Platelet content. Membrane glycoproteins of platelets include GPIa, GPIIb/IIIa (aIIbb3), or VLA-5 (fibrinogen receptor); GPIb/IX/V (vW and Mac-1 receptor); GPIc’-IIa or VLA-6 (laminin receptor); and a2b1 GPVI (Collagen receptor). Alpha granules contain P-selectin; platelet factor 4; transforming factor-b1; chemokines; proteoglycan; platelet-derived growth factor; a2-plasmin inhibitor; vitronectin; laminin; CD63; TGFbeta; CLEC-2; thrombospondin; fibronectin; B-thromboglobulin; vWF; fibrinogen; coagulation factors V, XI, and XIII; integrins; thrombocidins; proteases; thrombin; prothrombin; kininogens; immunoglobulin family receptors; leucine-rich repeat family receptors; and other proinflammatory and immune- modulating factors. Dense granules hold ADP, ATP, calcium, serotonin, histamine, dopamine, phosphate, eicosanoids. Receptors for primary agonist include P2X, P2Y 1 , and P2Y 12 (ADP); TPa-R and TPb-R (TXA 2 ); PAR-1 and PAR-4 (thrombin); PAFR (platelet-activating factor); 5-HT 2A 

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... recently, West Africa), with very high case fatality rates, ranging from 25% to 90% [38,39]. These viruses are most often transmitted by direct contact with infected animals or people. The natural reservoirs for filoviruses are thought to be bats [38,40]. Only two vaccines are licensed to prevent VHF: YF17D and Candid # 1, live-attenuated versions of the YF virus and JUNV. Unfortunately, the VHF-endemic areas are expanding, with the aggravation that treatments are limited or nonexistent. For this reason, it is important to continue research and development for new prevention and control options. VHF signs and symptoms range from asymptomatic infection to life-threatening disease. Initially, patients develop a nonspecific febrile syndrome (flu-like) that can include chills, malaise, headache, backache, arthralgia, myalgia, retro-orbital pain, anorexia, nausea, vomiting, diarrhea, cough, and sore throat [41–46]. At this stage the clinical signs resemble other infectious diseases, making early VHF clinical diagnoses impossible. Some cases progress to severe disease characterized by systemic vascular damage that, depending on the virus, will manifest as subcutaneous bleeding (flushing, conjunctivitis, peri-orbital edema, petechiae, or ecchymosis), positive tourni- quet test, hypotension and internal organ bleeding, hematem- esis, and melena [41–46]. The cause of bleeding in most VHF diseases is a disseminated intravascular coagulation (DIC) that depletes the coagulation factors, inducing massive plasma leakage, hypovolemia, and shock. A prolonged shock state leads to multiple organ failures and, in some cases, death. However, the loss of blood is rarely the cause of death (with the exception of filoviruses) [41–46]. The hallmark characteristic of all VHF is a decrease in platelet numbers and/or function. This decrease is usually accompanied by an increase in fibrinogen degradation products, prothrombin time (PT), and partial thromboplastin time (PTT). At least five days after the onset of fever, there is also a marked leucopenia and high viral loads that are associated with fatal outcomes. Another common finding is an increase in the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzymes, primarily due to liver damage [41–46]. Based on some clinical and laboratory findings (Table 3), different pathogenic mechanisms have been proposed for each VHF, including depletion of hepatic coagulation factors, cytokine storm, increased permeability by vascular endothelial growth factor, complement activation, and DIC. In spite of the differences seen with each VHF, there is a large body of evidence indicating that viral replication and host immune responses play an important role in determining disease severity and clinical outcome [47–50]. HF viruses can establish nonlytic replication, or ‘‘virus factories,’’ in monocytes and/or macrophage and DC [47,49]. Viral replication subverts the function of these cells, as well as the function of uninfected bystander cells, to undermine the innate immune response, such as interferon (IFN) production. This leads to uncontrolled viral replication and to a lack of specific antiviral responses in the host [47–50]. These cells might also act as vehicles to carry the virus to its replication sites, such as endothelium, liver, spleen, and other organs, leading to the pantropism of most hemorrhagic fever viruses [47–50]. Whereas the early replication stage is characterized by a lack of IFN production and IFN-responsive gene expression, later viremic stages reveal an unregulated release of pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF- a ), IL-10, IL-1R a , and soluble TNF-R, that could contribute to immunosuppression and increased vascular permeability, leading to hemorrhagic signs [51,52]. Thrombocytopenia (reduction of platelet numbers) and depressed immune responses are hallmarks of VHF, and platelets are involved in both processes; therefore, this review will focus on the