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Review ArticleThrombocytopenia in Dengue Interrelationship betweenVirus and the Imbalance between Coagulation and Fibrinolysisand Inflammatory Mediators

Elzinandes Leal de Azeredo1 Robson Q Monteiro2 and Luzia Maria de-Oliveira Pinto1

1Laboratorio de Imunologia Viral IOC FIOCRUZ Avenida Brasil 4365 Manguinhos 21040-360 Rio de Janeiro RJ Brazil2Instituto de Bioquimica Medica Leopoldo de Meis Universidade Federal do Rio de Janeiro 21941-599 Rio de Janeiro RJ Brazil

Correspondence should be addressed to Luzia Maria de-Oliveira Pinto lpintoiocfiocruzbr

Received 25 October 2014 Accepted 22 January 2015

Academic Editor Trai-Ming Yeh

Copyright copy 2015 Elzinandes Leal de Azeredo et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Dengue is an infectious disease caused by dengue virus (DENV) In general dengue is a self-limiting acute febrile illness followedby a phase of critical defervescence in which patients may improve or progress to a severe form Severe illness is characterizedby hemodynamic disturbances increased vascular permeability hypovolemia hypotension and shock Thrombocytopenia andplatelet dysfunction are common in both cases and are related to the clinical outcome Different mechanisms have beenhypothesized to explain DENV-associated thrombocytopenia including the suppression of bone marrow and the peripheraldestruction of platelets Studies have shown DENV-infected hematopoietic progenitors or bone marrow stromal cells Moreoveranti-platelet antibodies would be involved in peripheral platelet destruction as platelets interact with endothelial cells immunecells andor DENV It is not yet clear whether platelets play a role in the viral spread Here we focus on the mechanisms ofthrombocytopenia and platelet dysfunction in DENV infection Because platelets participate in the inflammatory and immuneresponse by promoting cytokine chemokine and inflammatory mediator secretion their relevance as ldquoimmune-like effector cellsrdquowill be discussed Finally an implication for platelets in plasma leakage will be also regarded as thrombocytopenia is associatedwith clinical outcome and higher mortality

1 Dengue General Aspects

Dengue viruses (DENVs) are the most important humanarboviruses worldwide and are transmitted by mosquitoes ofthe genusAedes in the formof four distinct serotypes (DENV-1 DENV-2 DENV-3 and DENV-4) Dengue causes seriousinfection in humans resulting in morbidity and mortalityin most tropical and subtropical areas of the world It isestimated that there are currently 50ndash100 million cases ofdengue every year worldwide including more than 500000reported cases of dengue hemorrhagic fever and dengueshock syndrome (DHFDSS) [1] DENVs are members of theFlaviviridae family which are single-stranded RNA viruses ofpositive polarity with approximately 11000 nucleotides andone phase open reading frame that encodes a single polypro-tein which is subsequently cleaved into three structural

proteins (C prMM and E) and seven nonstructural proteins(NS1 NS2A NS2b NS3 NS4A NS4B and NS5) [2 3] Thestructural proteins include a capsid protein (C) that bindsviral RNA [4] a Membrane protein (M) found in the matureviral particle and an envelope (E) protein that mediatesviral attachment membrane fusion and virion assembly[5] The E protein is the major structural protein exposedon the surface of the viral particle that triggers protectiveimmune responses in the host by eliciting the productionof neutralizing antibodies The E protein is composed of 3domains domain I contains the central region domain II isinvolved in virus-mediatedmembrane fusion and domain IIIinteracts with cell receptors and contains epitopes recognizedby neutralizing antibodies [6]

The nonstructural proteins are involved in viral transla-tion transcription and replication NS1 is a 46 kDa protein

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 313842 16 pageshttpdxdoiorg1011552015313842

2 Mediators of Inflammation

involved in viral RNA replication Notably NS1 is expressedon the surface of infected cells without forming part of thevirion [7] Serum levels of secreted NS1 (sNS1) positivelycorrelate with viral titers and have been a useful tool indengue infection diagnosis [8 9] Because it is expressedon the surface of infected cells NS1 triggers host immuneresponses Additionally NS1 has been shown to displaysoluble complement-fixing activity [10] and it was suggestedto be involved in dengue pathogenesis [11] NS2A is a 22 kDaprotein involved in RNA packaging and replication and itmay be involved in interferon type I antagonism [12 13]NS2B is a 14 kDa membrane-associated protein and servesas a cofactor for NS3 to form a viral protease complex [1415] NS3 is a multifunctional protein with serine proteasehelicasenucleoside triphosphate-NTPase activities and itis required for unwinding the double-stranded replicativeform of RNA It is also involved in processing the viralpolyprotein and RNA replication [7 16] NS4A and NS4B aresmall hydrophobic proteins that function as IFN-signalinginhibitors [12 13] Finally NS5 is a large multifunctional103 kDa protein that displays RNA-dependent polymeraseactivity and was recently identified as a potential type I IFNproduction antagonist [17 18]

All dengue serotypes are capable of causing disease witha wide spectrum of clinical manifestations ranging froman undifferentiated fever in a mild clinical form classicallyknown as dengue fever (DF) to the severe clinical andpotentially fatal DHFDSS [19] Initial symptoms are com-mon to all patients but the clinical manifestations of thesevere forms rapidly evolve with symptoms including highfever liver enlargement circulatory failure (hypotension andshock) edema cavity (pleural abdominal and cardiac) andinternal bleeding phenomenaThe severe forms are primarilycharacterized by plasma leakage and thrombocytopenia withor without hemorrhage The World Health Organization(WHO) classified the clinical presentations of DHF into fourseverity grades based on laboratory data Grade I fever withpositive tourniquet test Grade II plus mild spontaneousbleeding Grade III presence of weak and rapid pulse andGrade IV profound shock with undetectable pulse The lasttwo are considered DSS [20]

It has been difficult to apply the WHO classificationsin Central American countries and Latin America [21ndash23]suggesting that the disease classification into DF DHF andDSS may not be universally applicable for clinical manage-ment In this context Harris et al (2000) reported severalclinical DSS cases that could not be completely classified asabove Given the difficulty of classifying serious cases accord-ing to WHO criteria a new category has been proposedcalled Signs Associated with Dengue Shock which is verysimilar to DSS but without the presence of thrombocytopeniaand hemoconcentration [22] The Special Programme forResearch and Training in Tropical Diseases World HealthOrganization (TDRWHO) proposed a new classificationbased on the difficulties encountered in applying the criteriaof the WHO classification especially in Latin America Theclassification is based on current clinical criteria for severityof dengue cases understanding of the illness as a systemicand dynamic event and facilitating the approach to cases

and subsequent clinical management of patients Accordingto this criterion the new classification addresses three setsof clinical signs and symptoms (1) dengue without warningsigns (2) dengue with warning signs and (3) severe dengue[24 25] According to the new classification patients typicallydeveloped a sudden high-grade feverThis acute febrile phaseusually lasts 2ndash7 days and was often accompanied by facialflushing skin erythema generalized body ache myalgiaarthralgia and headache Anorexia nausea and vomitingwere also common These clinical features are characterizedas nonsevere dengue cases Monitoring for warning signsand other clinical parameters are crucial to recognizing theprogression to the critical phase and it may help distinguishnonsevere and severe dengue cases The warning signs wereassessed by the following clinical parameters abdominalpain or tenderness persistent vomiting clinical fluid accu-mulation mucosal bleed lethargy restlessness and liverenlargement gt2 cm associated with laboratory parameterssuch as increase in hematocrit (HCT) concurrent with rapiddecrease in platelet count Mild hemorrhagic manifestationssuch as petechiae and mucosal membrane bleeding (egnose and gums) may be present Patients require emer-gency treatment and urgent referral in the critical phaseof disease that is when they have severe dengue charac-terized by severe plasma leakage leading to dengue shockandor fluid accumulation with respiratory distress severehemorrhages shown by massive vaginal bleeding (in womenof childbearing age) and gastrointestinal bleeding andorsevere organ impairment (hepatic damage renal impairmentcardiomyopathy encephalopathy or encephalitis) [25] Mostpatients recovered from the illness uneventfully and survivedto hospital discharge

The mechanisms by which pathophysiologic changesoccur in dengue fever are still not fully understood Theinteraction of several factors seems to be responsible for thedevelopment of the severe disease These factors include thefollowing the virulence of the circulating strain the presenceof efficient or high density vector the wide circulation of thevirus and characteristics of the host as genetic factors eth-nicity presence of chronic diseases and subsequent DENVinfections [19 26] Natural infection by one of the fourDENVserotypes produces lasting immunity against reinfection bythe same serotype but heterotypic protection is temporaryandpartial thus allowing sequential infections Epidemiolog-ical studies have shown thatmost individualswhodevelop themore severe DHFDSS had been previously infected with adifferent serotypeThese observations indicate that a previousinfection with dengue is a risk factor for development ofsevere illness The antibody-dependent enhancement (ADE)mechanism was proposed by Halstead in the 1970s [27]

One striking finding that potentially plays a critical role indengue immunopathogenesis occurs during sequential infec-tions by heterologous DENV serotypes The phenomenonknown as the Original Antigenic Sin was first describedin relation to influenza responses involving B cells [28]and more recently in humans infected with DENV [29]According to the phenomenon of Original Antigenic SinT cells generated during primary infection with a viralserotype showed a low affinity for the serotype of the second

Mediators of Inflammation 3

infection which in turn leads to development of alteredimmune responses including decreased viral clearance Inaddition the extensive activation of cross-reactive memorycells promotes the aberrant release of cytokines (cytokinestorm) that contribute to disease severity [30]

2 Thrombocytopenia in Dengue

Thrombocytopenia has always been one of the criteria usedbyWHOguidelines as a potential indicator of clinical severity[20 31] In the most recent 2009 WHO guidelines thedefinitions generally describe a rapid decline in platelet countor a platelet count less than 150000 per microliter of blood[24]

A kinetic description of platelet count inDHFDF showeda significant decrease on the 4th day of the illness In factprevious studies reported DHF in adults without shock inwhich platelet counts mildly to moderately decreased on the3rd day until the 7th day of illness and reached normal levelson the 8th or 9th day [32ndash34] In children there is little cor-relation between platelet count and bleeding manifestationsor between platelet count and disease severity [35 36] Inadults a platelet count of 5 times 109 Lminus1 and packed cell volumegt50 are significantly associated with bleedingmanifestationsHowever a study enrolling 245 dengue patients showed nocorrelation between clinical bleeding and platelet count and81 nonbleeding patients had counts of less than 20 times 109 Lminus1[37] In contrast another study enrolling 225 dengue patientssuggested that bleeding occurred more often in patients withplatelet counts below 20 times 109 Lminus1 [38]

