You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Dengue virus nonstructural protein 1 activates platelets via Toll-like receptor 4, leading to thrombocytopenia and hemorrhage}
Article:
Abstract:
Dengue virus( DENV) infection, the most common mosquito-transmitted viral infection, can cause a range of diseases from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and shock syndrome. Thrombocytopenia is a major characteristic observed in both mild and severe dengue disease and is significantly correlated with the progression of dengue severity. Previous studies have shown that DENV nonstructural protein 1( NS1), which can be secreted into patients’ blood, can stimulate immune cells via Toll-like receptor 4( TLR4) and can cause endothelial leakage. However, it is unclear whether DENV NS1 can directly induce platelet activation or cause thrombocytopenia during DENV infection. In this study, we first demonstrated that DENV but not Zika virus cell culture supernatant could induce P-selectin expression and phosphatidylserine( PS) exposure in human platelets, both of which were abolished when NS1 was depleted from the DENV supernatant. Similar results were found using recombinant NS1 from all four serotypes of DENV, and those effects were blocked in the presence of anti-NS1 F( ab’) 2, anti-TLR4 antibody, a TLR4 antagonist( Rhodobacter sphaeroides lipopolysaccharide, LPS-Rs) and a TLR4 signaling inhibitor( TAK242), but not polymyxin B( an LPS inhibitor). Moreover, the activation of platelets by DENV NS1 promoted subthreshold concentrations of adenosine diphosphate( ADP)-induced platelet aggregation and enhanced platelet adhesion to endothelial cells and phagocytosis by macrophages. Finally, we demonstrated that DENV-induced thrombocytopenia and hemorrhage were attenuated in TLR4 knockout and wild-type mice when NS1 was depleted from DENV supernatant. Taken together, these results suggest that the binding of DENV NS1 to TLR4 on platelets can trigger its activation, which may contribute to thrombocytopenia and hemorrhage during dengue infection.
Introduction:
Dengue is the most widespread mosquito-borne viral infection, infecting approximately 390 million people and causing 500, 000 hospitalizations every year[1, 2]. Most infected patients have a mild, self-limited disease known as dengue without warning signs, but some can develop severe dengue, which is characterized by plasma leakage, fluid accumulation, severe bleeding, and organ impairment[3]. The underlying mechanisms by which mild dengue progresses to severe dengue are still unclear[4–6]. Thrombocytopenia, a common feature observed in both mild and severe dengue disease, is correlated with disease severity and is considered a predictive biomarker of severe dengue[7, 8]. However, the mechanisms that cause thrombocytopenia during dengue infection are not fully understood. Previous studies have demonstrated that elevated surface P-selectin and increased phosphatidylserine( PS) exposure among platelets are correlated with thrombocytopenia in dengue patients[9, 10]. The underlying mechanism of platelet activation has been analyzed by proteomic analysis of dengue patients’ platelets, and circulating histones in dengue patients’ plasma have been suggested to activate platelets[9]. In addition, dengue virus( DENV) infection can induce platelet activation and apoptosis[10]. However, it remains unclear which viral component, if any, participates in platelet activation and apoptosis. Dengue nonstructural protein 1( NS1) is a 48 kDa glycoprotein that can be expressed on the DENV-infected cell surface as a dimer and is the only viral protein secreted into the blood circulation, as a hexamer, in dengue patients[11]. The concentration of NS1 in the sera of DHF/DSS patients ranges from 0. 01–50 μg/ml, which correlates with disease severity[5, 12]. Recently, an increasing number of studies have shown that NS1 plays a critical role in dengue pathogenesis both in vitro and in vivo, which includes enhancing DENV replication/infection, directly inducing vascular leakage, and causing cytokine release from immune cells[13–18]. Furthermore, a previous study indicated that relative levels of NS1 antigen in dengue patients’ sera were negatively correlated with platelet count[19]. Recently, Modhiran et al. demonstrated that Toll-like receptor 4( TLR4), a well-known receptor of lipopolysaccharide( LPS), acts as the receptor of NS1 in immune cells[15, 20]. It is known that LPS can induce platelet activation and potentiate platelet aggregation via TLR4/MyD88 signal transduction[21]. Since both NS1 and LPS can activate cells through TLR4, in this study, we propose and test the hypothesis that NS1 can induce platelet activation and enhance aggregation through TLR4, leading to thrombocytopenia and hemorrhage during dengue infection.
