT levels appear elevated in infection and autoimmune diseases, suggesting that SSRI could be applicable for vascular and immune system modulation. Since platelets are the major 5-HT store in the blood, pharmacological blockage of 5-HT uptake in platelets increases the level of 5-HT in the blood plasma transiently. Unexpectedly, 5Htt−/− mice display reduced 5-HT levels in plasma [14]. In 5Htt−/− mice, elevated urinary 5-HIAA levels were detected suggesting a faster 5-HT metabolism in the peripheral blood. Consequently, platelet 5-HT uptake and storage play an important regulatory role for controlling systemic 5-HT metabolic cycles. Future studies are needed to specify the exact mechanisms of platelet-derived 5-HT on vascular and immune system modulation in normal physiology and diseases.
5-HT plasma concentration was analyzed in several pathological contexts. It became widely recognized that 5-HT is an independent risk factor for platelet aggregation and for thrombus formation in animal models (cf., Section 6) and human patients [19, 45–49]. Plasma 5-HT can support platelet aggregation and thrombus growth through 5-HT2A-dependent or independent signaling pathways. Pharmacological blockade of 5-HT2A receptor increases the 5-HT uptake rates in animal models of hypertension, as well as ex vivo platelet aggregation. Vikenes et al. detected a 10-fold increase of plasma 5-HT in patients undergoing angiography after admission for myocardial infarction [50]. In these patients, high plasma 5-HT was associated with cardiac events. In another study, more than 10-fold rise in 5-HT has been noticed in coronary vessels of patients following angioplasty. Importantly, in these patients the level of 5-HT in the systemic plasma was normal [51]. Together, these studies suggest that in vivo the interplay between circulating 5-HT and platelet function could be a predictive factor.
5-HT levels are drastically increased during myocardial ischemia, and blockade of the 5-HT2 receptor improves the outcome after myocardial infarction in different mouse models [52, 53]. 5-HT also enhances the survival of cardiomyocytes via the 5-HT2B receptor. In hepatic ischemia models, platelets promote tissue repair [54], and proliferation of hepatocytes was shown to be partly mediated by platelet 5-HT after liver ischemia [55]. 5-HT also contributes to intratumoral homeostasis by dysbalancing permeability factors [44]. 5-HT-induced growth of human hepatocellular carcinoma cells and specific blockade of the 5-HT2 receptor decreased recruitment of circulating tumor cells [56, 57]. It has been suggested that the inhibition of platelet granule contents might be effective to induce intratumoral bleeding, thereby decreasing tumor viability and growth. Additionally, plasma 5-HT levels are increased in patients with colorectal, liver, and intestinal cancers [58, 59].
Allergic airway inflammation provokes a local release of 5-HT in mouse models and human patients [41]. Interestingly, after challenge with an allergen, 5-HT increased 10-fold in broncho-alveolar lavage of predisposed patients, inducing asthmatic attacks. In line with these studies, 5-HT is known as a key regulator of pulmonary vascular resistance and vessel wall integrity [60, 61].
Selective serotonin reuptake inhibitors are commonly used drugs for the treatment of patients with severe depressive and anxiety disorders [62]. SSRIs were developed to selectively inhibit the uptake of 5-HT through the 5-HTT transporter in the brain, while having minimal side effects on DAT and NET proteins which can also transport 5-HT [63]. The action of SSRIs relies on the modulation of the allosteric region of the transporter, thereby leading to a conformational change and blocking of the uptake of 5-HT [63]. The uptake of 5-HT into neurons is very important for the clearance of the synaptic cleft, preventing firing rates and overstimulation of receptors [64]. This uptake and the later release are blocked upon treatment with SSRIs, such as fluvoxamine, fluoxetine, nortryptiline, citopram, and escitalopram [65]. The different SSRIs vary in kinetics being competitive and non-competitive inhibitors. Two distinct binding sites on 5-HTT have been identified, a low-affinity allosteric site, mediating the dissociation of SSRIs from their high-affinity site, which induces the blockade of 5-HT uptake [64].