Most clinical guidelines recommend that platelet transfu-sions be given to patients who develop serious hemorrhagicmanifestations or have very low platelet counts plateletcounts falling below 10ndash20 times 109 Lminus1 without hemorrhageor 50 times 109 Lminus1 with bleeding or hemorrhage The efficacyof platelet transfusions is controversial In a study of 106pediatric patients with DSS with thrombocytopenia andcoagulopathy there was no significant difference in hemor-rhage between patients who received preventive transfusionscompared to those who did not Patients who receivedtransfusion had a higher frequency of pulmonary edema andincreased length of hospitalization [39] Platelet transfusiondid not prevent the development of severe bleeding orshorten the time to bleeding cessation and was associatedwith significant side effects Thus according to the authorsplatelet transfusions should not be routinely performed in themanagement of dengue [40 41]

The mechanisms involved in thrombocytopenia andbleeding during DENV infection are not fully understoodSeveral hypotheses have been suggested to elucidate themechanism involved In this context DENV could directly orindirectly affect bone marrow progenitor cells by inhibitingtheir function [42] to reduce the proliferative capacity ofhematopoietic cells [43] Indeed there is evidence thatDENV can induce bone marrow hypoplasia during theacute phase of the disease [44] Besides platelets countsthe functional disruption of these cells is associated witha significant deregulation of the plasma kinin system andthe immunopathogenesis of dengue [45] In addition DENV

infection induces platelet consumption due to disseminatedintravascular coagulation (DIC) platelet destruction due toincreased apoptosis lysis by the complement system and bythe involvement of antiplatelet antibodies [46ndash48] Here wediscuss the relevance of platelets in physiology and theirimplication in dengue pathogenesis acting both as a victim ofinfection and an effector cell of the antiviral immune response

3 Platelets General Aspects

Platelets are the cellular effectors of primary hemostasis asthey contribute to thrombus formation at sites of vascularinjury a fundamental tenet in physiology and medicine[49ndash55] Platelets are anucleated circulating cells in mam-mals approximately 2 120583m in diameter and are derived frommegakaryocytes within the bone marrow [56 57] Thereare approximately 1012 platelets circulating in the blood ofan adult human and because the lifespan of an individualplatelet is only 8ndash10 days 1011 new platelets must be produceddaily from bonemarrowmegakaryocytes tomaintain normalplatelet counts (150ndash400times 109 platelets per liter of blood) [58]

The resting platelet plasma membrane is generallysmooth except for periodic invaginations delineating theentrances to the open canalicular system and a systemof folded membranes The canalicular system consists ofa complex network of intertwining membrane tubes thatpermeate the plateletrsquos cytoplasm Meanwhile the foldedmembrane systems allow the platelets to have a large surfacearea and readily take up proteins and molecules and re-release them upon activation The lentiform shape of theresting platelet is maintained by three major cytoskeletalcomponents the marginal microtubule coil the spectrin-based membrane skeleton and the actin-based cytoskeleton[59] When platelets encounter a damaged vessel wall theybecome activated and undergo a dramatic actin-mediatedshape change from smooth discoid to spiny spheres Thisprocess is initiated by a Ca+2 influx which promotes the for-mation of finger-like filopodia and pseudopods During thisreaction the number of receptors on the platelet membranefor adhesive and clotting proteins increases and activatedplatelets attract other platelets which clump together andultimately form a plug that seals the vascular leak [60]

At the resting state the platelet membrane is virtuallyimpermeable to Ca+2 Phosphatidylserine (PS) is a phos-pholipid in the internal hemilayer of the platelet mem-brane at rest and it is the main determinant of plateletprocoagulant activity When platelets are activated PS isexposed on the external hemilayer which is usually asso-ciated with the formation of platelet-derived microparti-cles (PMPs) which also have prothrombinase activity [61]Changes in intraplatelet Ca+2 concentration as a result ofCa+2 influx or mobilization of intracellular stores are fun-damental to the platelet activation response and precedeseveral activation responses such as shape change aggre-gation secretion and expression of procoagulant activityIn this process P-selectin (CD62P) translocates from the120572-granule membrane to the platelet membrane where itcontributes to platelet-leukocyte platelet-endothelium and


platelet-monocyte binding and thromboembolic tendency[62] In addition activated platelets release soluble CD154also called CD40 ligand or CD40L which can interact withvascular cells (including endothelial cells) and induce E-selectin (CD62E) and CD62P upregulation and IL-6 andTissue Factor (TF) release [63 64] In fact most circulatingsoluble CD154 in human plasma is generated from activatedplatelets and soluble CD154 levels may be an indicator of thedegree of platelet activation within the host [64]

Several biologically active molecules stored in intracellu-lar granules can be released into circulation or translocatedto the platelet surface to mediate other nonhemostatic func-tions Platelets have three major types of storage granules 120572-granules dense granules and lysosomes 120572-granules are themost abundant type of granule with 40ndash80 per platelet andthey derive their protein content by a combination of endo-cytosis and biosynthesis The proteins housed in 120572-granulesinclude coagulation factors chemokines adhesive proteinsmitogenic factors and angiogenic regulators Studies haveshown that platelets contain heterogeneous populations of 120572-granules that undergo differential patterns of release duringplatelet activation [65 66] In fact Sehgal and Storrie haveidentified two classes of120572-granules one that contains fibrino-gen and another that contains von Willebrand factor (vWF)[67] Therefore it is likely that there are distinct granulesubpopulations with differentially packaged immunomodu-latory substances in a specific manner to respond to differenttypes of tissue damage

Platelets contain several preformed molecules necessaryto mediate hemostasis In addition platelets contain largeamounts of mRNA and the translational machinery pack-aged during platelet formation can synthesize proteins dur-ing hemostatic and inflammatory events [68ndash71] Followingthrombin activation proteomic analyses have demonstratedthat platelets secrete more than 300 different proteins suchas interleukin-1 (IL-1) Toll-like receptors (TLRs) andCD154which are clearly involved in host defense processes PlateletCD154 expression may have an important role in linkingthe innate and adaptive immune responses and promotingprotective immunity [72]

It has also been suggested that platelets act as keyeffector cells in inflammation and the immune contin-uum [73 74] Platelets store and release many biologicallyactive substances including growth factors cytokines andchemokinesTheir impact on immune cells is associated withthe induction of leukocytes and progenitor cells to the siteof pathogen permeation or vascular injury inflow as well asendothelial cells Platelets interact with neutrophils mono-cytes and lymphocytes to activate them and they also formplatelet-leukocyte aggregates that immobilize pathogens andprevent their spreading Furthermore platelets can absorbpathogens to target the immune response against them It isalso assumed that the presence of surface receptors such asTLRs affects their initiation and activity in the immunologicalresponse [75]

Platelets express three (hem) immunoreceptor tyrosine-based activation motif- (ITAM-) coupled receptors gly-coprotein (GP) VI a receptor for collagen and lamininin the extracellular matrix that signals via the associated

ITAM-containing Fc receptor 120574 chain (FcR120574) Fc120574RIIA anITAM-containing receptor for immune complexes and C-type lectin-like receptor- (CLEC-) 2 a hemITAM-containingpodoplanin receptor expressed on select cell types such aspodocytes lymphatic endothelial cells and type I alveolarcells [76 77] The majority of studies on platelet ITAM sig-naling have focused on the role of GPVIFcR120574 in hemostasisand thrombosis at sites of vascular injury or plaque rupturePlatelets may be activated by different agonists includingthose that recognize G protein coupled receptors (GPCRs)and soluble ligands such as thrombin and ADP Moreoverimmunoreceptors such as GPVI trigger outside-in signalsto human platelets that result in rapid and in some casessustained functional responses These processes lead to theexpression of several cytokines chemokines and cell surfacemolecules that initiate and perpetuate hemostasis and alsoalert the immune system and induce leukocyte recruitmentto the injured tissue [60] FC120574RIIA is best known for itsrole in immune-mediated thrombocytopenia and throm-bosis Recently authors identified a critical role for GPVIand CLEC-2 in vascular integrity maintenance at sites ofinflammation [78]

4 Hemostasis

Hemostasis is a dynamic process regulated by several mech-anisms to prevent bleeding and includes two processes (1)primary hemostasis involving vascular constriction plateletactivation and aggregation (2) secondary hemostasis involv-ing the activation of coagulation mechanisms clot forma-tion and its subsequent dissolution by fibrinolysis Bloodcoagulation is initiated by exposure to membrane-boundTissue Factor (TF) which is constitutively expressed on thesurface of cells surrounding the vasculature (fibroblasts andmuscle cells) to form a hemostatic envelope that preventsexcessive bleeding after vascular injury [79ndash81] Monocytesand endothelial cells do not express TF but express itduring pathological conditions [82 83] and upon exposureto inflammatory cytokines such as TNF-120572 and IL1-120573 [84]

TF is the cellular receptor and cofactor for plasma factorVIIa The complex TF-VIIa catalyzes the conversion of factorX to Xa which further assembles into the prothrombinasecomplex formed by factor Xa factor Va factor II (pro-thrombin) and calcium thereby generating thrombin Inturn thrombin converts fibrinogen into fibrin The TF-VIIacomplex can also activate factor IX leading to assembly of theintrinsic tenase complex formed by factor IXa factor VIIIafactor X and Ca+2 which generates additional factor Xa toform an amplification loop [80] In addition to its procoag-ulant role TF exerts proinflammatory activity by activatingmembrane receptors sensitive to coagulation proteases suchas factor VIIa factor Xa and thrombin These receptorsknown as PARs (Protease Activated Receptors) are expressedin various tissues including endothelial cells mononuclearleukocytes [85] platelets fibroblasts smooth muscle cellsand others [86 87] PARs comprise a family of receptors(PAR1 PAR2 PAR3 and PAR4) that are uniquely activatedby the proteolytic cleavage of their extracellular portionThis cleavage unmasks a new N-terminus which serves


as a tethered ligand that binds to its second extracellulardomain resulting in a variety of cellular responses PAR1 canbe cleaved and activated by thrombin factor Xa plasminactivated protein C and matrix metalloproteinase 1 (MMP1)PAR2 can be activated by factor VIIa factor Xa tryptaseand trypsin but not thrombin PARs are involved in severalphysiological and pathological processes and are consideredto be a crucial link between coagulation and inflammation[86] PAR1 activation may lead to multiple signaling pathwayactivation including activation of PI3 kinase Src familytyrosine kinases and the ERK pathway and MAP kinasesPAR2 activation promotes the release of inositol triphosphate(IP3) and diacylglycerol (DAG) and subsequent increaseof intracellular calcium [88] Thus several pathways canbe activated such as protein kinase C and ERK cascadeof kinases and MAP The TF-factor VIIa complex activatesPAR2 to promote an increased inflammatory response inmacrophages (production of reactive oxygen species expres-sion of adhesion molecules and proinflammatory cytokines)and neutrophil infiltration In addition the ternary TF-factor VIIa-factor Xa complex can activate PAR1 and PAR2potentially enhancing the inflammatory response [89]

The release of cytokines such as TNF-120572 and IL-6 can leadto activation of the coagulation cascade by the TF pathway[90 91] In turn increased coagulation enzyme productionmay activate PAR receptors to increase proinflammatorycytokines and leukocyte migration to the infection site PARactivation is accompanied by adhesion molecule upregula-tion and proinflammatory cytokine production (eg TNF-120572IL-1120573 and IL-6) [91] Cytokines bind to specific receptors andtogether with coagulation enzymes perpetuate the inflam-matory response which promotes increased interaction ofactivated monocytes activated platelets and endothelial cells[81] The result is the convergence of signals leading toexacerbated TF expression to sustain coagulation Thereforethe processes of coagulation and inflammation are closelyrelated and coagulation may affect inflammation whichsubsequently modulates coagulation This bidirectional rela-tionship is mediated by PAR activation [88 91 92]