Discussion:
In this study, we demonstrated that DENV NS1 in the concentration, which is within the range of NS1 in dengue patients’ sera( 0. 01–50 μg/ml), could activate platelets and induce apoptosis in a dose-dependent manner through TLR4 signal transduction. In addition, the aggregation of NS1-activated platelets in response to subthreshold concentrations of ADP was enhanced. The adhesion of NS1-activated platelets to endothelial cells and the phagocytosis of these platelets by THP-1 cells were also increased. Because NS1 can also bind to endothelial cells and macrophage to cause their activation and cytokine release, all these effects induced by NS1 may contribute to platelet activation and thrombocytopenia during DENV infection( Fig 10)[31]. In previous studies, DENV supernatant was used to stimulate platelets, and the studies concluded that DENV infection of platelets induces platelet activation[9, 10]. However, because NS1 exists in the DENV supernatant, these results cannot exclude the possible effect of NS1 in the DENV supernatant as a cause of platelet activation. Therefore, we compared the effects of DENV supernatant on platelet activation with or without the presence of NS1. As shown in Fig 1, platelet activation induced by DENV supernatant was significantly abolished when NS1 was depleted. Furthermore, we demonstrated that the binding of NS1 to platelets was decreased when TLR4 was blocked by αTLR4 or in TLR4 knockout mice. Consistent with the binding results, blocking NS1 binding to TLR4 by antibodies or TLR4 antagonist also decreased the NS1-induced platelet activation. However, the blocking of TLR4 could not completely diminish the binding of NS1 and NS1-induced platelet activation; thus, we cannot rule out the possibility that other proteins, such as TLR2, may also be involved in NS1 binding to platelets[20, 24]. Next, we examined the properties of platelets after DENV NS1 stimulation. Like LPS, a well-known TLR4 ligand, DENV NS1 could enhance platelet aggregation in the presence of subthreshold concentrations of ADP[21]. Since NS1 could induce platelets to release ADP( S11 Fig), it is possible that the existence of these low-level ADP can only trigger platelet activation but not aggregation unless an extra-subthreshold concentration of ADP was added. In addition, previous studies have also shown that injecting mice with LPS induces profound thrombocytopenia due to the increased adherent capacity of platelets[32, 33]. In this study, we also found that the adhesion of NS1-activated platelets to endothelial cells and the phagocytosis of these platelets by THP-1 cells were also increased. Finally, we demonstrated that DENV-induced pathogenic signs, such as thrombocytopenia, prolonged bleeding time, and local skin hemorrhage in mice, were significantly abolished in NS1-depleted DENV supernatant, but not in UV-DENV supernatant, which had no detectable viral titer but still contained NS1. It is worth mentioning that we also measured the NS1 in mouse sera 3 days after infection, and the circulating NS1 levels in UV-DENV-inoculated mice were nearly clear. This result is consistent with our previous study, which shows that the half-life of DENV NS1 is 2 days. In addition, the results showed that the concentration of circulating NS1 in NS1-depleted DENV-infected mouse sera was lower than that in DENV-infected mouse sera, indicating that circulating NS1 is important to help DENV infection/replication[13]. These results also suggested that even when NS1 was depleted from DENV virus supernatant, DENV could still infect mice and produce de novo NS1; however, the concentration of circulating NS1 in mouse sera was not sufficient to cause pathologic signs in mice. Thrombocytopenia is a common feature observed in both mild and severe dengue disease and is correlated with disease severity. There are two possible mechanisms that can cause thrombocytopenia in dengue patients. One is increased platelet destruction and clearance from peripheral blood. The other is decreased production of platelets in the bone marrow[34]. The inhibition of megakaryocyte development in the bone marrow has been proposed as the key underlying mechanism causing thrombocytopenia in DENV-infected humanized mice[35]. Because the average platelet size is increased when the production of platelets is increased, a high mean platelet volume( MPV) generally indicates enhanced platelet destruction in