**A View of Platelets in Dengue A View of Platelets in Dengue**

 Tamiris Azamor da Costa Barros and Luzia Maria de-Oliveira-Pinto Luzia Maria de-Oliveira-Pinto

Tamiris Azamor da Costa Barros and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.73084

#### **Abstract**

Platelets were mainly associated with coagulation and hemostasis; however, other biological effects have been attributed to platelets, including angiogenesis, extracellular matrix synthesis, inflammation, and immune response. Dengue virus infection causes 200 million cases of severe flu-like illness annually, escalating to life-threatening hemorrhagic fever or shock syndrome. Some hypotheses are postulated for immunopathogenesis of dengue, including antibody enhancement theory, T-cell activation of cross-reactive memory, and original antigenic sin. All hypotheses, to some extent, induce an overproduction or a skewed profile of cytokine release, giving rise to the term cytokine storm/cytokine tsunami. Although thrombocytopenia is typical of both mild and severe diseases, the mechanism triggering platelet reduction is incompletely understood. In dengue, platelets are one of the major cell populations affected by direct and/or indirect mechanisms of infection. It is common to observe both thrombocytopenia and platelet dysfunction in dengue, both strongly related to the clinical outcome. Thus, platelets are frequently affected in dengue, either for alteration of their own functionality, for "silent transport" of virus, or as an anti-viral immune cell. In this way, we describe some of functional aspects of platelets on dengue, observing circulating mediators, intraplatelet proteins contents, morphology, activation markers, and ability to interact with dengue virus.

DOI: 10.5772/intechopen.73084

**Keywords:** dengue, platelets, thrombocytopenia, dysfunction of platelets, immunopathogenesis

#### **1. Introduction**

As the first cellular components accumulate at sites where there is vascular wall damage, platelets rapidly initiate events such as aggregation, exocytosis of granule constituents, adhesion protein expression, cytokine, and others inflammatory mediator's secretion and directly interact with endothelial cells and immune cells. In addition, they can perform the synthesis

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of new proteins through their complex post-transcription repertoire for post-activation translation, corroborating the existence of potential biological functions of platelets. In dengue, platelets are one of the major cell populations affected by direct and/or indirect mechanisms of infection.

dengue [1]. This classification was based on a multicenter study that treats the illness as a dynamic and systemic event. The new classification describes three sets of clinical signs and symptoms: (1) Dengue fever without signs of alarm (DF) characterized by nausea, vomiting, rash, myalgia, headache, arthralgia, and positive tourniquet test with no signs bleeding and leukopenia; (2) Dengue fever with warning signals (DFwWS) includes abdominal pain or tenderness, persistent vomiting, fluid accumulation, lethargy, agitation, hepatomegaly (increase > 2 cm), elevated serum transaminases, and decreased platelet count; and (3) Severe Dengue Fever characterized by severe plasma extravasation, leading to shock and fluid accumulation, accompanied by respiratory discomfort, severe bleeding, and involvement of organs such as the liver, central nervous system (with altered consciousness), heart, and other organs. This new classification proved to be more sensitive to the identification of severe forms, reducing the proportion of patients previously unclassifiable, which facilitated

A View of Platelets in Dengue

9

http://dx.doi.org/10.5772/intechopen.73084

Briefly, immunopathogenesis of dengue is postulated by some hypotheses, including antibody enhancement theory [11, 12], T-cell activation of cross-reactive memory, and original antigenic sin [13]. All hypotheses, to some extent, induce an overproduction or a skewed pro-

The humoral response is mainly directed to the prM, E, and NS1 proteins, whereas in the cases of secondary infection, the response against NS3 and NS5 is observed [15, 16]. It is believed that a primary infection can create effective, lasting protection against reinfection by the same serotype, but triggers short-term cross-protection against the other serotypes [17]. Neutralization of infection by specific antibodies may occur by inhibiting the entry of the virus through its specific receptors into the target cell [18] or by inhibiting viral fusion into the target cell cytoplasm [19]. On the other hand, epidemiological studies suggest that homologous immunity may increase the severity of the disease during a subsequent infection by a heterologous serotype [11]. It is believed that low neutralizing antibodies, those that induce cross-reaction, produced in response to a previous serotype, contribute to the pathogenesis of dengue by promoting the entry of the virus through Fcγ receptors into myeloid cells, a