Three main anticoagulant pathways regulate the coagula-tion reaction (1) the proteinC system (2) antithrombin (AT)and (3) Tissue Factor Pathway Inhibitor (TFPI) The proteinC pathway modulates both the inflammatory and hemostaticsystems [93] and is composed of four main constituentsprotein C endothelial protein C receptor (EPCR) protein Sand thrombomodulin Protein C is proteolytically activatedby thrombomodulin-bound thrombin on the endothelial cellsurface upon EPCR binding [94ndash96] Activated protein C(APC) acts with its cofactor protein S to proteolyticallydegrade the essential coagulation cofactors Va andVIIIa [97]Antithrombin belongs to the serpin family and is an inhibitorof thrombin factor IXa and factor Xa Notably the rate ofenzyme inhibition by antithrombin increases in the presenceof heparin [98] TFPI is a protease inhibitor that regulates theTF dependent pathway of blood coagulation as its primaryTFfactor VIIa complex inhibitor [99] TFPI acts in a two-step manner In the first step TFPI inactivates factor Xa toform a TFPIXa complex TFPI then inactivates TF-boundfactor VIIa Because TFPIfactor Xa complex formation is

a prerequisite for efficient factor VIIa inactivation the systemensures that some factor Xa generation occurs before thefactor VIIa-mediated initiation of the coagulation system isinhibited

Vascular damage is an expected effect of injury andinflammation as previously reviewed [100] Platelets havelong been recognized to support the endothelial semiper-meable function [101] attributed largely to the observationthat platelet activation results in the release of proangiogenicproteins and angiogenesis inhibitors as part of the negative-feedback mechanisms that limit the angiogenic processOverall proangiogenic molecules influence vascular cellmigration and proliferation and vessel organization and sta-bilization [100] The proangiogenic proteins include vascularendothelial growth factor (VEGF) hepatocyte growth factor(HGF) transforming growth factor beta (TGF-120573) basicfibroblast growth factor (bFGF) epidermal growth factor(EGF) platelet-derived growth factors (PDGF-A PDGF-Bor PDGF-C) other soluble cytokines (IL-8 angiopoietin andCXC chemokine ligand-12-CXCL12) and metalloproteasesMMP-1 MMP-2 and MMP-9 [102] For instance VEGFalso known as permeability factor increases endothelial per-meability causing plasma protein extravasation Moreovergrowth factors such as TGF-120573 PDGF brain-derived neu-tropic factor (BDNF) and insulin-like growth factor 1 (IGF-1) control extracellular matrix (ECM) production [103] andin turn trigger collagen synthesis and accumulation [104]The bioactive mediators and adhesive proteins expressed byactivated platelets facilitate homotypic interactions betweenplatelets and heterotypic interactions between platelets anddifferent immune cell populations For example activatedplatelets express CD62P and can promote lymphocyte rollingand adhesion on high endothelial venules [105] and activatedplatelets can mediate neutrophil adhesion to the endothe-lium and upregulate their proinflammatory functions Fur-thermore it is now known that platelet-expressed CD154(CD40L) can interact with CD40 on endothelial cells toinduce endothelial cell upregulation of intercellular adhesionmolecule 1 (ICAM-1) and vascular cell adhesion molecule 1(VCAM-1) and release of CC-chemokine ligand 2 (CCL2)thereby promoting leukocyte recruitment to inflammatorysites [106]

5 Bone Marrow Suppression Is a Cause ofThrombocytopenia in Dengue

Previous reports have shown that during the early phaseof disease bone marrow displays hypocellularity and atten-uation of megakaryocyte maturation [33 107] The precisemechanisms underlying DENV-induced bone marrow sup-pression during the acute phase remain unclear Howeverthree main factors have been suggested (1) direct lesionof progenitor cells by DENV (2) infected stromal cells (3)changes in bone marrow regulation [108] Thrombopoietin(TPO) is a cytokine that specifically regulates megakary-ocytopoiesis and platelet production by activating the TPOreceptor c-MPL (myeloproliferative leukemia virus onco-gene) [109 110] Because TPO is elevated when platelet


production decreases serum TPO levels may be a use-ful indicator of megakaryocytopoiesis in dengue [111] Infact Matondang et al showed that TPO levels significantlyincreased in adult DENV patients in which circulatingplatelets were markedly reduced and the TPO levels inverselyrelated to the platelet counts [112]

6 Increased Destruction of Platelets CausesThrombocytopenia in Dengue

Thrombocytopenia may also be due to (1) platelet consump-tion during ongoing coagulopathy process (2) activation ofthe complement system [113] or (3) increased peripheralsequestration [32 114] It has been shown in vitro that plateletsundergo increased phagocytosis by macrophages in patientswith secondary DENV infections by an uncharacterizedmechanism [115] It has also been demonstrated that DENVpatients develop anti-platelet antibodies of the IgM isotype[116] Notably antiplatelet IgM titers in patient sera werehigher in DHFDSS compared to DF Anti-platelet antibodiescause platelet lysis as measured using lactate dehydroge-nase activity assays In accordance with elevated IgM titersDHFDSS sera caused increased platelet lysis compared toDF patient sera In addition cytotoxicity was much higherin the presence of complement [116] Autoantibodies againstendothelial cells and blood-coagulation-related moleculeshave also been identified [47 117] In fact molecular mimicrybetween platelets endothelial cells or blood coagulationmolecules and dengue virus NS1 prM and E proteins mayexplain the cross-reactivity of anti-NS1 anti-prM or anti-E antibodies to host proteins and play a role in diseasepathogenesis Cross-reactive antibodies may cause plateletdysfunction endothelial cell damage coagulation defectsand macrophage activation which may contribute to someclinical features of DHF [118]

Some studies have shown platelet activation and apopto-sis in dengue-infected patients In this way platelet apoptosisplatelet phagocytosis and serum TPO levels significantlyincreased in patients during the acute and early conva-lescence phases compared to levels in patients during theconvalescence phase and in healthy volunteers suggestingaccelerated platelet clearance However this was overcomeby TPO-induced enhanced thrombopoiesis in these patients[119]

Another study later confirmed that platelets fromDENV-infected patients exhibited classic signs of the intrinsicpathway of apoptosis which include increased surface PSexposure mitochondrial depolarization and caspase-9 andcaspase-3 activation Moreover all of these changes wereobserved when platelets from healthy subjects were directlyexposed to DENV in vitro which may contribute to throm-bocytopenia development in dengue patients [46]

7 Are Platelets Directly Infected byDengue Virus

The detection of DENV antigens on the surface and inplatelet-containing immune complexes from skin biopsy

specimens has been well documented [120ndash122] In additionthe association of DENV with platelets in vitro has beenreported Reverse transcription polymerase chain reaction(RT-PCR) and electron transmission microscopy (EM) anal-yses have been performed in plasma and platelets from33 hospitalized DENV-infected children [123] Dengue viralRNAwas detected in the platelets and plasma by conventionalRT-PCR and EM which confirmed the presence of dengueviral-like particles inside platelets isolated from patientsThese data suggested that the presence of DENV in plateletsmight be associated with platelet dysfunction However noevidence for competent DENV replication has been demon-strated in enriched preparations of platelets from DENV-infected patients [124] More recently a prospective obser-vational study using blood samples from dengue-confirmedpatients as well as rhesus monkeys (RM) experimentallyinfectedwithDENV revealed thatDENVantigenwas presentin small vesicles of varying size and more frequently inanucleated cells associated with platelets DENV RNA wasobserved in a highly enriched CD61(+) cell population frominfected RM during the acute stageThese results suggest thatvirus-containing CD61(+) cells are directly linked to plateletdysfunction and low platelet count characteristics of denguepatients [125]

8 The Balance between CoagulationFibrinolysis and Anticoagulant Pathwaysin Dengue

Both coagulation and fibrinolysis are activated during acutedengue infection leading to alterations in their parameters[126ndash129] The kinetic profile of the circulating markers ofcoagulopathy such as D-dimer (DD) [130] activated partialthromboplastin time (aPTT) [130 131] and prothrombintime (PT) [127 130] demonstrated that these parametersare increased in patients in the acute phase of the diseaseImportantly circulating TF levels were significantly higherduring the febrile phase especially in FHD [129] and SCDpatients [127] followed by a gradual normalization duringthe convalescent phase The circulating levels of the naturalanticoagulants protein C protein S and antithrombin aresignificantly reduced during the early disease stages TotalTFPI levels were moderately elevated during the acute phasebut not after hematocrit correction Higher Plasminogenactivator inhibitor-1 (PAI-1) levels and lower protein S levelswere associated with an increased severity of bleeding [127]

Initially evidence showed that prekallikrein factor XIIand complement C3 levels were significantly lower in DHFpatients compared to fever control patients Notably thelowest mean levels were observed in dengue patients withshock However bradykinin concentrations decreased andmean activity levels of kallikrein inhibitors did not changein dengue patients [45] Funahara et al showed that allDHF patients had manifestations of acute DIC in whichthey detected transient prolonged aPTT and PT decreasedplatelet counts fibrinogen prothrombin factor VIII plas-minogen and antithrombin activities [48 132] DIC is asevere acute subacute or chronic dysregulation of hemo-static and fibrinolytic processes occurring as a secondary


complication in a variety of diseases including cancer andsepsisThemechanisms that trigger DIC are primarily relatedto increased expression of the clotting initiator protein TFinto circulation and endothelial injury [133]The initiation ofDIC leads to enhanced fibrin formation platelet activationand microthrombus deposition in microcirculation whichmay contribute to systemic organ failure Remarkably con-sumption of blood coagulation factors and platelets com-monly lead to paradoxical hemorrhagic disturbances dueto consumption of these hemostatic factors [94 134] Thesame group later postulated that acute DIC occurring inDHF is associated with increased vascular permeability [132]These parameters have been confirmed by another study thatshowed that PTT and PT act as indices in predicting bleedingand outcome in DHF as mortality was 6-fold higher inpatients with platelet counts lt50000microliters comparedto patients with platelet counts gt50000microliters [135]More recently a study confirmed the predictive value of thehemogram (ie peripheral white cell count platelet count)coagulation profile (ie PT aPTT) and blood chemistry (iealanine aminotransferase (ALT) and aspartate aminotrans-ferase (AST)) in DFDHF diagnosis [136]

