**3. Deregulation of the platelets' role in dengue: Hemostasis, site of virus, and immune cells**

#### **3.1. Deregulation between pro-and anticoagulant mechanisms in dengue**

Hemostasis is a dynamic process in which physical and biochemical mechanisms promote blood clotting in a fast and regulated way [110]. Platelet adhesion and activation are mediated through the interactions between GP Ib-IX-V receptors with vWF and GP VI with subendothelial collagen [111]. Alternatively, when the vascular endothelium is damaged, collagen sites are exposed, facilitating platelet adhesion at these specific sites. Platelets adhere to molecules in the subendothelial tissues at the lesion site, where they aggregate and interact with leukocytes and endothelial cells, thus initiating the coagulation cascade [112]. The coagulation factors present in the bloodstream are involved in a tightly controlled activation sequence, resulting in the formation of thrombin and subsequently fibrin. These factors circulate as zymogens, which require processing by proteolysis to be activated. According to the new model of blood clotting, it occurs in three overlapping stages: initiation phase, amplification phase, and propagation phase [113].

During acute DENV infection, coagulation and fibrinolysis are activated, leading to coagulation changes and fibrinolytic parameters that may lead to disseminated intravascular coagulation (DIC) [114–116]. Funahara et al. reported that dengue patients with DIC had decreased platelet counts, transient prolongations of partial thromboplastin time (PTT) and prothrombin time (PT), and decreased levels of fibrinogen, prothrombin activity, factor VIII, antithrombin III, and plasminogen [117]. DIC leads to platelet activation, formation of fibrin, and deposition of small clots in the microcirculation, possibly contributing to organic failure. Notably, the consumption of clotting factors usually leads to paradoxical hemorrhagic disorders due to their consumption [118]. Later, it has been demonstrated that acute DIC that occurs in patients with DHF is associated with increased vascular permeability [117]. Thus, parameters such as platelet count, PTT, and PT present predictive value in the diagnosis of severe dengue [119].

levels of DHF patients with shock decreased significantly than those of normal controls and

In addition to exerting an effector role, platelets influence the production of cytokines by peripheral mononuclear cells. Activated platelets exhibit anti-inflammatory properties related to the CD40 and CD40L interaction, leading to increased IL-10 production and inhibition of TNF-α by monocytes [104]. The authors also verified that the interaction of apoptotic monocytes and platelets regulates the secretion of IL-10 through the recognition of platelet phosphatidylserine. It appears that IL-10 secretion requires only monocyte-platelet contact, but not phagocytosis, indicating that activated and apoptotic platelets aggregate to monocytes during infection [86]. Azeredo et al. found that IL-10 levels were correlated with low platelet counts [34]. Platelets are the major source of TGF-β1 in the human body [105]. One study has shown that circulating levels of TGF-β1 are significantly lower in patients with DHF than in controls [106]. Patients with immune thrombocytopenia have low levels of TGF-β1 in the circulation. However, after therapy to restore normal platelet count, their TGF-β1 levels return to levels

The innate immune system recognizes infection and changes in cellular homeostasis to initiate responses to clear pathogens and repair tissue damage. Toll-like receptors (TLRs) are part of the innate immune system, key players that modulate the inflammatory response and tumor dynamics. Many investigators have confirmed the expression of TLR1-9 both human and murine platelets [108]. Other major complex involved in these processes is the inflammasome, a multimeric protein complex that activates pro-caspase-1, which then proceeds to cleave multiple substrates including the pro-inflammatory cytokines IL-1β and IL-18 [109]. The presence of the nucleotide-binding domain leucine rich repeat containing protein (NLRP3) inflammasome in platelets activated after infection by DENV has been described, inducing the production of IL-1β by platelets and platelet-derived microparticles of dengue patients [89].

**3. Deregulation of the platelets' role in dengue: Hemostasis, site of** 

Hemostasis is a dynamic process in which physical and biochemical mechanisms promote blood clotting in a fast and regulated way [110]. Platelet adhesion and activation are mediated through the interactions between GP Ib-IX-V receptors with vWF and GP VI with subendothelial collagen [111]. Alternatively, when the vascular endothelium is damaged, collagen sites are exposed, facilitating platelet adhesion at these specific sites. Platelets adhere to molecules in the subendothelial tissues at the lesion site, where they aggregate and interact with leukocytes and endothelial cells, thus initiating the coagulation cascade [112]. The coagulation factors present in the bloodstream are involved in a tightly controlled activation sequence, resulting in the formation of thrombin and subsequently fibrin. These factors circulate as zymogens, which require processing by proteolysis to be activated. According to the new model of blood clotting, it occurs in three overlapping stages: initiation phase, amplification phase, and propagation phase [113].