The role of T cells during dengue infection is still controversial, with studies supporting either an immunoprotective or immunopathological role [21]. Pioneer studies proposed that T cells have a detrimental role during secondary dengue infections in a process termed "original antigenic sin." Based on this theory, cross-reactive T cells generated during primary infection, which recognize secondary-infected DENV serotype with low affinity, are poorly functional but prone to inducing immunopathology [13]. Thus, as cross-reactive memory, T cells are present in increased numbers and have a low activation threshold. They may outcompete their naïve subsets that have high affinity for secondary-infected serotype with an overall detrimental outcome for protective immunity [22]. Collectively, studies showed that dengue infection elicits a broad-specific T cell response that peaks around day 8–10 from fever onset [23, 24]. Dengue-specific CD8+ T cells are present at higher frequencies compared to their CD4+ counterparts and preferentially target nonstructural proteins NS3, NS4b, and NS5, while CD4+ T cells are mainly directed toward the capsid envelope and the secreted protein NS1 [23].

file of cytokine release, giving rise to the term cytokine storm/cytokine tsunami [14].

phenomenon known as antibody-dependent infection (ADE) [20].

the clinical management of patients [10].

#### **1.1. An overview of immunopathogenesis of dengue**

Dengue is one of the arboviruses transmitted by mosquitoes of the genus *Aedes* in a humanmosquito-human cycle. It is endemic in more than 120 countries, where 50 to 100 million infections occur each year, which correspond to 55% of the world population live at risk of infection. Therefore, dengue has the greatest impact on public health worldwide with higher morbidity, albeit fortunately with low mortality rate [1]. The etiologic agent is the dengue virus (DENV), which has four antigenically distinct viral serotypes, the DENV 1 to 4. DENVs share between 65 and 75% homology in their RNA sequences. As a member of the *Flaviviridae* family, the DENV consists of an envelope formed by a lipid bilayer derived from the endoplasmic reticulum of the host cell into which the envelope (E) and membrane (M) proteins are inserted. The viral particle is spherical in shape and approximately 50 nm in diameter. Below the viral envelope is a nucleocapsid composed of an icosahedral viral capsid formed by the capsid protein (C) and complexed to a single-stranded RNA molecule with positive polarity [2, 3]. Viral RNA DENV is approximately 10.7 Kb and is modified at its 5' end by the addition of the cap structure but is devoid of the poly-A tail at the 3' end and has a single open reading frame encoding a protein precursor polyprotein viral infection. This precursor protein is cleaved by both host cell proteases and viral protease, yielding 10 proteins: structural C, pre-Membrane (prM)/ M, E, and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The translated structural proteins are incorporated into the viral particles during their maturation, while the nonstructural proteins are involved in the replication and/ or assembly of the virions. The 3'and 5' noncoding regions are also important for viral replication [4–6].

Dengue fever is generally an acute disease, with a broad spectrum of clinical manifestations ranging from a clinically inapparent infection, an undifferentiated acute febrile illness, dengue fever (DF), to more severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The first symptoms of DF and DHF are indistinguishable, but DHF is associated with hemorrhagic manifestations, plasma extravasation, and thrombocytopenia (counts below 150,000 platelets/mm<sup>3</sup> ). Thrombocytopenia is not necessarily restricted to severe forms of dengue, since it is possible to see small bleeding in mild infections. DSS is distinguished from DHF by the presence of cardiovascular or other organs impairment, which occurs when loss of plasma to interstitial spaces results in shock. In general, DSS is a serious disease, with mortality rates of up to 20%, but may also be less than 1% in places with sufficient resources and with clinical experience [7, 8]. Symptomatic disease typically follows three phases: an initial febrile phase lasting 3 to 7 days; a critical phase around the defervescence during which complications may appear in some patients; and a spontaneous recovery phase [9].