Because hemostasis depends on the balance betweencoagulation and fibrinolysis some coagulation parameters(platelet count and aPTT) and fibrinolytic parameters (tissueplasminogen activator tPA and PAI-1) have been evaluatedin DHFDSS and DF patients DF patients show throm-bocytopenia aPTT prolongation and increased tPA levelsindicating activation of coagulation and fibrinolysis Theseparameters indicate more severe activation of coagulationand fibrinolysis in DHFDSS patients In the convalescentstage an increase in the PAI-1 level and platelet countwith a concomitant decrease in tPA level and return tonormal aPTT has been reported in both DHFDSS andDF patients Therefore the activation of coagulation andfibrinolysis during the acute stage of DENV infection is offsetby increased platelet and PAI-1 during convalescent stageAltogether these results suggest that the degree of DENVinfection-induced coagulation and fibrinolysis activation isassociated with disease severity [137]

Activation in blood coagulation and fibrinolysis arefrequently observed during viral hemorrhagic fevers andsepsis Increased TF expression has been detected in mono-cytesmacrophages in primates experimentally infected withEbola virus suggesting a role in the development of coagula-tion disorders during infection [138] Remarkably TF inhibi-tion reduces lethality in experimental virus infectionmodelswith reduced inflammation and coagulation processes [139]Several studies have suggested that increased TF expressionhas an important role in dengue pathogenesis (Figure 1)Using primary human endothelial cells (EC) infected withDENV isolated from DHFDSS cases Jiang et al showed anincrease in TF mRNA expression associated with a reductionin TFPI mRNA expression [140] Moreover Huerta-Zepedaet al showed that DENV upregulates PAR-1 and TF inactivated endothelium [141] These data are consistent withevidence of increased TF plasma levels in DHF DENVpatients [127] Our group further demonstrated increased TFexpression on monocytes from severe dengue patients In

fact TFmonocyte surface expressionwas inversely correlatedwith platelet count [142] Interestingly we also found signifi-cantly higher circulatingTFPI levels in severe dengue patients(unpublished data) The release of cytokines such as TNF-120572and IL-6 can lead to activation of the coagulation cascadeby the TF pathway [90 91] In turn further productionof coagulation enzymes may activate PAR receptors thusamplifying the increase in proinflammatory cytokines andleukocyte migration to the infection site PAR activation isaccompanied by upregulation of adhesion molecules andproduction of proinflammatory cytokines (eg TNF-120572 IL1-120573 and IL-6) [91] which have reportedly been found inDENVinfection [143ndash147] Cytokines bind to specific receptors andtogether with coagulation enzymes (and vice versa) sustainthe inflammatory response which promotes increased inter-action of activated monocytes activated platelets and ECsThe result is a convergence of signals leading to exacerbatedTF expression to sustain coagulationTherefore the processesof coagulation and inflammation are closely related and thisbidirectional relationship is mediated by PAR activation [91]

Moreover levels of TNF-120572 thrombomodulin and vWFwere significantly increased inDENVpatients with andwith-out bleeding than in healthy controls However plasma tPAand D-dimer levels were significantly increased in patientswith bleeding The thrombin generation test showed thatpatients with bleeding complications had reduced thrombinformation [148] Other interesting data demonstrated thatsecreted DENV NS1 might bind to prothrombin and inhibitits activation which in turn may contribute to aPTT prolon-gation and hemorrhage in DHF patients [149] Furthermoredata have suggested that DENV-induced plasminogen cross-reactive Abs enhance plasminogen conversion to plasminwhich could contribute to hyperfibrinolysis in DHFDSSpatients [150]

9 Platelet Dysfunction in Dengue

Anumber of studies have documented platelet dysfunction inDENV infection In this context the suppression of plateletaggregation was demonstrated during the acute phase ofDHF in both shock and nonshock patients with a simul-taneous increase in release of beta-thromboglobulin (BTG)and platelet factor 4 (PF4) from platelets into plasma [114]Production of platelet activating factor (PAF) thromboxaneB2 (TxB2) and prostaglandin D2 (PGD2) was measured inmononuclear leukocytes (MNLs) from nonimmune and pre-viously DENV-1-infected donors when infected in vitro withDENV-2 The authors found that MNLs regardless of pri-mary or heterologous secondary exposure to DENV-2 couldrelease significant amounts of PAF TxB2 and PGD2 PAF butnot TxB2 or PGD2 levels released byMNLs were significantlyhigher in those obtained from previously DENV-1-infecteddonors [151] The effect of DENV-2 on the morphologyand physiological activation profile was measured in normalhuman platelets Their results showed that DENV-2 activatesplatelets with an increase in P-selectin (CD62P) expressionand fibrinogen-binding property Additionally atomic forcescanning and transmission electron microscopy analysis


Prothrombin ThrombinFibrinogen Fibrin

Antithrombin

Activated protein C

Protein CActivated monocytes

Inflammatory mediators


PARs

TM

Activated platelets

sTF

Proteolitic cleavage

Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573

Figure 1 Coagulation and inflammatory response activation during dengue DENV infection is often associated with coagulation disordersThe coagulation system is activated by the host immune responseThe activation of coagulation system leads to the generation of thrombin andintracellular signaling through PAR platelet and monocyte activation and recruitment and increased TF expression on the endothelium andmonocytes The following anticoagulant pathways balance this system the protein C system antithrombin (AT) and Tissue Factor PathwayInhibitor (TFPI) Increased CD62P expression in platelets from dengue patients indicates platelet activation and therefore favors homo-and heterotypic interactions with immune cells and endothelial cells Additionally CD40L (CD154) expressing platelets can interact withCD40 on endothelial cells inducing upregulation of adhesion molecule (eg ICAM-1) chemokine secretion (eg CCL2) and leukocyterecruitment Evidence of increased circulating TF levels in dengue patients and increased expression in monocytes from severe patientssuggests its importance in disease pathogenesisThe release of inflammatory cytokines leads to activation of the coagulation cascade throughthe TF pathway Coagulation factors (eg thrombin) activate PAR and amplify proinflammatory cytokine production (eg TNF-120572 IL-1120573IL-6 and CCL2) and leukocyte migration to the infection site EPCR endothelial protein C receptor TM thrombomodulin

revealed typical activation-related morphological changessuch as altered platelet membrane architecture degranu-lation presence of filopodia and dilatation of the opencanalicular system in DENV-2-exposed platelets but not incontrols [152] It is well known that nitric oxide (NO) playsa physiological role in maintaining hemostasis regulatingvasodilatation and the aggregation recruitment and adhe-sion of platelets to the vascular endothelium [153] Previouslyhigh nitrite or nitrate concentrations were observed in DFpatients when compared with DHF patients and controlswhile there were no differences between values from DHFpatients and controls [154] Although platelets can generateNO through stimulation of NO synthase [155 156] this samestudy performed experiments to determine NO productionafter coculture of human platelets with active and inactive

forms of DENV-2 Virus-platelet interactions did not con-tribute to increased NO levels in the culture supernatantssuggesting that platelets are not a source of NO during DF[154] Authors have previously shown that increased rates ofL-arginine transport and the cationic amino acid l-argininethat generated NO via oxidation catalyzed by a family ofnitric oxide synthase (NOS) enzymes were associated withenhanced NOS activity elevated plasma fibrinogen levelsand reduced platelet aggregation in DENV patients [157]Recent findings from the same group provided the firstevidence of platelet l-arginine-NO pathway upregulation inDHF despite no changes in NOS enzyme protein levels indi-cating that this disease affects not only platelet productionfrom bone marrow and its peripheral destruction but also itsfunction [158]


10 Platelets as Effector Cells inDENV Infection

Acutedengue isassociatedwithplatelet activationwith increasedexpression of activated fibrinogen receptor (120572IIb1205733) the lyso-somalmarker CD63 and the120572-granulemarker CD62P Uponmaximal platelet activation byTRAP (thrombin receptor ago-nist peptide-6) platelet function defects were observed witha significantly reduced (120572IIb1205733) and CD63 expression andreduced platelet-monocyte and platelet-neutrophil interac-tions [159] Beyond its direct effector role studies have inves-tigated the influence of platelets on cytokine production bynormal humanmononuclear cellsTheir findings suggest thatactivated platelets have anti-inflammatory properties relatedto the interaction between CD40L and CD40 by enhancingIL-10 production and inhibiting TNF-120572production bymono-cytes [160] In addition authors found that interaction ofmonocytes with apoptotic platelets mediates IL-10 secretionthrough PS recognition in platelet-monocyte aggregatesMoreover IL-10 secretion required platelet-monocyte con-tact but not phagocytosis demonstrating that activated andapoptotic platelets aggregate with monocytes during dengueinfection and signal specific cytokine responses that maycontribute to dengue pathogenesis [161]

The TLRs belong to a family of pattern recognitionreceptors (PRRs) and are key players in the innate responseagainst pathogens These receptors are expressed in manycell types including B cells monocytes dendritic cells (DCs)macrophages and neutrophils [162] Interestingly differentgroups described the expression of TLR1-9 on both humanand mouse platelets Certain platelet-expressed TLRs arefunctional (eg TLR-4) and can regulate sepsis inducedthrombocytopenia and TNF-120572 production in vivo [163ndash168]Recently the presence of the nucleotide-binding domainleucine rich repeat containing protein (NLRP3) inflamma-some was found to be associated with caspase-1 activationand synthesis of IL-1120573 in platelets activated upon DENVinfection Moreover the authors demonstrated an increasedaccumulation of IL-1120573 in platelets and platelet-derived MPsfrom dengue patients [169]

Data published by our group showed strong evidencethat increased TNF-120572 levels correlate with hemorrhagicmanifestations while increased IL-10 levels correlate withlow platelet counts in a cohort of Brazilian DENV-infectedpatients [145] A more recent study found that circulatingTGF-1205731 concentrations on admissionwere significantly lowerin DHF than in controls [170] Interestingly platelets expressthe largest amount of TGF-120573 in the body [171] Althoughthe role of TGF-120573 in platelet-mediated hemostasis is unclearcirculating platelets appear to be important for regulatingblood levels of TGF-120573 Patients with immune thrombocy-topenia have low levels of circulating TGF-120573 but their TGF-120573levels recover after therapy to restore normal platelet counts[172 173]

11 Do Platelets Increase the Risk ofEndothelium Vascular Permeability inSevere Dengue Disease

Previous work by Butthep et al showed that diverse cellssuch as platelets white blood cells neutrophils lymphocytes

and large lymphocytes but not basophils and eosinophilswere preferentially bound to dengue-infected endothelialcells compared to control endothelial cells It was suggestedthat the increased binding of platelets to endothelial cellcould contribute to thrombocytopenia in DHF patients[174] Protein disulfide isomerase (PDI) an endoplasmicreticulum protein localizes on the platelet surface [175] andis involved in the regulation of integrin-mediated plateletaggregation as anti-PDI Abs blocked platelet adhesion andaggregation [176] Previous studies demonstrated that plateletmembrane PDI is recognized by anti-DENV NS1 Abs andrecently Rachman et al observed similar kinetics of NS1and PDI antibodies [177] Anti-DENV NS1 reduced thePDI enzymatic activity and ADP-stimulated platelet aggre-gation The DENV NS1 amino acid residues 311ndash330 (311ndash330) represent a dominant epitope with sequence homol-ogy to the thioredoxin domain of PDI [178] In contrastalthough dengue patient sera inhibited platelet aggregationthere is no correlation between NS1 antibodies and PDIantibodies with platelet aggregation dysfunction suggest-ing that other mechanisms could be involved in plateletaggregation inhibition [177] While some studies confirm thelack of association between VEGF and severity of illnessin dengue virus infection others have documented signifi-cantly higher VEGF levels in patients with DHF [179ndash182]TGF-120573 showed a very significant and positive correlationwith platelet counts consistent with platelet release [183]Angiopoietins are other key regulators of vascular integrityand are stored in platelets Dengue-associated thrombocy-topenia and endothelial activation are associated with animbalance in angiopoietin-2 angiopoietin-1 plasma levelsIn fact the authors demonstrated that there was an inversecorrelation between angiopoietin-1 and markers of plasmaleakage and a positive correlation between angiopoietin-2 and markers of plasma leakage in DHFDSS patients[184] Hottz et al showed that dengue-infected patientswith signs of increased vascular permeability were stronglyassociated with a higher percentage of IL1-120573-positive plateletsand IL1-120573-rich platelet-derived MPs as well as caspase-1activation compared to patients with no evidence of alteredvascular barrier functionThese results were confirmedwhenMPs from DENV-2-exposed platelets caused an increase inendothelial cell permeability thatwas blocked by IL-1Ra [185]