**3.1. Deregulation between pro-and anticoagulant mechanisms in dengue**

production

DHF patients without shock patients, supposing that failure or inadequate TXB2

may eventually lead to shock [103].

14 Thrombocytopenia

found in healthy controls [107].

**virus, and immune cells**

The mechanisms that trigger DIC are mainly related to endothelial lesions and increased circulating TF levels [118]. Several studies have suggested that increased TF expression plays an important role in the pathogenesis of dengue. Huerta-Zepeda et al. showed that DENV regulates levels of protease-activated receptor type 1 (PAR-1) and TF in the activated endothelium [120]. These data are reinforced by the evidence of increased plasma levels of TF in dengue patients, and the expression of TF in monocytes was inversely correlated with platelet counts [121, 122]. Our previous data found that dengue patients with a good outcome showed decreased circulating levels of TF than those with a poor outcome (Severe). Similarly to TF, tissue factor pathway inhibitor (TFPI) levels were significantly lower in patients with a good outcome, but increased TFPI plasma levels were observed in severe patients. We also demonstrated that TF and TFPI levels were significantly higher among patients with hemorrhagic manifestations. In addition, DENV-1 or -2 patients were more likely to have increased levels of TF than DENV-4 patients [123]. Activation of PAR-1 is accompanied by positive regulation of adhesion molecules and production of proinflammatory cytokines [124]. The coagulation enzymes generated in DENV infection can activate PAR-1 receptors, thus increasing the increase of pro-inflammatory cytokines and leukocyte migration. These cytokines, along with coagulation enzymes (and *vice versa*), perpetuate the inflammatory response, which promotes increased interaction between activated monocytes, activated endothelial cells, and platelets. The result is a convergence of signals that lead to exacerbated expression of TF. Therefore, the coagulation and inflammation processes are closely related and establish a bidirectional relationship mediated by the activation of PARs [125].

Since hemostasis depends on the balance between coagulation and fibrinolysis, Huang et al. evaluated some coagulation parameters (platelet count and PTT) as well as fibrinolytic parameters (tPA and PAI-1) in patients with DHF and DF. Patients with DF show thrombocytopenia, PTT prolongation, and increased tPA levels, indicating coagulation activation and fibrinolysis. However, the parameters used indicated much more severe activation of coagulation and fibrinolysis in patients with DHF. In the convalescent phase, there is an increase in PAI-1 and platelet counts with concomitant decline in tPA levels and normalization of PTT, both in patients with DHF and in DF. According to the authors, the activation of coagulation and fibrinolysis during the acute phase of DENV infection is compensated by the increase of platelets and PAI-1 during the convalescence phase. These results suggest that the degree of activation of coagulation and fibrinolysis induced during dengue infection is associated with the severity of the disease [114].

#### **3.2. Platelets as target cells for DENV replication**

Platelets contain receptors related to DENV entry, such as Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) [126]. *In vitro* infection by DENV induces activation, mitochondrial dysfunction, and platelet apoptosis through mechanisms dependent on the DC-SIGN receptor [127]. A study detected DENV RNA by conventional reverse transcription polymerase chain reaction (RT-PCR) and DENV-like particles by electron microscopy in patients' platelets [128] and later confirmed the presence of viral antigen in platelets by immunofluorescence and confocal microscopy [129]. It is still unclear whether platelets are involved in the spread of viral infection. Recent data from our group have confirmed the *in vitro* interaction of DENV with platelets, leading to subsequent morphological modifications characteristic of platelet activation, such as presence of membrane extensions (filopodia), loss of cytoplasmic content and dilation of the membrane system [Azamor and Oliveira submitted]. A study using blood cells from dengue patients and rhesus monkeys experimentally infected revealed DENV antigens present in vesicles of varying sizes and often in nuclear cells like platelets. The DENV RNA was detected in a highly enriched population of rhesus platelet-characteristic CD61+ cells in the acute phase of infection, indicating that the virus may be directly linked to dysfunction and low platelet counts [130]. More recent and for the first time, authors demonstrated in fact that platelets directly bind DENV saturably and produce infectious virus. Interestingly, at 37 and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days, with production of viral NS1 protein. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. DC-SIGN and heparan sulfate proteoglycan (HSP) were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism [131]. Finally, very recent analyze revealed that DENV works as the primary driver of platelet activation and also enters and replicates in platelets but does not result in a productive infection of platelets. Moreover, the DENV-exposed/DENV antigen-positive platelets associate with CD14+CD16+ monocytes may mediate platelet clearance from the circulation [132].