A simplified categorization for the classification of dengue severity was proposed by the World Health Organization (WHO) in 2009 in which DHF and DSS were grouped as severe dengue [1]. This classification was based on a multicenter study that treats the illness as a dynamic and systemic event. The new classification describes three sets of clinical signs and symptoms: (1) Dengue fever without signs of alarm (DF) characterized by nausea, vomiting, rash, myalgia, headache, arthralgia, and positive tourniquet test with no signs bleeding and leukopenia; (2) Dengue fever with warning signals (DFwWS) includes abdominal pain or tenderness, persistent vomiting, fluid accumulation, lethargy, agitation, hepatomegaly (increase > 2 cm), elevated serum transaminases, and decreased platelet count; and (3) Severe Dengue Fever characterized by severe plasma extravasation, leading to shock and fluid accumulation, accompanied by respiratory discomfort, severe bleeding, and involvement of organs such as the liver, central nervous system (with altered consciousness), heart, and other organs. This new classification proved to be more sensitive to the identification of severe forms, reducing the proportion of patients previously unclassifiable, which facilitated the clinical management of patients [10].

of new proteins through their complex post-transcription repertoire for post-activation translation, corroborating the existence of potential biological functions of platelets. In dengue, platelets are one of the major cell populations affected by direct and/or indirect mechanisms

Dengue is one of the arboviruses transmitted by mosquitoes of the genus *Aedes* in a humanmosquito-human cycle. It is endemic in more than 120 countries, where 50 to 100 million infections occur each year, which correspond to 55% of the world population live at risk of infection. Therefore, dengue has the greatest impact on public health worldwide with higher morbidity, albeit fortunately with low mortality rate [1]. The etiologic agent is the dengue virus (DENV), which has four antigenically distinct viral serotypes, the DENV 1 to 4. DENVs share between 65 and 75% homology in their RNA sequences. As a member of the *Flaviviridae* family, the DENV consists of an envelope formed by a lipid bilayer derived from the endoplasmic reticulum of the host cell into which the envelope (E) and membrane (M) proteins are inserted. The viral particle is spherical in shape and approximately 50 nm in diameter. Below the viral envelope is a nucleocapsid composed of an icosahedral viral capsid formed by the capsid protein (C) and complexed to a single-stranded RNA molecule with positive polarity [2, 3]. Viral RNA DENV is approximately 10.7 Kb and is modified at its 5' end by the addition of the cap structure but is devoid of the poly-A tail at the 3' end and has a single open reading frame encoding a protein precursor polyprotein viral infection. This precursor protein is cleaved by both host cell proteases and viral protease, yielding 10 proteins: structural C, pre-Membrane (prM)/ M, E, and nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The translated structural proteins are incorporated into the viral particles during their maturation, while the nonstructural proteins are involved in the replication and/ or assembly of the virions. The 3'and 5' noncoding regions are also

Dengue fever is generally an acute disease, with a broad spectrum of clinical manifestations ranging from a clinically inapparent infection, an undifferentiated acute febrile illness, dengue fever (DF), to more severe forms, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The first symptoms of DF and DHF are indistinguishable, but DHF is associated with hemorrhagic manifestations, plasma extravasation, and thrombocytopenia (counts

of dengue, since it is possible to see small bleeding in mild infections. DSS is distinguished from DHF by the presence of cardiovascular or other organs impairment, which occurs when loss of plasma to interstitial spaces results in shock. In general, DSS is a serious disease, with mortality rates of up to 20%, but may also be less than 1% in places with sufficient resources and with clinical experience [7, 8]. Symptomatic disease typically follows three phases: an initial febrile phase lasting 3 to 7 days; a critical phase around the defervescence during which

A simplified categorization for the classification of dengue severity was proposed by the World Health Organization (WHO) in 2009 in which DHF and DSS were grouped as severe

complications may appear in some patients; and a spontaneous recovery phase [9].

). Thrombocytopenia is not necessarily restricted to severe forms

of infection.

8 Thrombocytopenia

**1.1. An overview of immunopathogenesis of dengue**

important for viral replication [4–6].

below 150,000 platelets/mm<sup>3</sup>

Briefly, immunopathogenesis of dengue is postulated by some hypotheses, including antibody enhancement theory [11, 12], T-cell activation of cross-reactive memory, and original antigenic sin [13]. All hypotheses, to some extent, induce an overproduction or a skewed profile of cytokine release, giving rise to the term cytokine storm/cytokine tsunami [14].