There is evidence that targeting 5-HT receptors or using serotonin-like molecules is effective in the treatment of non-neuronal diseases. The use of tricyclic antidepressants, but not SSRIs, is associated with an increased risk of myocardial infarction. SSRIs have shown no cardiac toxicity, even in patients with heart disease. Several epidemiologic studies reported lower cardiovascular morbidity and mortality in patients treated with SSRI [66–68].
Depression is a significant risk factor for ischemic heart and cerebrovascular disease as well as mortality following myocardial infarction. The potential effects of SSRIs upon the cardiovascular system may therefore play an important role. These drugs had potential benefit in hypertensive patients after myocardial infarction and hypertensive responses to depression were reduced in patients who had been prescribed SSRIs [30]. In blood samples of depressive patients taking fluoxetine, the platelet aggregation response to submaximal collagen stimulation was decreased [69]. In this study, a significant decrease in 5-HT concentration was observed in platelet-rich plasma associated with the use of fluoxetine but not with the tricyclic antidepressant amitryptiline. It is intriguing whether lowered platelet 5-HT content translates into less 5-HT release during platelet activation in patients with thrombotic diseases. Enhanced platelet reactivity was observed in patients suffering from depression and chronic heart disease due to the upregulated β-thromboglobulin (β-TG) and platelet factor 4 (PF4) levels [70]. Lowered PF4 and β-TG levels have been observed upon treatment with SSRI paroxetine [71], suggesting that reduced platelet aggregation in vivo may impact coronary artery-related mortality. SSRI treatment also decreases platelet reactivity in patients with heart failure. Other SSRIs, sertraline, and N-desmethylsertraline were also shown to dampen platelet responses [72].
SSRIs have been shown to increase the risk of bleeding in patients with liver cirrhosis and liver failure. Importantly, SSRIs may also directly increase gastric acidity with ulcerogenic effect resulting in GI bleeding. The risk of SSRI-associated GI bleeding is increased with the concurrent use of nonsteroidal anti-inflammatory drugs, anticoagulants, and antiplatelet agents, and is decreased by concurrent proton pump inhibitors [73, 74]. In conclusion, SSRIs appear to be protective against cardiovascular diseases and may enhance the risk for GI bleeding. However, to date this evidence is not yet conclusive.
Over the past decades, the functions of peripheral 5-HT have received increasing attention. It has been shown that peripheral 5-HT plays a major role in a variety of important processes, including hemostasis and immune defense. This has been addressed by using Tph1−/− mice, which lack peripheral 5-HT in the circulation, due to the lack of the enzyme that converts hydroxylases tryptophan to 5-HT in the gut [75]. In humans, abolished or decreased level of TPH1 is associated with impulsive behavior, aggression, irritable bowel syndrome, anxiety, and other pathologies [76–79]. Genetic ablation of TPH1 function in mice not only leads to several disorders, such as mild anemia, cardiomyopathy, and diabetes, but also to other defects in hemostasis, erythropoiesis, pulmonary hypertension, and lung regeneration. The lack of 5-HT in this mouse model is associated with decreased neutrophil recruitment to inflammatory sites, diabetics, and mild anemia [37, 80].
Recent studies using wild-type mice infused with 5-HT or Tph1−/− mice have demonstrated that peripheral 5-HT is required for platelet aggregation [14]. Additionally, in vivo 5-HT infusion generates hyperreactive platelets with reduced bleeding time and shortened occlusion time of the carotid arteries in wild-type mice. 5-Htt−/− mice have prolonged bleeding time, reflecting the increased bleeding risk described to occur using long-term SSRI treatment in human patients. In comparison to this relatively mild hemostatic defect, 5Htt−/− mice were not able to form occlusive thrombi in response to mechanical injury of the abdominal aorta as compared to wild-type animals [14].