patients[36]. In dengue patients, MPV is usually either high or normal. Therefore, excessive platelet destruction may be the main reason for thrombocytopenia in dengue patients[37, 38]. Indeed, platelets from DENV-infected patients exhibit increased activation and apoptosis profiles compared to healthy donors and patients with non-dengue febrile illness[10]. However, the mechanisms that cause platelet activation and apoptosis leading to thrombocytopenia are not fully understood. In this study, we demonstrated that platelets activated by DENV NS1 were prone to aggregation, attachment to endothelial cells, and phagocytosis by immune cells. Therefore, we suggest that NS1 is one of the key factors that may contribute to thrombocytopenia during DENV infection. While previous studies have shown little difference in the presence or absence of circulating NS1 between primary and secondary infections, secondary infections are overwhelmingly associated with more severe disease[39]. We could not exclude the possibility that other immune-related factors, such as autoantibodies against platelets, may also contribute to thrombocytopenia during DENV infection, especially during secondary infection[40–42]. Nevertheless, these results may explain why prophylactic platelet transfusion could not reduce the risk of clinical bleeding and thrombocytopenia in dengue patients because transfused platelets will be activated and cleared due to the presence of circulating NS1 and antiplatelet antibodies in dengue patients[43]. It is known that NS1 proteins from different flaviviruses reveal 50% to 80% homology; they may have conserved functions as well as unique characteristics, leading to different modes of flavivirus pathogenesis[44, 45]. Therefore, in this study, we compared the platelet-activating effect of NS1 from DENV and another flavivirus, ZIKV. The results demonstrated that DENV NS1 was much more potent than ZIKV NS1 at the same concentration in terms of inducing platelet activation, which may explain why DENV is the most prevalent flavivirus infection with clinical manifestations of thrombocytopenia. Vascular leakage, thrombocytopenia, and cytokine storm are thought to play important roles in the complexity of dengue pathogenesis[45]. Previous studies have demonstrated that DENV NS1 can also bind to endothelial cells to induce vascular leakage and stimulate immune cells to cause a cytokine storm[45]. Thus, DENV NS1 has been considered to be a viral toxin, which is a potential corner piece in the puzzle of dengue pathogenesis[46]. In this study, we further demonstrated that NS1 may also contribute to thrombocytopenia, another characteristic clinical feature of dengue infection. It is known that activated/apoptotic platelets could further modulate monocyte inflammatory responses and endothelium hyperpermeability to cause vascular leakage during DENV infection[47, 48]. Consequently, platelet activation/apoptosis induced by DENV NS1 can not only cause thrombocytopenia but also enhance the inflammatory response and vascular leakage in dengue patients. Together, these results suggest that DENV NS1 plays a central role in the complex interplay of dengue pathogenesis. Therefore, NS1 may represent an important target that should be a focus of attention in the development of therapeutic drugs and vaccines against dengue infection[49].
Over the past 50 years , dengue has been a continuing global threat , with no effective vaccine or specific antiviral drug . Dengue infection causes a wide range of outcomes , from fever-like symptoms to severe dengue hemorrhagic fever . Thrombocytopenia , a reduction in platelet count , is a common feature observed in both mild and severe dengue and is correlated with disease severity . In this study , we used dengue viral supernatant or DENV recombinant NS1 protein to stimulate human-isolated platelets . We found that DENV NS1 could directly activate platelets through TLR4 and could further enhance platelet aggregation , adhesion to endothelial cells and phagocytosis by macrophages , which could lead to thrombocytopenia . We also proved that both NS1 and TLR4 are critical for DENV-induced thrombocytopenia and hemorrhage using a DENV-induced hemorrhagic mouse model . Our study reveals a new pathogenic role of NS1 during dengue infection and highlights that NS1 should be a topic of attention in the development of therapeutic drugs and vaccines against dengue infection .