The humoral response is mainly directed to the prM, E, and NS1 proteins, whereas in the cases of secondary infection, the response against NS3 and NS5 is observed [15, 16]. It is believed that a primary infection can create effective, lasting protection against reinfection by the same serotype, but triggers short-term cross-protection against the other serotypes [17]. Neutralization of infection by specific antibodies may occur by inhibiting the entry of the virus through its specific receptors into the target cell [18] or by inhibiting viral fusion into the target cell cytoplasm [19]. On the other hand, epidemiological studies suggest that homologous immunity may increase the severity of the disease during a subsequent infection by a heterologous serotype [11]. It is believed that low neutralizing antibodies, those that induce cross-reaction, produced in response to a previous serotype, contribute to the pathogenesis of dengue by promoting the entry of the virus through Fcγ receptors into myeloid cells, a phenomenon known as antibody-dependent infection (ADE) [20].

The role of T cells during dengue infection is still controversial, with studies supporting either an immunoprotective or immunopathological role [21]. Pioneer studies proposed that T cells have a detrimental role during secondary dengue infections in a process termed "original antigenic sin." Based on this theory, cross-reactive T cells generated during primary infection, which recognize secondary-infected DENV serotype with low affinity, are poorly functional but prone to inducing immunopathology [13]. Thus, as cross-reactive memory, T cells are present in increased numbers and have a low activation threshold. They may outcompete their naïve subsets that have high affinity for secondary-infected serotype with an overall detrimental outcome for protective immunity [22]. Collectively, studies showed that dengue infection elicits a broad-specific T cell response that peaks around day 8–10 from fever onset [23, 24]. Dengue-specific CD8+ T cells are present at higher frequencies compared to their CD4+ counterparts and preferentially target nonstructural proteins NS3, NS4b, and NS5, while CD4+ T cells are mainly directed toward the capsid envelope and the secreted protein NS1 [23].

Moreover, studies have demonstrated that high concentrations of circulating cytokines, mainly released by T cells, monocytes, macrophages, and endothelial cells from patients, would be involved in the pathogenesis of dengue [24]. Initially, antiviral mechanisms of innate immune response mediated by interferons (IFNs), mainly produced by dendritic cells (DCs), monocytes, macrophages, and natural killer (NKs) cells, are involved in initial infection control. The antiviral activity of type I IFNs (IFN-α/β) is initiated hours after infection and promotes inhibition of viral replication of infected cells, activation of the antiviral state by uninfected cells, and stimulation of the antiviral activity of the cells NK and CD8+ T lymphocytes [25, 26]. DENV proteins such as NS4B and NS5 have been shown to inhibit IFN-α/-β signaling [27–29]. However, *in vitro* and *in vivo* studies have demonstrated that DENV is capable of activating the production of IFN-α by human plasmacytoid dendritic cells (pDC) [30]. The IFN-γ (or IFN-type II), a cytokine involved with Th1 profile, is produced primarily by T lymphocytes, NK cells, and to a lesser extent by macrophages. The IFN-γ, like other IFNs, has an antiviral effect and promotes increased expression of human leukocyte antigen (HLA) class I and II molecules and stimulates antigen presenting and cytokine production by antigen-presenting cells (APCs) [31]. Kurane et al. reported higher levels of IFN-γ in the serum of patients with DHF and DF compared to healthy subjects, but IFN-γ levels were still higher after defervescence in patients with DHF. According to the authors, these results suggest that IFN-γ would play an important role in infection control; however, high levels of this cytokine after defervescence, together with increased T cell activation, would contribute to the pathogenesis of DHF [32]. TNF-α is another cytokine that appears to play an important role in dengue. TNF-α is produced by mononuclear phagocytes, neutrophils, lymphocytes, and NK cells. The interaction of TNF-α and endothelial cells promotes induction of adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (E-CD62), being strongly involved in vascular damage, septic shock, and anti-tumor immunity [31]. In dengue, TNF-α appears to be involved in vascular damage, and authors observed an increased permeability and morphological changes in endothelial cells treated in vitro with TNF-α [33]. Studies have shown elevated plasmatic cytokines in dengue, such as IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13, and IL-18, transforming growth factor-beta-receptor (TGF-β) [34–40]. Chaturvedi et al. reported that DF patients had higher levels of IFN-γ and IL-2, whereas the majority of DHF patients had IL-4, IL-6, and IL-10 elevation, on the 4th and 8th days of the disease, coinciding with the defervescence phase [41]. Pretreatment of monocytes/macrophages with Th2 profile cytokines (IL-4 or IL-13) increased the susceptibility of these cells to DENV infection [42]. Plasma levels of IL-10 were correlated with thrombocytopenia in dengue patients [34, 43]. High production of TNF-α, IL-1β, IL-12, IL-17, soluble IL-1 receptor type 1 protein (sST2), and tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), as well as apoptosis in DENV-infected monocyte/macrophages cultures, was also observed. It has been shown, therefore, that beneficial or deleterious biomarkers may be present in dengue, regardless of the severity of the disease [44].