action is that of TLRs. Among the 10 TLRs identified in humans, six are expressed in platelets, the TLR-1, -2, -4, -6, -8, and -9. TLR-4 has been shown to modulate sepsis and inducer of TNF-α *in vivo* [134]. TLR-2 modulates IL-1β RNA processing, inducing the production of IFN type 1 and other inflammatory cytokines [135]. The set of inflammatory, hemostatic, angiogenic, and coagulators reactions are multicellular events that include chemotaxis, adhesion, interactions between leukocytes, endothelial cells, and platelets in the walls of blood vessels. Platelets contribute to these interactions through secretion of adhesion proteins and regulation of chemokine synthesis by leukocytes and endothelial cells [136]. Molecules such as IL-1β, PAF, and P-CD62 stand out as the main mediators derived from platelets, able of activating leukocytes [137]. Studies have shown that activated platelets induce increased IL-10

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17

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

Several studies have indicated that DENV infection leads to the activation of endothelial cells, which increase the expression on the surface of the E-CD62 molecule. E-CD62, as well as P-CD62, are adhesion molecules responsible for platelet adhesion to endothelial cells [113, 138]. In addition to the endothelium, P-CD62 is expressed on the surface of activated platelets, promoting their interaction with leukocytes and formation of aggregates between platelets and monocytes and/ or neutrophils in primates [139]. Platelet-monocyte aggregates are also observed in DENV-infected patients, leading to the synthesis of IL-1β, CXCL8/ IL-8, CCL4/ MCP-1, and IL-10 by monocytes [107]. The formation of cell aggregates results in an increase in the inflammatory response in dengue, as well as contributing to the generation of thrombocytopenia both by the physical retention of cells, by lowering the number of circulating cells and by the induction of cell death [140]. In 1992, Butthep et al. showed that platelets, as well as neutrophils and lymphocytes, preferentially bind to endothelial cells exposed to DENV, compared to cells not exposed to DENV. It has been suggested that increased platelet endothelial cell binding may contribute to thrombocytopenia in dengue patients [141]. Protein disulfide isomerase (PDI), an endoplasmic reticular protein, is located on the surface of platelets [142] and is involved in the regulation of integrin-mediated platelet aggregation, since anti-PDI antibodies block platelet adhesion and aggregation [143]. Previous studies have demonstrated that platelet surface PDI can be recognized by anti-NS1 antibodies. Rachman and co-workers observed a similar kinetic profile between anti-NS1 and PDI antibodies [144]. PDI enzyme activity and platelet aggregation were reduced with anti-NS1 action. Amino acid residues 311–330 (P311–330) of NS1 represent an epitope that shares sequence homology with the PDI thiorretoxin domain [145]. In contrast, although the serum of dengue patients inhibits platelet aggregation, there is no correlation between anti-NS1 and PDI with platelet aggregation dysfunction, suggesting that other mechanisms may be involved in the inhibition of platelet aggregation [144, 145]. Platelets are responsible for the maintenance of vascular integrity due to the constitutive release of pro-angiogenic cytokines and growth factors. The α-granule-derived molecules such as angiopoietin-1, α and β-catenins, and PAF bind to specific receptors on the surface of endothelial cells, causing intracellular signaling that stabilizes the intercellular adhesion junctions [146]. Angiopoietins, key molecules of vascular integrity, are also stored in platelets. Both dengue-associated thrombocytopenia and endothelial activation are associated with an imbalance in the ratio of angiopoietin-2: angiopoietin-1 plasmatic. Studies have shown that there is an inverse correlation between angiopoietin-1 and plasma extravasation markers but a direct correlation between angiopoietin-2 and markers of plasma

expression and decreased TNF-α by monocytes [101].