Platelets contribute to the progression of infarct growth after transient brain ischemia by thrombo-inflammation with platelet-immune cell interactions. SSRI treatment of stroke patients has been described to enhance brain function recovery, indicating a therapeutic benefit of the direct blockade of 5-HTT function. Neuroblast proliferation and cell migration have been shown to be enhanced and associated with increased microvessel density during SSRI treatment, explaining the possible role of 5-HTT in tissue repair after ischemic insults [81–83]. 5Htt−/− mice have been studied in the tMCAO (transient intraluminal filament model of middle cerebral artery occlusion) model of ischemic stroke. Unexpectedly, these mice developed similar brain infarcts to wild-type controls and the 5-HTT neurological outcome was indistinguishable [14]. In line with this study, SSRI treatment could not reduce infarct size or cerebral edema in mice [82], suggesting that this treatment cannot protect neurons or other cells in the ischemic brain. Altogether, these results indicate that SSRI treatment may have a long-term effect in the ischemic brain tissue which positively influences post-stroke recovery. Further investigation is necessary to understand the specific role of peripheral and brain 5-HT in thrombo-inflammation during stroke and infarct progression.
5-HT is an ancient molecule that is better known for its functions in the brain than in the periphery. However, literature describing the contribution of peripheral 5-HT, including platelet 5-HT, is rapidly growing. It became evident that platelet 5-HT has a complex role involving many bidirectional interactions with tissue microenvironment to regulate platelet and immune cell functions. SSRI treatment in animal models appears to improve thrombotic and inflammatory diseases. Further fundamental and preclinical studies are needed for a better understanding of platelet 5-HT functions in humans. In conclusion, targeting thrombo-immune-modulatory functions of platelet serotonin may provide new important therapeutic approaches.
As novel COVID-19 testing develops, saliva has become of increasing interest as an alternate biological sample for rapid testing [1]. The appeal in saliva-based testing lies within the ease of which samples are collected, as well as patient comfort throughout the collection process [2]. Yacoubian Jr., Wish, and Perez (2001) found that the benefits in the ease of saliva collection were multifaceted. These benefits include the uncomplicated nature of collection, which, coupled with a low risk of direct contact and contamination, makes salivary diagnostics an attractive alternative to biological sample collection where contamination may be more challenging to avoid, such as with blood or urine analyses. For these reasons, saliva-based testing has become an increasingly popular choice in the creation of novel forms of diagnostic testing. With this, it has become increasingly important to delineate the characteristics of saliva viscosity due to its effects on the movement and interactions of the substances and molecules found within it. In the context of this study, viscosity refers to internal friction of a fluid, which is marked by the resistance of a fluid to flow [3].
While viscosity can affect the interactions and molecules within saliva is important to note in developing diagnostic tests, salivary viscosity itself can also be seen as an important factor in maintaining oral and overall health. A study by Katsuhiro Kitada and Takahiko Oho (2011) found that an increase in saliva viscosity decreases the bacterial co-aggregation between Streptococcus oralis and Actinomyces naeslundii [4]. Under normal circumstances, co-aggregation can prevent bacterial infection in the oral cavity, as co-aggregated bacteria may be swallowed before forming attachments within the oral cavity. The study indicated that increasing saliva viscosity decreased formation of these co-aggregated bacteria, which may allow for further health problems, such as pneumonia or other infections that may be brought on by the aspiration of oral bacteria or microorganisms [4]. The demonstrated health implications surrounding salivary viscosity further suggests the importance of developing protocols to accurately measure salivary viscosity following saliva collection.
The characteristics of salivary viscosity, namely the presence of aggregates, variations in temperature, sample handling, and time elapsed between sample collection and testing, serve as points of interest in the creation of laboratory protocols for salivary-based rapid diagnostic testing. Understanding how external factors affect saliva viscosity are important in generating guidelines for proper sample handling in saliva testing to ensure consistent and reliable results.
Multiple studies demonstrated in the literature reflect the variability of saliva viscosity. The 1998 Rantonen and Meurman study concluded that salivary viscosity can be dependent on the method of its production. Particularly, whether secreted by the submandibular, sublingual, or palatal glands [5]. Although the study demonstrated that the quantity of mucin within each saliva sample of differing origin did not change, the species of mucin did. Particularly, it was demonstrated that the saliva stemming from the sublingual glands demonstrated more elasticity than those of the submandibular and palatal glands, which would affect the viscosity of the saliva. In addition, the 2016 study by Antoon Ligtenberg, Erwin Liem, Henk Brand, and Enno Veerman found that acute exercise correlated with a significant increase of saliva viscosity when collected shortly thereafter [6]. These findings were parallel with the Rodica Murineanu, Corina Stefanescu, Agripina Zaharia, Carolina Davidescu, and Sorin Popsor (2011) study that found medication, general illness, and acrylic dentures to all correlate with a change in saliva viscosity [7]. This study suggested medication and disease state may affect saliva viscosity. For example, complete acrylic dentures were specifically found to correlate with an increase in salivary viscosity. It is also interesting to note the apparent correlation between salivary viscosity and dental cavities. A 2014 study by Animireddy et al found that in a sample of 75 school children, the cavity-free children had on average higher salivary viscosity than their counterparts [8]. These findings delineate some of the known variability to saliva viscosity discussed in the literature, which further demonstrate the necessity of qualifying the properties and behavior of saliva viscosity.