platelet’s role in VHF pathogenesis. Platelets, also known as thrombocytes, are small, 2–3 m m, enucleated cell fragments derived from the cytoplasm of large, 100 m m, megakaryocytes (MKs) located in the bone marrow (Figure 2) [53]. Each MK sheds thousands of platelets into the blood stream, making them the second-most-abundant cell type in peripheral blood. Platelets’ most-known function is to maintain the integrity of the vascular system [53]; however, their function as immune elements is becoming more evident. Under specific stimulus, such as tissue damage, platelets are activated by extracellular matrix proteins, such as collagen and von Willebrand factor. Their irregular shapes swell to a compact spherical form with projections containing glycoprotein receptors that help them to attach to each other and to cells at wound sites [54]. After activation, platelets release granule contents and coagulation factors that further enhance their activation and cell attachment (Figure 3). This process results in the formation of an effective plug at the site of injury that supports formation of a fibrin network and stops bleeding [55]. There are some pathological processes in which the normal number of circulating platelets can decrease (thrombocytopenia), increase (thrombocytosis), or diminish in function (thrombasthe- nia) with or without hemostatic manifestations. The study of those conditions showed that in addition to their role in homeostasis, platelets are important mediators of inflammation and innate immunity [56]. Sub-products generated from microorganisms, from complement fixation reactions, or from expression of receptors such as Fc and C3 a /C5 a can attract platelets to the site of infection or injury where they are activated by thrombin and bind to vascular endothelial cells and leukocytes. Platelets can also bind pathogens directly, recognizing them through Toll-like receptors (TLRs), and can alert other cells via the innate immune response [57]. Platelets can directly release antimicrobial factors such as platelet factor 4 (PF-4), RANTES, connective tissue activating peptide 3 (CTAP-3), platelet basic protein, thymosin b -4 (T b -4), fibrinopeptide B (FP-B), fibrinopeptide A (FP-A), superox- ide, hydrogen peroxide, and hydroxyl free radicals. Additionally, platelets participate in antibody-dependent cell cytotoxicity against microbial pathogens [58]. In conclusion, any pathogenic state affecting platelets will not only impact hemostasis but also will modify the immune response to infection. Although there are some advances in understanding the specific platelet activation mechanisms induced by some microorganisms, little is known about these interactions and their role in the pathogenesis of VHF. Several microorganisms or molecules derived from them interact with platelets and affect their function [55,59,60]. Virus–platelet interactions were initially described around 50 years ago with some in vitro experiments using influenza virus and other myxoviruses [61,62]. Since then, other viruses have been reported to interact not only with platelets but also with MKs [63– 68]. From these interactions, platelet numbers or functions can be compromised, and little is known about the exact mechanisms. In the case of VHF, recent publications described pathogenic mechanisms involving the role of platelets in homeostasis as well as their role in the initial immune responses in animals and human beings. The next paragraphs will describe different ways in which hemorrhagic fever viruses affect platelets. There are four major causes of thrombocytopenia or platelet malfunction induced by viruses. These include direct effects of viruses on platelets, immunological platelet destruction, impaired megakaryopoiesis, and MK destruction (Table 4) [68]. In the VHF diseases, the most common mechanism of thrombocytopenia is platelet disappearance from damaged tissues or generalized virus- induced DIC, in which coagulation factors are depleted. This common pattern does not seem to apply to LASV. Additionally, there is some evidence that viruses not belonging to the hemorrhagic fever group (such as varicella, herpes zoster, smallpox, rubella, measles, cytomegalovirus, rotavirus, adenovirus, and HIV), under specific circumstances, can also cause hemostatic problems and can be associated with DIC [66,69–72]. More work is necessary to clarify the role of the platelet–virus interaction in coagulopathies and in DIC induced by both hemorrhagic and nonhemorrhagic viruses. Platelets bind viruses through different receptors, such as b -3 integrins or TLRs, and platelets are known to express TLR2, TLR4, and TLR9 [73,74]. In severe sepsis, there are coagulation abnormalities and DIC that are thought to be due to TLR signaling in platelets [57]. Bacterial stimulation of platelet TLR2 increased P-selectin surface expression, activation of integrin a IIb b 3 , generation of reactive oxygen species, and formation of platelet–neutrophil heterotypic aggregates in human whole ...