#### **3.3. Platelets as immune cells**

Platelets, when interacting physically with leukocytes and endothelial cells, act as a kind of signaling of inflammation and immune response. When stimulated and activated, these cells initiate events, such as aggregation, formation of microparticles and exosomes [133], expression of adhesion proteins and receptors, and exocytosis of constituents present in their granules [76]. Exocytosis of α-dense granules and lysosomes releases cytokines and biological mediators with various immunological and inflammatory functions. In addition to secreting soluble mediators, platelets express receptors involved with the immune defense, such as Fc receptors that are able to recognize IgG, IgE, and IgA classes. These receptors may confer rudimentary antibacterial activities of platelets, such as the secretion of antimicrobial peptides and phagocytosis against direct interaction with bacteria, viruses, protozoa, or helminths. These immunoreceptors influence platelet adhesion activity through the modulation of integrin production [76]. Another set of receptors present on platelets with immunological action is that of TLRs. Among the 10 TLRs identified in humans, six are expressed in platelets, the TLR-1, -2, -4, -6, -8, and -9. TLR-4 has been shown to modulate sepsis and inducer of TNF-α *in vivo* [134]. TLR-2 modulates IL-1β RNA processing, inducing the production of IFN type 1 and other inflammatory cytokines [135]. The set of inflammatory, hemostatic, angiogenic, and coagulators reactions are multicellular events that include chemotaxis, adhesion, interactions between leukocytes, endothelial cells, and platelets in the walls of blood vessels. Platelets contribute to these interactions through secretion of adhesion proteins and regulation of chemokine synthesis by leukocytes and endothelial cells [136]. Molecules such as IL-1β, PAF, and P-CD62 stand out as the main mediators derived from platelets, able of activating leukocytes [137]. Studies have shown that activated platelets induce increased IL-10 expression and decreased TNF-α by monocytes [101].

**3.2. Platelets as target cells for DENV replication**

16 Thrombocytopenia

Platelets contain receptors related to DENV entry, such as Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) [126]. *In vitro* infection by DENV induces activation, mitochondrial dysfunction, and platelet apoptosis through mechanisms dependent on the DC-SIGN receptor [127]. A study detected DENV RNA by conventional reverse transcription polymerase chain reaction (RT-PCR) and DENV-like particles by electron microscopy in patients' platelets [128] and later confirmed the presence of viral antigen in platelets by immunofluorescence and confocal microscopy [129]. It is still unclear whether platelets are involved in the spread of viral infection. Recent data from our group have confirmed the *in vitro* interaction of DENV with platelets, leading to subsequent morphological modifications characteristic of platelet activation, such as presence of membrane extensions (filopodia), loss of cytoplasmic content and dilation of the membrane system [Azamor and Oliveira submitted]. A study using blood cells from dengue patients and rhesus monkeys experimentally infected revealed DENV antigens present in vesicles of varying sizes and often in nuclear cells like platelets. The DENV RNA was detected in a highly enriched population of rhesus platelet-characteristic CD61+ cells in the acute phase of infection, indicating that the virus may be directly linked to dysfunction and low platelet counts [130]. More recent and for the first time, authors demonstrated in fact that platelets directly bind DENV saturably and produce infectious virus. Interestingly, at 37 and 25°C, platelets replicated the positive sense single-stranded RNA genome of DENV by up to ∼4-fold over 7 days, with production of viral NS1 protein. The infectivity of DENV intrinsically decayed in vitro, which was moderated by platelet-mediated generation of viable progeny. DC-SIGN and heparan sulfate proteoglycan (HSP) were implicated as coreceptors because only the combination of anti-DC-SIGN and low-molecular-weight heparin prevented binding. Thus, expression of antigen encoded by DENV is a novel consideration in the pathogen-induced thrombocytopenia mechanism [131]. Finally, very recent analyze revealed that DENV works as the primary driver of platelet activation and also enters and replicates in platelets but does not result in a productive infection of platelets. Moreover, the DENV-exposed/DENV antigen-positive platelets associate with

CD14+CD16+ monocytes may mediate platelet clearance from the circulation [132].

Platelets, when interacting physically with leukocytes and endothelial cells, act as a kind of signaling of inflammation and immune response. When stimulated and activated, these cells initiate events, such as aggregation, formation of microparticles and exosomes [133], expression of adhesion proteins and receptors, and exocytosis of constituents present in their granules [76]. Exocytosis of α-dense granules and lysosomes releases cytokines and biological mediators with various immunological and inflammatory functions. In addition to secreting soluble mediators, platelets express receptors involved with the immune defense, such as Fc receptors that are able to recognize IgG, IgE, and IgA classes. These receptors may confer rudimentary antibacterial activities of platelets, such as the secretion of antimicrobial peptides and phagocytosis against direct interaction with bacteria, viruses, protozoa, or helminths. These immunoreceptors influence platelet adhesion activity through the modulation of integrin production [76]. Another set of receptors present on platelets with immunological