12 Conclusion

Thrombocytopenia coagulopathy and vasculopathy arehematological abnormalities related to platelet and endothe-lial dysfunction generally observed in severe dengue Amongthe causes and consequences previous data have suggestedan imbalance between clotting versus fibrinolysis systems asDIC In aminority of patients with severe or prolonged shockthe abnormalities may be profound and in combinationwith severe thrombocytopenia and the secondary effects ofhypoxia and acidosis may result in true DIC and majorhemorrhage DIC is primarily triggered by TF release andmay induce PAR membrane receptor activation on circu-lating monocytes and vascular endothelial cells in dengue


patients forming a crucial link between coagulation andinflammation Despite limited clinically significant bleedingand only mild alterations in the results of coagulation screen-ing tests children with DSS had significant abnormalities inall of the major pathways of the coagulation cascade Thelow circulating levels of proteins C S and antithrombinare likely related to leakage of these proteins through thevascular endothelium and correlate with the severity ofshock Elevated levels of TF thrombomodulin and PAI-1 reflect endothelial platelet andor monocyte activationand may be a secondary response to direct activation offibrinolysis by the dengue virus Comorbidities in denguepatients result in complications leading to deaths Anotherstudy reported that dengue patients with allergies or diabetesare 25 times more at risk of developing DHF Likewisea higher frequency of complications has been reported indengue patients suffering from hepatitis Hyperferritinemiawas described to be associated with clinical disease severityin children and is currently used as a laboratory markerfor dengue In fact the coagulation and fibrinolytic systemsare highly activated in dengue patients with hyperferritine-mia Furthermore cytokines and coagulation mediatorsother bioactive mediators and adhesive proteins perpetuatethe inflammatory response promoting increased interactionbetween immune cells platelets and ECs contributing tothrombocytopenia Moreover it has been suggested thatthrombocytopenia arises from both decreased productionof cells from bone marrow associated with an increasedperipheral destruction of platelets The cross-reactive anti-bodies anti-NS1 prM and E viral proteins against plateletsendothelial cells or coagulatory molecules may cause plateletdysfunction endothelial cell damage coagulation defectsand macrophage activation Impaired platelet function couldincrease the risk of vascular fragility leading to hemorrhageand contributing to plasma leakage in DHFDSS There arefewer studies in dengue about platelets as effector immunecells To date no scientific studies have suggested that plateletgranule constituents can amplify inflammation and vascularpermeability alteration during dengue Several mechanismsare involved in thrombocytopenia and platelet dysfunction indengue indicating the complexity of dengue immunopatho-genesis

Conflict of Interests

The authors declare that they have no conflict of interests

References

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[2] B J Geiss H Stahla A M Hannah H H Gari and S MKeenan ldquoFocus on flaviviruses current and future drug targetsrdquoFuture Medicinal Chemistry vol 1 no 2 pp 327ndash344 2009

[3] S Green and A Rothman ldquoImmunopathological mechanismsin dengue and dengue hemorrhagic feverrdquo Current Opinion inInfectious Diseases vol 19 no 5 pp 429ndash436 2006

[4] C-J Chang H-W Luh S-H Wang H-J Lin S-C Leeand S-T Hu ldquoThe heterogeneous nuclear ribonucleoprotein K(hnRNP K) interacts with dengue virus core proteinrdquoDNA andCell Biology vol 20 no 9 pp 569ndash577 2001

[5] Y Modis S Ogata D Clements and S C Harrison ldquoStructureof the dengue virus envelope protein after membrane fusionrdquoNature vol 427 no 6972 pp 313ndash319 2004

[6] W D Crill and G-J J Chang ldquoLocalization and characteriza-tion of flavivirus envelope glycoprotein cross-reactive epitopesrdquoJournal of Virology vol 78 no 24 pp 13975ndash13986 2004

[7] S C Weaver and N Vasilakis ldquoMolecular evolution of dengueviruses contributions of phylogenetics to understanding thehistory and epidemiology of the preeminent arboviral diseaserdquoInfection Genetics andEvolution vol 9 no 4 pp 523ndash540 2009

[8] F M Kassim M N Izati T TgRogayah Y M Apandi and ZSaat ldquoUse of dengue NS1 antigen for early diagnosis of denguevirus infectionrdquo Southeast Asian Journal of Tropical Medicineand Public Health vol 42 no 3 pp 562ndash569 2011

[9] M da Rocha Queiroz Lima R M R Nogueira H G Schatz-mayr A M B de Filippis D Limonta and F B dos SantosldquoA new approach to dengue fatal cases diagnosis NS1 antigencapture in tissuesrdquo PLoS Neglected Tropical Diseases vol 5 no5 Article ID e1147 2011

[10] P Somnuke R E Hauhart J P AtkinsonM S Diamond and PAvirutnan ldquoN-linked glycosylation of dengue virus NS1 proteinmodulates secretion cell-surface expression hexamer stabilityand interactions with human complementrdquo Virology vol 413no 2 pp 253ndash264 2011

[11] D H Libraty P R Young D Pickering et al ldquoHigh circulatinglevels of the dengue virus nonstructural protein NS1 earlyin dengue illness correlate with the development of denguehemorrhagic feverrdquo The Journal of Infectious Diseases vol 186no 8 pp 1165ndash1168 2002

[12] J L Munoz-Jordan G G Sanchez-Burgos M Laurent-Rolleand A Garcia-Sastre ldquoInhibition of interferon signaling bydengue virusrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 100 no 24 pp 14333ndash143382003

[13] M Jones A Davidson L Hibbert et al ldquoDengue virus inhibitsalpha interferon signaling by reducing STAT2 expressionrdquoJournal of Virology vol 79 no 9 pp 5414ndash5420 2005

[14] P Erbel N Schiering A DrsquoArcy et al ldquoStructural basis for theactivation of flaviviralNS3 proteases fromdengue andWestNilevirusrdquoNature Structural andMolecular Biology vol 13 no 4 pp372ndash373 2006

[15] D Leung K Schroder HWhite et al ldquoActivity of recombinantdengue 2 virusNS3protease in the presence of a truncatedNS2Bco-factor small peptide substrates and inhibitorsrdquo Journal ofBiological Chemistry vol 276 no 49 pp 45762ndash45771 2001

[16] H Li S Clum S You K E Ebner and R Padmanabhan ldquoTheserine protease and RNA-stimulated nucleoside triphosphataseand RNA helicase functional domains of dengue virus type 2NS3 converge within a region of 20 amino acidsrdquo Journal ofVirology vol 73 no 4 pp 3108ndash3116 1999

[17] M-P Egloff D Benarroch B Selisko J-L Romette and BCanard ldquoAn RNA cap (nucleoside-21015840-O-)-methyltransferasein the flavivirus RNA polymerase NS5 crystal structure andfunctional characterizationrdquo The EMBO Journal vol 21 no 11pp 2757ndash2768 2002

[18] M Mazzon M Jones A Davidson B Chain and M JacobsldquoDengue virus ns5 inhibits interferon-a signaling by blocking


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[38] R N Makroo V Raina P Kumar and R K Kanth ldquoRole ofplatelet transfusion in the management of dengue patients in atertiary care hospitalrdquo Asian Journal of Transfusion Science vol1 no 1 pp 4ndash7 2007

[39] L C S Lum M E-A Abdel-Latif A Y T Goh P W KChan and S K Lam ldquoPreventive transfusion in dengue shocksyndromemdashis it necessaryrdquo Journal of Pediatrics vol 143 no 5pp 682ndash684 2003

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[47] C-F Lin S-W Wan H-J Cheng H-Y Lei and Y-S LinldquoAutoimmune pathogenesis in dengue virus infectionrdquo ViralImmunology vol 19 no 2 pp 127ndash132 2006

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[50] J E Freedman ldquoMolecular regulation of platelet-dependentthrombosisrdquo Circulation vol 112 no 17 pp 2725ndash2734 2005

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[54] J Rivera M L Lozano L Navarro-Nunez and V VicenteldquoPlatelet receptors and signaling in the dynamics of thrombusformationrdquo Haematologica vol 94 no 5 pp 700ndash711 2009

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[66] G C White II and R Rompietti ldquoPlatelet secretion indis-criminately spewed forth or highly orchestratedrdquo Journal ofThrombosis andHaemostasis vol 5 no 10 pp 2006ndash2008 2007

[67] S Sehgal and B Storrie ldquoEvidence that differential packagingof the major platelet granule proteins von Willebrand factorand fibrinogen can support their differential releaserdquo Journal ofThrombosis and Haemostasis vol 5 no 10 pp 2009ndash2016 2007

[68] S Lindemann and M Gawaz ldquoThe active platelet translationand protein synthesis in an anucleate cellrdquo Seminars in Throm-bosis and Hemostasis vol 33 no 2 pp 144ndash150 2007

[69] P N Shashkin G T Brown A Ghosh G K Marathe and TMMcIntyre ldquoLipopolysaccharide is a direct agonist for plateletRNA splicingrdquo The Journal of Immunology vol 181 no 5 pp3495ndash3502 2008

[70] A N Wicki A Walz S N Gerber-Huber R H Wenger RVornhagen and K J Clemetson ldquoIsolation and characteri-zation of human blood platelet mRNA and construction ofa cDNA library in lambda gt11 Confirmation of the plateletderivation by identification of GPIb coding mRNA and cloningof a GPIb coding cDNA insertrdquo Thrombosis and Haemostasisvol 61 no 3 pp 448ndash453 1989

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[74] A S Weyrich and G A Zimmerman ldquoPlatelets signaling cellsin the immune continuumrdquo Trends in Immunology vol 25 no9 pp 489ndash495 2004

[75] A Trzeciak-Ryczek B Tokarz-Deptuła and W DeptułaldquoPlateletsmdashan important element of the immune systemrdquoPolishJournal of Veterinary Sciences vol 16 no 2 pp 407ndash413 2013