Beyond the variability of salivary viscosity, the level of normal viscosity is very different from that of other commonly used human biofluids in diagnostic testing. This is an important factor to note in the development of such tests, especially when considering technologies previously developed for other biofluids. The viscosity of normal cerebrospinal fluid, for example, is remarkably close to that of water, which is 1.00 cSt at 20°C [9, 10] Similarly, the kinematic viscosity of urine is 1.07 cSt at the same temperature [11]. These examples are lower than the kinematic viscosity of normal blood, which is around 3.65 cSt at 21.2°C [12]. While there is variability within the viscosities of these human biofluids, they are far lower than what we expect of human saliva, an important challenge to overcome in developing diagnostic testing.
Due to the interest in point-of-care saliva-based diagnostic testing, and based on the current literature demonstrating potential variabilities in saliva viscosity and associated causes, it is rather surprising that the literature on salivary viscosity characterization for protocol creation is rather sparse. This study hopes to address some of the gaps in the literature pertaining to salivary properties by exploring how viscosity changes upon freezing and subsequent thawing, and how it changes over time with consecutive trials, using the Cannon-Fenske experimental protocol, with the goal of aiding in the development of laboratory protocols pertaining to salivary-based diagnostic testing.
Based on the previous literature at hand, the research questions of this study are as follows:
How does the viscosity of collected saliva change over time with subsequent trials? How does the viscosity of collected saliva change after freezing and subsequent thawing?
In the absence of detailed information in the research literature, this study seeks to better understand the specific properties of saliva viscosity, and how saliva viscosity reacted to factors that are integral in the creation of lab protocols; specifically, how the samples are stored. It is not uncommon for biological samples to be frozen or cooled, and this makes sense with respect to slowing down bacterial contamination and maintaining biological molecules of interest. It is well understood that many human proteins, enzymes, vitamins, degrade over time [9] and that the degradation over time can be diminished by freezing or cooling samples beyond given temperature degradation thresholds, to allow for long-term storage.
Changes within the aggregates commonly found in human saliva, such as mucins, may also be affected by temperature and shear forces. Enzymes, such as salivary alpha-amylase, and hormones, such as cortisol, will degrade over time unless this process is inhibited, typically by freezing samples to −20 degrees Celsius, or below [10]. However, it is important to determine the effects of sub zero temperatures on viscosity itself, as the viscosity of the saliva may be impactful in how the aggregates are measured via point-of-care salivary biosensors. This was reflected in the 2013 Robles et al. study which found that the most consistent and reliable salivary alpha-amylase biosensor data was obtained from frozen and centrifuged passive saliva samples, rather than samples that were collected as fresh, passive, drool [11]. The authors hypothesize this discrepancy to be due to various factors of the saliva itself (such as mucin molecules), which may interfere with the device in question by preventing close binding to the sensor surface, as it attempts to detect quantities of salivary alpha-amylase. Also, this hypothesis reflects the prediction that salivary viscosity is an important factor in molecular measurements. While the aggregation effect was disadvantageous when in reference to the assessing quantities of the enzyme salivary alpha-amylase, it may actually be preferable when measuring quantities of different gases within saliva samples. This further delineates the importance of taking a closer look at physical salivary properties, in order to approach proposed handling methodologies appropriately, depending on the given purpose of the saliva sampling.