**3.3. Platelets as immune cells**

Several studies have indicated that DENV infection leads to the activation of endothelial cells, which increase the expression on the surface of the E-CD62 molecule. E-CD62, as well as P-CD62, are adhesion molecules responsible for platelet adhesion to endothelial cells [113, 138]. In addition to the endothelium, P-CD62 is expressed on the surface of activated platelets, promoting their interaction with leukocytes and formation of aggregates between platelets and monocytes and/ or neutrophils in primates [139]. Platelet-monocyte aggregates are also observed in DENV-infected patients, leading to the synthesis of IL-1β, CXCL8/ IL-8, CCL4/ MCP-1, and IL-10 by monocytes [107]. The formation of cell aggregates results in an increase in the inflammatory response in dengue, as well as contributing to the generation of thrombocytopenia both by the physical retention of cells, by lowering the number of circulating cells and by the induction of cell death [140]. In 1992, Butthep et al. showed that platelets, as well as neutrophils and lymphocytes, preferentially bind to endothelial cells exposed to DENV, compared to cells not exposed to DENV. It has been suggested that increased platelet endothelial cell binding may contribute to thrombocytopenia in dengue patients [141]. Protein disulfide isomerase (PDI), an endoplasmic reticular protein, is located on the surface of platelets [142] and is involved in the regulation of integrin-mediated platelet aggregation, since anti-PDI antibodies block platelet adhesion and aggregation [143]. Previous studies have demonstrated that platelet surface PDI can be recognized by anti-NS1 antibodies. Rachman and co-workers observed a similar kinetic profile between anti-NS1 and PDI antibodies [144]. PDI enzyme activity and platelet aggregation were reduced with anti-NS1 action. Amino acid residues 311–330 (P311–330) of NS1 represent an epitope that shares sequence homology with the PDI thiorretoxin domain [145]. In contrast, although the serum of dengue patients inhibits platelet aggregation, there is no correlation between anti-NS1 and PDI with platelet aggregation dysfunction, suggesting that other mechanisms may be involved in the inhibition of platelet aggregation [144, 145]. Platelets are responsible for the maintenance of vascular integrity due to the constitutive release of pro-angiogenic cytokines and growth factors. The α-granule-derived molecules such as angiopoietin-1, α and β-catenins, and PAF bind to specific receptors on the surface of endothelial cells, causing intracellular signaling that stabilizes the intercellular adhesion junctions [146]. Angiopoietins, key molecules of vascular integrity, are also stored in platelets. Both dengue-associated thrombocytopenia and endothelial activation are associated with an imbalance in the ratio of angiopoietin-2: angiopoietin-1 plasmatic. Studies have shown that there is an inverse correlation between angiopoietin-1 and plasma extravasation markers but a direct correlation between angiopoietin-2 and markers of plasma extravasation in patients with DHF/DSS [147]. Hottz et al. demonstrated that DENV-infected patients who showed signs of increased vascular permeability demonstrated a higher percentage of platelets and platelet-derived microparticles (MP) expressing IL-1β and caspase-1 activator compared to patients who had no evidence change in vascular permeability. These results were confirmed in experiments in which platelet-derived MPs exposed to DENV caused an increase in the permeability of endothelial cells that was blocked by IL-1Ra [127].

DF dengue fever or dengue fever without signs of alarm

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DHF dengue hemorrhagic fever DSS dengue shock syndrome

WHO World Health Organization

ADE antibody-dependent infection

pDC plasmacytoid dendritic cells

HLA human leukocyte antigen APCs antigen-presenting cells

ICAM-1 intracellular adhesion molecule-1 VCAM-1 vascular cell adhesion molecule-1

TGF-β transforming growth factor-beta-receptor

sTRAIL tumor necrosis factor-related apoptosis-inducing ligand

sST2 soluble IL-1 receptor type 1 protein

DENV-EIII DENV-envelope protein domain III

RGD sequence Arginine-Glycine-Asparagine peptide sequence

PT profound thrombocytopenia

PAF platelet-activating factor

vWF von Willebrand factor ADP adenosine diphosphate

TXA2 thromboxane A2 GP glycoprotein

TPO thrombopoietin

P-CD62 P-selectin

IFNs interferons

NKs natural killer cells

DCs dendritic cells

IFN-γ or IFN-type II

IFN-α/β type I IFNs

E-CD62 E-selectin

DFwWS dengue fever with warning signals

More recently, proteome analysis related to platelet activating signaling from platelets from dengue patients demonstrated an increase of PAR-4 (F2RL3), G protein subunits (GNA12 and GNA14), and p38 MAPK (MAPK14), in which potentially contributing to increased platelet activation during dengue infection. Moreover, dengue patients had increased P-CD62 surface expression on platelets from patients presenting dengue with warning signs and severe dengue syndromes compared to mild dengue. In agreement, they observed exhaustion of the granule-stored chemokine PF4/CXCL4 in platelets from patients with dengue [148], similarly to another study reported that patients with severe dengue have lower levels of PF4/ CXCL4 in plasma when compared to mild dengue patients [149].
