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Current Opinion in Hematology:
doi: 10.1097/MOH.0000000000000073
HEMOSTASIS AND THROMBOSIS: Edited by Joseph E. Italiano and Jorge A. Di Paola

Understanding platelets in malaria infection

Morrell, Craig N.

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Author Information

Aab Cardiovascular Research Institute, University of Rochester School of Medicine, Rochester, New York, USA

Correspondence to Craig N. Morrell, DVM, PhD, University of Rochester, Box CVRI, 601 Elmwood Avenue, Rochester, NY 14642, USA. Tel: +1 585 276 9921; e-mail: Craig_Morrell@urmc.rochester.edu

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Abstract

Purpose of review

Platelets are most identified as the cellular mediator of thrombosis. It is becoming increasingly evident that platelets also have complicated roles in vascular inflammatory and infectious diseases. Platelets have been linked to initiating or accelerating the pathogenesis of diverse pathologies, such as atherosclerosis acute and chronic transplant rejection, arthritis, influenza, and malaria infection. Platelets may also have protective roles in killing microbes, such as bacteria. Malaria kills over 500 000 people per year, so understanding the multifaceted roles for platelets in malaria infection is of critical importance.

Recent findings

Recent literature has on the surface made the role of platelets in malaria infection somewhat confusing, with seemingly contradictory studies indicating a protective role for platelets in malaria infection by direct parasite killing, although others have indicated that platelets have an adverse proinflammatory role. However, what can appear to be mechanistic discrepancies are likely best explained through a better understanding and appreciation of platelet immune functions, especially in the context of the disease outcome or model systems used.

Summary

In this review, we will first briefly highlight platelet immune cell functions. We will then discuss how platelet immune and inflammatory functions may affect responses to malaria infection in a disease outcome and animal model context.

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INTRODUCTION

Our understanding that platelets do much more than form blood clots is continually expanding [1–9]. With loss of the endothelial cell barrier and exposure of the subendothelium, there is initiation of the coagulation cascade. The exposed subendothelium and coagulation activation leads to platelets becoming localized to the site of vascular lesion, activated, and adhering with eventual thrombus formation. Platelets also interact with an intact and inflamed endothelium. Endothelial cells release von Willebrand's factor in response to inflammatory stimuli, and platelets adhere to von Willebrand's factor, localizing platelets to the site of inflammation. Adhesion molecules expressed on endothelial cells at sites of inflammation, such as P-selectin and intercellular adhesion molecule, can also localize platelets. This leads to foci of platelet deposition without forming occlusive thrombi. The release of platelet inflammatory molecules further increases endothelial cell inflammation. Platelets also interact with leukocytes, and platelet–leukocyte aggregates (PLA) are commonly used as a measure of ongoing vascular inflammation. Platelet P-selectin and GPIbα have particularly important roles in platelet–leukocyte adhesion through interactions with PSGL-1 and Mac-1, respectively [10]. PLAs are more than just markers of inflammation; platelets also increase leukocyte inflammatory molecule expression and leukocyte adhesion, further amplifying immune responses [11,12]. Platelets have been shown in both mouse models and human disease to accelerate the development of vascular inflammatory diseases, such as atherosclerosis. In an ApoE–/– mouse model of atherosclerosis, platelets interacted with an intact endothelial cell layer in the region of an atherosclerotic lesion leading to platelet-derived chemokine deposition and monocyte recruitment [13]. Human population studies have confirmed that circulating activated platelets and PLAs are increased in those with atherosclerosis.

An activated platelet releases a large number of preformed granule constituents, secretes new activation-induced inflammatory molecules, and expresses many surface molecules that recruit, localize, and activate immune cells. Platelets have three types of granules: alpha (α), dense, and lysosomal. Alpha granules, in particular, contain highly abundant inflammatory molecules that either become surface expressed or are released into the extracellular environment following platelet activation [14]. These include P-selectin, platelet factor 4 (PF4/CXCL4), proplatelet basic protein (PPBP, which is broken down after release into β-thromboglobulin and then NAP-2), MIP-1α, and TGF-β. Activated platelets also produce and release cyclooxygenase-derived products (thromboxane), as well as IL-1α, and IL-1β, each of which has inflammatory roles in many vascular diseases. Platelet-derived immune molecules are also needed for normal immune development. PF4 has a central role in maintaining T-helper cell homeostasis. Mice that are genetically deficient in PF4 or platelets have increased numbers of T helper 17 type of CD4+ T-cells and an exaggerated T helper 17 response to infection [2].