As previously mentioned, we hoped to better understand the properties of saliva viscosity in regards to different methodologies in sample preservation or usage. For this reason, two cycles of laboratory trials were conducted, with the aim of determining how saliva viscosity was affected. The first study aim is to determine how time alone affects saliva samples. The second trial aims at determining whether freezing and subsequent thawing affect the viscosity of the sample, as well. Both phases of data collection were done at a Biomedical Engineering laboratory, at Arizona State University, Tempe, and human saliva samples were collected within this department.
Participants were instructed to not eat within an hour of sample collection, and were then asked to drink approximately 100 mL of water immediately prior to saliva collection. This was to prevent short-term dehydration effects from confounding our variables. In addition, this aided in the ease of saliva collection. Participants were then asked to collect approximately 15 mL of passive saliva over a span of 25 minutes, into a plastic vial. The goal for saliva collection included diminishing the amount of air bubbles trapped within the saliva, by collecting the saliva very carefully, slowly, and with as little movement as possible. Foam-like saliva that is saturated with small air bubbles was not included in the overall 15 mL amount of passive saliva collected.
The viscosity of the collected saliva sample was measured using a Size 350 Cannon-Fenske apparatus, with a capillary radius of 0.045 cm, a shear rate of 2.08 1/s at 10cSt, and a viscometer constant of 0.5 cSt/s, which was cleaned and dried prior to commencing the viscosity protocol. The saliva sample was then poured into the apparatus, and allowed to flow through, while efflux time was measured concomitantly, indicating the time required for the meniscus of the viscous fluid to flow between the designated markings. This viscometer procedure was replicated 10 times consecutively for each collected sample, after which each sample was frozen and subsequently thawed the following day, at which point the viscometer procedure was performed again. A series of viscosity measurements of a 50% glycerol/water control solution was tested in the same manner to act as a control variable.
The kinematic viscosity of each trial was calculated using the efflux time and viscosity constant in the following relationship:
This procedure reflected the first half of the research questions, as to how saliva viscosity changes with time [12]. By comparing the time elapsed for the viscosity of human saliva with the glycerol/water solution, we are able to visualize how viscosity properties may be affected by the presence of the aggregates in unprocessed human saliva, which are not present in the glycerol/water mixture.
Data was collected for each measurement of salivary viscosity. The initial graph (Figure 1) represents the methodology behind the first research question; how does salivary viscosity behave over time? Figure 2 represents the viscosity of saliva (as average kinematic viscosity), with freshly collected samples of saliva, compared to with samples that were frozen, and subsequently, thawed for analysis.
This figure evaluates how salivary viscosity changes with time over subsequent trials following collection. This arbitrary time is demonstrated in sequential trials, as trials were completed one after the other following collection. This saliva kinematic viscosity is contrasted with that of a 50% glycerol water solution, acting as a control. In the passive saliva trials, we see a stark decrease in kinematic viscosity with each subsequent trial, however, the glycerol/water solution shows a slight variation within an expected range given the experimental apparatus and simple laboratory control of room temperature (22 C).
This figure demonstrates the statistically significant discrepancy between fresh and thawed passive saliva samples at 22 C. each bar represents an average of 10 trials. One outlier was removed from the thawed trial group as it was handled outside the guidelines of the lab protocol.
There are several points of interest with regards to the conclusions drawn from the experimental design that reflect the difference in how fresh saliva behaves, compared to fresh saliva, and compared to saliva that had been frozen and re-thawed. Such comparisons are reflected in the literature by the 2019 study by Johannsen et al. which found that salivary viscosity measurements varied depending on whether the saliva was untreated or subject to magnet-beating prior to viscosity measurements at low shear rates [13].
One aspect that is of interest is whether there is a methodology that can guide how fresh saliva can be processed in order to have a consistent flow property or ensure that the major aggregates, presumably due to entangled mucin chains, can be minimized quickly so that a rapid test can be performed (Figure 3).
This figure demonstrates the cross equation fit, using the limits of η∞ and η0. The independent variable is demonstrated to be 1/(1 + aT).
Bansil et al. [14] provide a molecular interpretation on how the structure of mucin leads to entanglements among the biopolymers in solution and create a range of viscosity effects depending upon concentration and mucin type. They suggest that a dilute solution of mucin generally has viscoelastic behavior that depends upon shear rate. A more general approach to the viscosity behavior of biopolymer solutions is given by Picout and Ross-Murphy [15] who provide experimental verification of the Cross equation:
with lambda being a time constant and gamma the shear rate.