[76] S PWatson JM J Herbert andA Y Pollitt ldquoGPVI andCLEC-2 in hemostasis and vascular integrityrdquo Journal of Thrombosisand Haemostasis vol 8 no 7 pp 1456ndash1467 2010

[77] A Kasirer-Friede M L Kahn and S J Shattil ldquoPlateletintegrins and immunoreceptorsrdquo Immunological Reviews vol218 no 1 pp 247ndash264 2007

[78] Y Boulaftali P R Hess T M Getz et al ldquoPlatelet ITAM signal-ing is critical for vascular integrity in infammationrdquoThe Journalof Clinical Investigation vol 123 no 2 pp 908ndash916 2013

[79] R S Kasthuri S L Glover J Boles and N MacKman ldquoTissuefactor and tissue factor pathway inhibitor as key regulators ofglobal hemostasis measurement of their levels in coagulationassaysrdquo Seminars in Thrombosis and Hemostasis vol 36 no 7pp 764ndash771 2010

[80] V Daubie R Pochet S Houard and P Philippart ldquoTissuefactor a mini-reviewrdquo Journal of Tissue Engineering and Regen-erative Medicine vol 1 no 3 pp 161ndash169 2007

[81] A P Owens III and N Mackman ldquoTissue factor and thrombo-sis the clot starts hererdquo Thrombosis and Haemostasis vol 104no 3 pp 432ndash439 2010

[82] P M Fernandez and F R Rickles ldquoTissue factor and angiogen-esis in cancerrdquo Current Opinion in Hematology vol 9 no 5 pp401ndash406 2002

[83] EM EgorinaM A Sovershaev G Bjoslashrkoslashy et al ldquoIntracellularand surface distribution of monocyte tissue factor applicationto intersubject variabilityrdquo Arteriosclerosis Thrombosis andVascular Biology vol 25 no 7 pp 1493ndash1498 2005

[84] B Osterud and E Bjorklid ldquoTissue factor in blood cells andendothelial cellsrdquo Frontiers in Bioscience vol 4 no 1 pp 289ndash299 2012

[85] M L Lopez N Soriano-Sarabia G Bruges et al ldquoExpressionpattern of protease activated receptors in lymphoid cellsrdquoCellular Immunology vol 288 no 1-2 pp 47ndash52 2014

[86] V S Ossovskaya and N W Bunnett ldquoProtease-activated recep-tors contribution to physiology and diseaserdquo PhysiologicalReviews vol 84 no 2 pp 579ndash621 2004


[87] E Camerer W Huang and S R Coughlin ldquoTissue factor- andfactor X-dependent activation of protease-activated receptor 2by factor VIIardquo Proceedings of the National Academy of Sciencesof the United States of America vol 97 no 10 pp 5255ndash52602000

[88] R Ramachandran and M D Hollenberg ldquoProteinases andsignalling pathophysiological and therapeutic implications viaPARs and morerdquo British Journal of Pharmacology vol 153supplement 1 pp S263ndashS282 2008

[89] W Ruf ldquoProtease-activated receptor signaling in the regulationof inflammationrdquo Critical Care Medicine vol 32 no 5 supple-ment pp S287ndashS292 2004

[90] J P Chen andTM Cosgriff ldquoHemorrhagic fever virus-inducedchanges in hemostasis and vascular biologyrdquo Blood Coagulationamp Fibrinolysis vol 11 no 5 pp 461ndash483 2000

[91] M Levi T van der Poll and H R Buller ldquoBidirectional relationbetween inflammation and coagulationrdquo Circulation vol 109no 22 pp 2698ndash2704 2004

[92] W Ruf ldquoEmerging roles of tissue factor in viral hemorrhagicfeverrdquo Trends in Immunology vol 25 no 9 pp 461ndash464 2004

[93] A R Rezaie ldquoRegulation of the protein C anticoagulant andantiinflammatory pathwaysrdquo Current Medicinal Chemistry vol17 no 19 pp 2059ndash2069 2010

[94] J Petaja ldquoInflammation and coagulation An overviewrdquoThrom-bosis Research vol 127 supplement 2 pp S34ndashS37 2011

[95] T van der Poll J D D de Boer and M Levi ldquoThe effect ofinflammation on coagulation and vice versardquo Current Opinionin Infectious Diseases vol 24 no 3 pp 273ndash278 2011

[96] M Levi ldquoThe coagulant response in sepsis and inflammationrdquoHamostaseologie vol 30 no 1 pp 10ndash16 2010

[97] M Levi and T van der Poll ldquoInflammation and coagulationrdquoCritical Care Medicine vol 38 no 2 supplement pp S26ndashS342010

[98] S T Olson and I Bjork ldquoRegulation of thrombin activity byantithrombin and heparinrdquo Seminars in Thrombosis andHemostasis vol 20 no 4 pp 373ndash409 1994

[99] S A Maroney and A E Mast ldquoTissue factor pathway inhibitorand bacterial infectionrdquo Journal ofThrombosis andHaemostasisvol 9 no 1 pp 119ndash121 2011

[100] I Andia and M Abate ldquoPlatelet-rich plasma underlying biol-ogy and clinical correlatesrdquo Regenerative Medicine vol 8 no 5pp 645ndash658 2013

[101] J F Danielli ldquoCapillary permeability and oedema in theperfused frogrdquo Journal of Physiology vol 98 no 1 pp 109ndash1291940

[102] A T Nurden P Nurden M Sanchez I Andia and E AnitualdquoPlatelets and wound healingrdquo Frontiers in Bioscience vol 13no 9 pp 3532ndash3548 2008

[103] I Andia M Sanchez and N Maffulli ldquoTendon healing andplatelet-rich plasma therapiesrdquo Expert Opinion on BiologicalTherapy vol 10 no 10 pp 1415ndash1426 2010

[104] A J Hayes and J R Ralphs ldquoThe response of foetal annulusfibrosus cells to growth factors modulation of matrix synthesisby TGF-1205731 and IGF-1rdquoHistochemistry and Cell Biology vol 136no 2 pp 163ndash175 2011

[105] T G Diacovo K D Puri R A Warnock T A Springer andU H von Andrian ldquoPlatelet-mediated lymphocyte delivery tohigh endothelial venulesrdquo Science vol 273 no 5272 pp 252ndash255 1996

[106] P Andre L Nannizzi-Alaimo S K Prasad and D R PhillipsldquoPlatelet-derivedCD40L the switch-hitting player of cardiovas-cular diseaserdquo Circulation vol 106 no 8 pp 896ndash899 2002

[107] L K KhoHWulur and THimawan ldquoBlood and bonemarrowchanges in dengue haemorrhagic feverrdquo Paediatrica Indone-siana vol 12 no 1 pp 31ndash39 1972

[108] T Srichaikul ldquoDisseminated intravascular coagulation indengue haemorrhagic feverrdquo The Southeast Asian Journal ofTropical Medicine and Public Health vol 18 no 3 pp 303ndash3111987

[109] F J de Sauvage P E Hass S D Spencer et al ldquoStimulationof megakaryocytopoiesis and thrombopoiesis by the c-Mplligandrdquo Nature vol 369 no 6481 pp 533ndash538 1994

[110] A L Gurney K Carver-Moore F J de Sauvage and M WMoore ldquoThrombocytopenia in c-mpl-deficient micerdquo Sciencevol 265 no 5177 pp 1445ndash1447 1994

[111] E Putintseva G Vega and L Fernandez ldquoAlterations inthrombopoiesis in patients with thrombocytopenia producedby dengue hemorrhagic feverrdquo Nouvelle Revue FrancaisedrsquoHematologie vol 28 no 5 pp 269ndash273 1986

[112] A V Matondang D Widodo I Zulkarnain et al ldquoThe correla-tion between thrombopoietin andplatelet count in adult dengueviral infection patientsrdquo Acta medica Indonesiana vol 36no 2 pp 62ndash69 2004

[113] C Krishnamurti R A Peat M A Cutting and S W RothwellldquoPlatelet adhesion to dengue-2 virus-infected endothelial cellsrdquoTheAmerican Journal of Tropical Medicine and Hygiene vol 66no 4 pp 435ndash441 2002

[114] T Srichaikul S Nimmannitya T Sripaisarn M Kamolsilpaand C Pulgate ldquoPlatelet function during the acute phase ofdengue hemorrhagic feverrdquo The Southeast Asian Journal ofTropical Medicine and Public Health vol 20 no 1 pp 19ndash251989

[115] S Honda M Saito E M Dimaano et al ldquoIncreased phago-cytosis of platelets from patients with secondary dengue virusinfection by human macrophagesrdquo The American Journal ofTropical Medicine and Hygiene vol 80 no 5 pp 841ndash845 2009

[116] C F Lin H Y Lei C C Liu et al ldquoGeneration of IgM anti-platelet autoantibody in dengue patientsrdquo Journal of MedicalVirology vol 63 no 2 pp 143ndash149 2001

[117] H-Y Lei T-M Yeh H-S Liu Y-S Lin S-H Chen and C-CLiu ldquoImmunopathogenesis of dengue virus infectionrdquo Journalof Biomedical Science vol 8 no 5 pp 377ndash388 2001

[118] Y-S Lin T-M Yeh C-F Lin et al ldquoMolecular mimicrybetween virus and host and its implications for dengue diseasepathogenesisrdquo Experimental Biology and Medicine vol 236 no5 pp 515ndash523 2011

[119] M T G Alonzo T L V Lacuesta EMDimaano et al ldquoPlateletapoptosis and apoptotic platelet clearance by macrophages insecondary dengue virus infectionsrdquo The Journal of InfectiousDiseases vol 205 no 8 pp 1321ndash1329 2012

[120] K Oishi M Saito C A Mapua and F F Natividad ldquoDengueillness clinical features and pathogenesisrdquo Journal of Infectionand Chemotherapy vol 13 no 3 pp 125ndash133 2007

[121] M Saito K Oishi S Inoue et al ldquoAssociation of increasedplatelet-associated immunoglobulins with thrombocytopeniaand the severity of disease in secondary dengue virus infec-tionsrdquo Clinical and Experimental Immunology vol 138 no 2pp 299ndash303 2004

[122] S Boonpucknavig O Vuttiviroj C Bunnag N Bhamarapra-vati and S Nimmanitya ldquoDemonstration of dengue antibodycomplexes on the surface of platelets from patients with denguehemorrhagic feverrdquo The American Journal of Tropical Medicineand Hygiene vol 28 no 5 pp 881ndash884 1979


[123] S Noisakran K Chokephaibulkit P Songprakhon et al ldquoA re-evaluation of the mechanisms leading to dengue hemorrhagicfeverrdquo Annals of the New York Academy of Sciences vol 1171supplement 1 pp E24ndashE35 2009

[124] S Noisakran R V Gibbons P Songprakhon et al ldquoDetectionof dengue virus in platelets isolated from dengue patientsrdquoSoutheast Asian Journal of Tropical Medicine and Public Healthvol 40 no 2 pp 253ndash262 2009

[125] S Noisakran N Onlamoon K Pattanapanyasat et al ldquoRole ofCD61+ cells in thrombocytopenia of dengue patientsrdquo Interna-tional Journal of Hematology vol 96 no 5 pp 600ndash610 2012