[45–47]. Thrombocytopenia occurs when platelet formation (thrombopoiesis) is insufficient to balance physiological or pathological platelet consumption. Thrombocytopenia may occur in patients with either mild or severe cases of dengue infection and are associated in the early days of dengue infection [1]. The WHO guidelines for 2009 reaffirmed that a rapid decline or

ing. Together, the functional disturbance associated with deregulation of the plasma quinine

The kinetic observation of platelet counts in dengue patients showed a mild to moderate decrease in the 3rd to the 7th day, a significant decrease on day 4, reaching normal levels in the 8th or 9th day of the disease [50, 51]. Profound thrombocytopenia (nadir platelet count ≤20,000/mm<sup>3</sup>

significantly more likely to detect early warning signs and longer hospital stays, but profound thrombocytopenia was not affected by DENV serotypes, coinfections, and secondary DENV infections [52]. However, a study involving 245 dengue patients showed no relationship between bleeding and platelet count, while 81 nonbleeding patients had a score below 20,000/mm<sup>3</sup>

In contrast, another study involving 225 dengue patients suggested that bleeding occurred more frequently in patients with PT [54]. Most clinical guidelines recommend platelet transfusion in patients with dengue who develop severe bleeding or platelet counts below 10–20,000/mm<sup>3</sup>

However, another study confirms that platelet transfusion does not prevent the development of severe bleeding or shorten coagulation time [55], and in severe dengue disease with hemorrhagic

Previous published data indicated that DENV can induce thrombocytopenia through bone marrow suppression, lysis of megakaryocytes, and/or peripheral destruction of platelets [56]. Three main mechanisms seem to be involved, although partially explained, such as a direct lesion of progenitor cells by DENV, infected stromal cells, and modification of bone marrow regulation [51]. In fact, studies have shown a hypocellularity in bone marrow and inhibition of megakaryocyte maturation [51, 57]. *In vitro* studies using an adventitious reticular cell line, which are bone marrow stromal cells, incubated with DENV found DENV antigens in the perinuclear region of these cells [58]. These interactions lead to a modification in the cytokine profile produced in the bone marrow, as in the case of TGF-β capable of inhibiting the differentiation of multipotent stem cells into megakaryocyte precursor cells, leading to inhibition of the cell differentiation process [59, 60]. Another cytokine, the thrombopoietin (TPO), regulates megakaryocytopoiesis and platelet production specifically through the activation of myeloproliferative leukemia virus oncogene (c-MPL), the TPO receptor [61, 62]. When platelet counts fall, circulating levels of TPO increase and may function as a useful indicator of megakaryocytopoiesis in dengue [63, 64]. Recently, authors showed that mice inoculated with recombinant DENV-envelope protein domain III (DENV-EIII)-suppressed megakaryopoiesis of progenitor cells from murine bone marrow and human cord blood in vitro, similarly to those observed with DENV infection. Additional analyses suggested that autophagy impairment and apoptosis are involved in DENV-EIII-mediated suppression of megakaryopoiesis. Thus, these data suggest that, even without viral replication, the binding of DENV-EIII to the

manifestations, the need for intensive care was not significantly associated with PT [52].

system is related with the immunopathogenesis of dengue [1, 48, 49].

**megakaryocytes and/or peripheral destruction of platelets**

cell surface is sufficient to suppress megakaryopoiesis [65].

**2.1. Thrombocytopenia induced by bone marrow suppression, lysis of** 

of blood are one of the indicators of clinical dengue worsen-

) was

A View of Platelets in Dengue

11

http://dx.doi.org/10.5772/intechopen.73084

[53].

.

platelet count below 150,000/mm<sup>3</sup>