Comparing the Cross equation to the data shown in this study in Figure 2 suggests that repeated shearing with a Cannon-Fenske viscometer is a cumulative effect, so one may conjecture the following modified forms of the Cross equation could explain the apparent viscosity change in saliva after repeated shearing due to the capillary flow within the viscometer.
or
where n is an integer and k is the number of repeat measurements in the first equation and T is the total elapsed time of shear for the combined repeat measurements. It is not as important to determine which equation may be better at predicting data than to understand how to use the concept to prepare saliva samples in a variety of ways rather than relying solely on freezing or centrifugation which can be cumbersome and time consuming.
The potential utility of these modified Cross equations is in determining a way to rapidly shear saliva samples using a simple microfluidic device or mixer, rather than subjecting the saliva to freezing or flowing the saliva through a longer tube to simulate the cumulative shear thinning shown in Figure 2. The shear rate experienced in the Cannon Fenske viscometer used for these experiments is on the order of 2 s−1, so based on the Figure 2 data of approximately a total time of 80 seconds of shearing is needed in order to reach a stable and minimum kinematic viscosity a device capable of deliver a shear rate of 200 s−1 which is well within the reach of portable and low cost homogenizers [16, 17, 18, 19].
Salivary viscosity can be an important parameter to consider when designing diagnostic devices for rapid testing. Consideration should be given to the fact that not only is saliva viscoelastic, but its apparent viscosity can change by mild shearing over a period of time. Shearing at rates as low as 2 s−1 can decrease its kinematic viscosity by more than half, which could change some kinetics of enzyme action, sensor signal development, or diffusive transport. After approximately a total time of 80 seconds of shearing at 2 s−1 can lead to a stable and minimum kinematic viscosity. The concept of biopolymer viscosity behavior being modeled by the Cross equation suggests that a device capable of delivering shear rates of 200 s−1 and above may be able to modify the mucin superstructure sufficiently to provide saliva samples with consistent apparent viscosities. Microfluidic devices or low cost handheld homogenizers could very quickly deliver the needed shearing action in order to provide a more consistent saliva sample, in terms of its viscous properties.
The institutions involved in the findings for this paper are Arizona State University, Tempe, and New Mexico State University. The data was collected at a Arizona State University Biomedical Engineering laboratory in Tempe, Arizona. This project was done under the supervision of Professor Antonio A. Garcia, who is now Associate Dean of Engineering at New Mexico State University in Las Cruces, New Mexico.
The authors declare no conflict of interest.
Special thanks to Professor Antonio A. Garcia, Associate Dean of Engineering at New Mexico State University. His mentorship is invaluable.
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',metaTitle:"Horizon 2020 Compliance",metaDescription:"General requirements for Open Access to Horizon 2020 research project outputs are found within Guidelines on Open Access to Scientific Publication and Research Data in Horizon 2020. The guidelines, in their simplest form, state that if you are a Horizon 2020 recipient, you must ensure open access to your scientific publications by enabling them to be downloaded, printed and read online. Additionally, said publications must be peer reviewed. ",metaKeywords:null,canonicalURL:null,contentRaw:'[{"type":"htmlEditorComponent","content":"Publishing with IntechOpen means that your scientific publications already meet these basic requirements. It also means that through our utilization of open licensing, our publications are also able to be copied, shared, searched, linked, crawled, and mined for text and data, optimizing our authors' compliance as suggested by the European Commission.
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\n\nMetadata for all publications is also automatically deposited in IntechOpen's OAI repository, making them available through the Open Access Infrastructure for Research in Europe's (OpenAIRE) search interface further establishing our compliance.
\n\nIn other words, publishing with IntechOpen guarantees compliance.
\n\nRead more about Open Access in Horizon 2020 here.
\n\nWhich scientific publication to choose?
\n\nWhen choosing a publication, Horizon 2020 grant recipients are encouraged to provide open access to various types of scientific publications including monographs, edited books and conference proceedings.
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