[126] A L Rothman and F A Ennis ldquoImmunopathogenesis ofdengue hemorrhagic feverrdquoVirology vol 257 no 1 pp 1ndash6 1999

[127] B A Wills E E Oragui A C Stephens et al ldquoCoagulationabnormalities in dengue hemorrhagic fever serial investiga-tions in 167 Vietnamese children with Dengue shock syn-dromerdquo Clinical Infectious Diseases vol 35 no 3 pp 277ndash2852002

[128] B Wills T V Ngoc N T H Van et al ldquoHemostatic changesin Vietnamese children with mild dengue correlate with theseverity of vascular leakage rather than bleedingrdquoTheAmericanJournal of Tropical Medicine and Hygiene vol 81 no 4 pp 638ndash644 2009

[129] D Sosothikul P Seksarn S Pongsewalak U Thisyakorn andJ Lusher ldquoActivation of endothelial cells coagulation and fib-rinolysis in children with Dengue virus infectionrdquo Thrombosisand Haemostasis vol 97 no 4 pp 627ndash634 2007

[130] M L Avila-Aguero C R Avila-Aguero S L Um A Soriano-Fallas A Canas-Coto and S B Yan ldquoSystemic host inflam-matory and coagulation response in the Dengue virus primo-infectionrdquo Cytokine vol 27 no 6 pp 173ndash179 2004

[131] T-S Ho S-M Wang Y-S Lin and C-C Liu ldquoClinicaland laboratory predictive markers for acute dengue infectionrdquoJournal of Biomedical Science vol 20 no 1 article 75 2013

[132] Y Funahara K Ogawa N Fujita andYOkuno ldquoThree possibletriggers to induce thrombocytopenia in dengue virus infectionrdquoSoutheast Asian Journal of Tropical Medicine and Public Healthvol 18 no 3 pp 351ndash355 1987

[133] E F Mammen ldquoDisseminated intravascular coagulation(DIC)rdquo Clinical Laboratory Science vol 13 no 4 pp 239ndash2452000

[134] R L Bick ldquoDisseminated intravascular coagulation a review ofetiology pathophysiology diagnosis and management Guide-lines for carerdquo Clinical and AppliedThrombosisHemostasis vol8 no 1 pp 1ndash31 2002

[135] MN Chua RMolanidaM deGuzman and F Laberiza ldquoPro-thrombin time and partial thromboplastin time as a predictorof bleeding in patients with dengue hemorrhagic feverrdquo TheSoutheast Asian Journal of Tropical Medicine and Public Healthvol 24 supplement 1 pp 141ndash143 1993

[136] J-W Liu I-K Lee L Wang R-F Chen and K D YangldquoThe usefulness of clinical-practice-based laboratory data infacilitating the diagnosis of dengue illnessrdquo BioMed ResearchInternational vol 2013 Article ID 198797 11 pages 2013

[137] Y H Huang C C Liu S T Wang et al ldquoActivation ofcoagulation and fibrinolysis during dengue virus infectionrdquoJournal of Medical Virology vol 63 no 3 pp 247ndash251 2001

[138] T W Geisbert L E Hensley T Larsen et al ldquoPathogenesis ofEbola hemorrhagic fever in cynomolgus macaques evidencethat dendritic cells are early and sustained targets of infectionrdquoThe American Journal of Pathology vol 163 no 6 pp 2347ndash2370 2003

[139] T W Geisbert L E Hensley P B Jahrling et al ldquoTreatmentof Ebola virus infection with a recombinant inhibitor of factorVIIatissue factor a study in rhesus monkeysrdquoThe Lancet vol362 no 9400 pp 1953ndash1958 2003

[140] Z Jiang X Tang R Xiao L Jiang and X Chen ldquoDenguevirus regulates the expression of hemostasis-related moleculesin human vein endothelial cellsrdquo The Journal of Infection vol55 no 2 pp e23ndashe28 2007

[141] A Huerta-Zepeda C Cabello-Gutierrez J Cime-Castillo etal ldquoCrosstalk between coagulation and inflammation duringDengue virus infectionrdquo Thrombosis and Haemostasis vol 99no 5 pp 936ndash943 2008

[142] E L de Azeredo C F Kubelka L M Alburquerque et alldquoTissue factor expression on monocytes from patients withsevere dengue feverrdquo Blood Cells Molecules and Diseases vol45 no 4 pp 334ndash335 2010

[143] L M O Pinto S A Oliveira E L A Braga R M R Nogueiraand C F Kubelka ldquoIncreased Pro-inflammatory Cytokines(TNF-120572 and IL-6) and Anti-inflammatory Compounds (sTN-FRp55 and sTNFRp75) in Brazilian patients during exanthe-matic dengue feverrdquo Memorias do Instituto Oswaldo Cruz vol94 no 3 pp 387ndash394 1999

[144] C F Kubelka P A Borges F F VonSydow and E LampeldquoAnalysis of tumor necrosis factor-alpha serum level in Brazilianpatients with Dengue-2rdquo Memorias do Instituto Oswaldo Cruzvol 90 no 6 pp 741ndash742 1995

[145] E L Azeredo SM O ZagneM A Santiago et al ldquoCharacteri-sation of lymphocyte response and cytokine patterns in patientswith dengue feverrdquo Immunobiology vol 204 no 4 pp 494ndash5072001

[146] S Green D W Vaughn S Kalayanarooj et al ldquoEarly immuneactivation in acute dengue illness is related to development ofplasma leakage and disease severityrdquo The Journal of InfectiousDiseases vol 179 no 4 pp 755ndash762 1999

[147] D Hober A-S Delannoy S Benyoucef D de Groote andP Wattre ldquoHigh levels of sTNFR p75 and TNF120572 in dengue-infected patientsrdquoMicrobiology and Immunology vol 40 no 8pp 569ndash573 1996

[148] F A Orsi R N Angerami B M Mazetto et al ldquoReducedthrombin formation and excessive fibrinolysis are associatedwith bleeding complications in patients with dengue fever acase-control study comparing dengue fever patients with andwithout bleeding manifestationsrdquo BMC Infectious Diseases vol13 no 1 article 350 2013

[149] S-W Lin Y-C Chuang Y-S Lin H-Y Lei H-S Liu andT-M Yeh ldquoDengue virus nonstructural protein NS1 binds toprothrombinthrombin and inhibits prothrombin activationrdquoJournal of Infection vol 64 no 3 pp 325ndash334 2012

[150] Y C Chuang H Y Lei Y S Lin H S Liu H L Wu and T MYeh ldquoDengue virus-induced autoantibodies bind to plasmino-gen and enhance its activationrdquo Journal of Immunology vol 187no 12 pp 6483ndash6490 2011

[151] K D Yang C S Lee and M F Shaio ldquoA higher productionof platelet activating factor in ex vivo heterologously secondarydengue-2 virus infectionsrdquoActaMicrobiologica et ImmunologicaHungarica vol 42 no 4 pp 403ndash407 1995

[152] K Ghosh S Gangodkar P Jain et al ldquoImaging the interactionbetween dengue 2 virus and human blood platelets using atomicforce and electron microscopyrdquo Journal of Electron Microscopy(Tokyo) vol 57 no 3 pp 113ndash118 2008


[153] S Moncada and E A Higgs ldquoThe discovery of nitric oxide andits role in vascular biologyrdquoBritish Journal of Pharmacology vol147 supplement 1 pp S193ndashS201 2006

[154] N Valero L M Espina G Anez E Torres and J A MosqueraldquoShort report increased level of serum nitric oxide in patientswith denguerdquo The American Journal of Tropical Medicine andHygiene vol 66 no 6 pp 762ndash764 2002

[155] L R Queen B Xu K Horinouchi I Fisher and A Ferroldquo1205732-Adrenoceptors activate nitric oxide synthase in humanplateletsrdquo Circulation Research vol 87 no 1 pp 39ndash44 2000

[156] J R Sheu Y C KanW CHung C H Lin andMH Yen ldquoTheantiplatelet activity of tetramethylpyrazine is mediated throughactivation of NO synthaserdquo Life Sciences vol 67 no 8 pp 937ndash947 2000

[157] A C Mendes-Ribeiro M B Moss M A S Siqueira et alldquoDengue fever activates the L-arginine-nitric oxide pathwayan explanation for reduced aggregation of human plateletsrdquoClinical and Experimental Pharmacology amp Physiology vol 35no 10 pp 1143ndash1146 2008

[158] C Matsuura T L Moraes J B Barbosa et al ldquoNitric oxideactivity in platelets of dengue haemorrhagic fever patients theapparent paradoxical role of ADMA and l-NMMArdquo Transac-tions of the Royal Society of Tropical Medicine and Hygiene vol106 no 3 pp 174ndash179 2012

[159] M Michels B Alisjahbana P G de Groot et al ldquoPlatelet func-tion alterations in dengue are associated with plasma leakagerdquoThrombosis and Haemostasis vol 112 no 2 pp 352ndash362 2014

[160] S Gudbrandsdottir H C Hasselbalch and C H NielsenldquoActivated platelets enhance IL-10 secretion and reduce TNF-120572 secretion by monocytesrdquoThe Journal of Immunology vol 191no 8 pp 4059ndash4067 2013

[161] E D Hottz I M Medeiros-de-Moraes A Vieira-de-Abreuet al ldquoPlatelet activation and apoptosis modulate monocyteinflammatory responses in denguerdquoThe Journal of Immunologyvol 193 no 4 pp 1864ndash1872 2014

[162] C A Janeway Jr and R Medzhitov ldquoInnate immune recogni-tionrdquo Annual Review of Immunology vol 20 pp 197ndash216 2002

[163] G Andonegui S M Kerfoot K McNagny K V J Ebbert KD Patel and P Kubes ldquoPlatelets express functional Toll-likereceptor-4rdquo Blood vol 106 no 7 pp 2417ndash2423 2005

[164] F Cognasse H Hamzeh P Chavarin S Acquart C Geninand O Garraud ldquoEvidence of Toll-like receptor molecules onhuman plateletsrdquo Immunology and Cell Biology vol 83 no 2pp 196ndash198 2005

[165] R Aslam E R Speck M Kim et al ldquoPlatelet Toll-like receptorexpression modulates lipopolysaccharide-induced thrombocy-topenia and tumor necrosis factor-alpha production in vivordquoBlood vol 107 no 2 pp 637ndash641 2006

[166] P Patrignani C di Febbo S Tacconelli et al ldquoReducedthromboxane biosynthesis in carriers of toll-like receptor 4polymorphisms in vivordquo Blood vol 107 no 9 pp 3572ndash35742006

[167] J W Semple R Aslam M Kim E R Speck and J Freed-man ldquoPlatelet-bound lipopolysaccharide enhances Fc receptor-mediated phagocytosis of IgG-opsonized plateletsrdquo Blood vol109 no 11 pp 4803ndash4805 2007

[168] G Zhang J Han E J Welch et al ldquoLipopolysaccharidestimulates platelet secretion and potentiates platelet aggregationvia TLR4MyD88 and the cGMP-dependent protein kinasepathwayrdquo Journal of Immunology vol 182 no 12 pp 7997ndash80042009

[169] E D Hottz J F Lopes C Freitas et al ldquoPlatelets medi-ate increased endothelium permeability in dengue throughNLRP3-inflammasome activationrdquo Blood vol 122 no 20 pp3405ndash3414 2013

[170] K Djamiatun S M H Faradz T E Setiati M G Netea AJ A M van der Ven and W M V Dolmans ldquoIncrease ofplasminogen activator inhibitor-1 and decrease of transforminggrowth factor-1205731 in children with dengue haemorrhagic feverin Indonesiardquo Journal of Tropical Pediatrics vol 57 no 6 pp424ndash432 2011

[171] R K Assoian A Komoriya C A Meyers D M Millerand M B Sporn ldquoTransforming growth factor-120573 in humanplatelets Identification of a major storage site purification andcharacterizationrdquo Journal of Biological Chemistry vol 258 no11 pp 7155ndash7160 1983

[172] P O Andersson D Stockelberg S Jacobsson and H Waden-vik ldquoA transforming growth factor-beta1-mediated bystanderimmune suppression could be associated with remission ofchronic idiopathic thrombocytopenic purpurardquo Annals ofHematology vol 79 no 9 pp 507ndash513 2000

[173] P-O Andersson A Olsson and HWadenvik ldquoReduced trans-forming growth factor-1205731 production by mononuclear cellsfrom patients with active chronic idiopathic thrombocytopenicpurpurardquoBritish Journal ofHaematology vol 116 no 4 pp 862ndash867 2002

[174] P Butthep A Bunyaratvej and N Bhamarapravati ldquoDenguevirus and endothelial cell a related phenomenon to thrombo-cytopenia and granulocytopenia in dengue hemorrhagic feverrdquoThe Southeast Asian Journal of Tropical Medicine and PublicHealth vol 24 supplement 1 pp 246ndash249 1993

[175] K Chen T C Detwiler and D W Essex ldquoCharacterization ofprotein disulphide isomerase released from activated plateletsrdquoBritish Journal of Haematology vol 90 no 2 pp 425ndash431 1995

[176] D W Essex and M Li ldquoProtein disulphide isomerase mediatesplatelet aggregation and secretionrdquo British Journal of Haematol-ogy vol 104 no 3 pp 448ndash454 1999

[177] A Rachman A R Harahap and R M Widhyasih ldquoThe roleof anti-dengue virus NS-1 and anti-protein disulfide isomeraseantibodies on platelet aggregation in secondary dengue infec-tionrdquo Acta medica Indonesiana vol 45 no 1 pp 44ndash48 2013

[178] H-J Cheng H-Y Lei C-F Lin et al ldquoAnti-dengue virusnonstructural protein 1 antibodies recognize protein disulfideisomerase on platelets and inhibit platelet aggregationrdquo Molec-ular Immunology vol 47 no 2-3 pp 398ndash406 2009

[179] RC S Seet AW L ChowAM LQuek Y-HChan andECH Lim ldquoRelationship between circulating vascular endothelialgrowth factor and its soluble receptors in adults with denguevirus infection a case-control studyrdquo International Journal ofInfectious Diseases vol 13 no 5 pp e248ndashe253 2009

[180] P Sathupan A Khongphattanayothin J Srisai K Srikaewand Y Poovorawan ldquoThe role of vascular endothelial growthfactor leading to vascular leakage in children with dengue virusinfectionrdquo Annals of Tropical Paediatrics vol 27 no 3 pp 179ndash184 2007

[181] A Srikiatkhachorn C Ajariyakhajorn T P Endy et al ldquoVirus-induced decline in soluble vascular endothelial growth receptor2 is associated with plasma leakage in dengue hemorrhagicfeverrdquo Journal of Virology vol 81 no 4 pp 1592ndash1600 2007

[182] C-S Tseng H-W Lo H-C Teng W-C Lo and C-G KerldquoElevated levels of plasma VEGF in patients with dengue hem-orrhagic feverrdquo FEMS Immunology and Medical Microbiologyvol 43 no 1 pp 99ndash102 2005


[183] G N Malavige L-C Huang M Salimi L Gomes S DJayaratne and G S Ogg ldquoCellular and cytokine correlates ofsevere dengue infectionrdquo PLoS ONE vol 7 no 11 Article IDe50387 2012

[184] MMichels A J AM van der Ven K Djamiatun et al ldquoImbal-ance of angiopoietin-1 and angiopoetin-2 in severe dengueand relationship with thrombocytopenia endothelial activa-tion and vascular stabilityrdquo The American Journal of TropicalMedicine and Hygiene vol 87 no 5 pp 943ndash946 2012


Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


involved in viral RNA replication Notably NS1 is expressedon the surface of infected cells without forming part of thevirion [7] Serum levels of secreted NS1 (sNS1) positivelycorrelate with viral titers and have been a useful tool indengue infection diagnosis [8 9] Because it is expressedon the surface of infected cells NS1 triggers host immuneresponses Additionally NS1 has been shown to displaysoluble complement-fixing activity [10] and it was suggestedto be involved in dengue pathogenesis [11] NS2A is a 22 kDaprotein involved in RNA packaging and replication and itmay be involved in interferon type I antagonism [12 13]NS2B is a 14 kDa membrane-associated protein and servesas a cofactor for NS3 to form a viral protease complex [1415] NS3 is a multifunctional protein with serine proteasehelicasenucleoside triphosphate-NTPase activities and itis required for unwinding the double-stranded replicativeform of RNA It is also involved in processing the viralpolyprotein and RNA replication [7 16] NS4A and NS4B aresmall hydrophobic proteins that function as IFN-signalinginhibitors [12 13] Finally NS5 is a large multifunctional103 kDa protein that displays RNA-dependent polymeraseactivity and was recently identified as a potential type I IFNproduction antagonist [17 18]

All dengue serotypes are capable of causing disease witha wide spectrum of clinical manifestations ranging froman undifferentiated fever in a mild clinical form classicallyknown as dengue fever (DF) to the severe clinical andpotentially fatal DHFDSS [19] Initial symptoms are com-mon to all patients but the clinical manifestations of thesevere forms rapidly evolve with symptoms including highfever liver enlargement circulatory failure (hypotension andshock) edema cavity (pleural abdominal and cardiac) andinternal bleeding phenomenaThe severe forms are primarilycharacterized by plasma leakage and thrombocytopenia withor without hemorrhage The World Health Organization(WHO) classified the clinical presentations of DHF into fourseverity grades based on laboratory data Grade I fever withpositive tourniquet test Grade II plus mild spontaneousbleeding Grade III presence of weak and rapid pulse andGrade IV profound shock with undetectable pulse The lasttwo are considered DSS [20]

It has been difficult to apply the WHO classificationsin Central American countries and Latin America [21ndash23]suggesting that the disease classification into DF DHF andDSS may not be universally applicable for clinical manage-ment In this context Harris et al (2000) reported severalclinical DSS cases that could not be completely classified asabove Given the difficulty of classifying serious cases accord-ing to WHO criteria a new category has been proposedcalled Signs Associated with Dengue Shock which is verysimilar to DSS but without the presence of thrombocytopeniaand hemoconcentration [22] The Special Programme forResearch and Training in Tropical Diseases World HealthOrganization (TDRWHO) proposed a new classificationbased on the difficulties encountered in applying the criteriaof the WHO classification especially in Latin America Theclassification is based on current clinical criteria for severityof dengue cases understanding of the illness as a systemicand dynamic event and facilitating the approach to cases

and subsequent clinical management of patients Accordingto this criterion the new classification addresses three setsof clinical signs and symptoms (1) dengue without warningsigns (2) dengue with warning signs and (3) severe dengue[24 25] According to the new classification patients typicallydeveloped a sudden high-grade feverThis acute febrile phaseusually lasts 2ndash7 days and was often accompanied by facialflushing skin erythema generalized body ache myalgiaarthralgia and headache Anorexia nausea and vomitingwere also common These clinical features are characterizedas nonsevere dengue cases Monitoring for warning signsand other clinical parameters are crucial to recognizing theprogression to the critical phase and it may help distinguishnonsevere and severe dengue cases The warning signs wereassessed by the following clinical parameters abdominalpain or tenderness persistent vomiting clinical fluid accu-mulation mucosal bleed lethargy restlessness and liverenlargement gt2 cm associated with laboratory parameterssuch as increase in hematocrit (HCT) concurrent with rapiddecrease in platelet count Mild hemorrhagic manifestationssuch as petechiae and mucosal membrane bleeding (egnose and gums) may be present Patients require emer-gency treatment and urgent referral in the critical phaseof disease that is when they have severe dengue charac-terized by severe plasma leakage leading to dengue shockandor fluid accumulation with respiratory distress severehemorrhages shown by massive vaginal bleeding (in womenof childbearing age) and gastrointestinal bleeding andorsevere organ impairment (hepatic damage renal impairmentcardiomyopathy encephalopathy or encephalitis) [25] Mostpatients recovered from the illness uneventfully and survivedto hospital discharge

The mechanisms by which pathophysiologic changesoccur in dengue fever are still not fully understood Theinteraction of several factors seems to be responsible for thedevelopment of the severe disease These factors include thefollowing the virulence of the circulating strain the presenceof efficient or high density vector the wide circulation of thevirus and characteristics of the host as genetic factors eth-nicity presence of chronic diseases and subsequent DENVinfections [19 26] Natural infection by one of the fourDENVserotypes produces lasting immunity against reinfection bythe same serotype but heterotypic protection is temporaryandpartial thus allowing sequential infections Epidemiolog-ical studies have shown thatmost individualswhodevelop themore severe DHFDSS had been previously infected with adifferent serotypeThese observations indicate that a previousinfection with dengue is a risk factor for development ofsevere illness The antibody-dependent enhancement (ADE)mechanism was proposed by Halstead in the 1970s [27]

One striking finding that potentially plays a critical role indengue immunopathogenesis occurs during sequential infec-tions by heterologous DENV serotypes The phenomenonknown as the Original Antigenic Sin was first describedin relation to influenza responses involving B cells [28]and more recently in humans infected with DENV [29]According to the phenomenon of Original Antigenic SinT cells generated during primary infection with a viralserotype showed a low affinity for the serotype of the second




















4 Hemostasis

































Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































4 Hemostasis

































Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Hemostasis

































Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Antithrombin

Activated protein C




PARs

TM

Activated platelets

sTF


Endothelium

sTFPI

sTM

DENV

PAR activation

X Xa

V Va

Coagulation

Activated

PAR activation

TMTF

VIIa

TFPI

PAR-2PAR-1

ICAM-1

sCD62P

sCD40L

CD62P

TXA2 IL-1120573CCL2

CD40L

CD40

PAR activation

TF

VIIaPAR-2

PAR-1

Activated

EPCR

EPCR

endothelial cells

endothelial cells

activation

TF VIIa

TNF-120572 IL-6 and IL-1120573

TNF-120572 IL-6 and IL-1120573












12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























12 Conclusion






References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















References






































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



































































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
































































































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




























































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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