Platelets are an abundant source of microparticles. Platelet-derived microparticles accelerate thrombosis and the chronic inflammatory disease rheumatoid arthritis [3]. Microparticles are also microRNA (miRNA) rich. miRNA is a class of small noncoding RNA that regulates gene expression. There is mounting evidence of cell–cell communication via miRNA transfer and that platelet-derived miRNA may affect endothelial cell gene expression patterns [15]. Whether platelet-derived miRNA influences vascular and immune cell expression in malaria is unknown, but is intriguing to speculate as red blood cell (RBC) miRNA may limit parasite growth [16].

Taken together, there is a great deal of evidence that platelets are much more than the cellular mediator of thrombosis. Platelets are also immune and inflammatory cells that communicate with other cells in the vasculature. This has a direct influence on both health and disease pathogenesis.  

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MALARIA AND PLATELETS

Malaria infection is caused by the protozoa parasite Plasmodium and is transmitted by mosquito bites. Sporozoites grow, divide, and mature to the merozoite stage in the liver. The merozoites exit the liver and enter the bloodstream in which they infect RBCs. In the RBCs, merozoites begin a cycle of asexual replication, lyse the RBC, and release more merozoites into the bloodstream to begin new cycles of RBC infection and multiplication. Merozoite release and RBC lysis lead to anemia as infection continues and RBC destruction exceeds production demands. The major Plasmodium species infecting humans are Plasmodium falciparum and Plasmodium vivax, with infections less commonly caused by Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Uncomplicated malaria typically has episodes of illness that are cyclical and consist of cold, shivering, fever, headache, sweat, and fatigue. Severe malaria is a more rare, but serious, form of malaria complicated by organ failure and includes severe anemia, hemoglobinuria, respiratory distress, and in the most severe and complicated form, cerebral vascular compromise. Cerebral malaria is most commonly seen in P. falciparum infection of children less than 5 years old. Mouse models of uncomplicated malaria include the use of Plasmodium yoelii and Plasmodium chabaudi. In each model, mice develop peak parasitemia of about 40% infected RBC. These parasites are commonly used to study the immune responses to infection to develop new methods of prevention and treatment. Plasmodium berghei ANKA is used as a model of severe cerebral malaria in mice, often called experimental cerebral malaria (ECM). In C57/Bl6 mice, P. berghei causes immune-driven cerebral vascular injury, hemorrhage, and thrombosis that resembles stroke-like lesions in the brains of infected mice [17,18]. Although no mouse model is a perfect recapitulation of human disease, they serve as useful models to begin to dissect interactions between malaria infection and the host response, including platelets. This means that attention must be paid to the mouse and parasite strains used when interpreting and integrating studies. The model systems have a large impact on platelet-mediated outcomes, and each model is valuable in understanding platelet interactions with both infected RBCs and the immune response to infection.

Both platelet activation and thrombocytopenia occur very early postinfection in both humans and mouse models [19–21]. Mechanisms for malaria-related thrombocytopenia are not completely understood, but have been shown to be due to increased platelet destruction, rather than decreased production [22].

Thrombocytopenia may involve platelet activation and clearance, shedding of platelet receptors (including GPIb), platelet apoptosis, and immune-mediated platelet destruction [23–25]. Platelet activation is in part driven by infected RBC expression of PfEMP1 on its membrane surface that interacts with CD36 on platelets, leading to platelet activation and the release of platelet-derived inflammatory molecules [4]. Immune cells are activated by malaria infection, and there is increased release of inflammatory and thrombotic mediators postinfection that activates platelets, including thromboxane and ADP. The effect of platelet activation on malaria pathogenesis has been described as adverse in models of cerebral malaria and protective in models of uncomplicated malaria. This can be confusing to those unfamiliar with models and malaria disease forms, and therefore warrants closer examination in each context.

Many initial studies into platelets and malaria focused on cerebral malaria because of its potentially devastating outcomes, and its cerebral vascular pathology has evidence of hemorrhage and thrombosis. For reasons not well understood, young children infected with P. falciparum are at much greater risk of developing cerebral vascular compromise compared with adults. Autopsy of brains from those that die of cerebral malaria have evidence of multifocal stroke like lesions and retinal hemorrhage [26]. Histologic examination typically demonstrates infected RBC (iRBC) sequestration in the small vessels of the brain and retina, fibrin thrombi, and perivascular hemorrhages [27,28]. Unlike uninfected RBC, sequestered iRBC is rarely found outside the vasculature indicating that iRBC bind to endothelial cells to become localized within the vessel. The inflammatory response of the host may initiate and accelerate iRBC brain vascular localization.

In 2006, a ‘unifying hypothesis for the genesis of cerebral malaria’ that placed platelets in the center of cerebral malaria pathogenesis was proposed [29]. It was proposed that parasite-mediated platelet activation induces inflammatory responses and endothelial cell stimulation, and upregulated endothelial cell adhesion molecule expression. Endothelial cell adhesion molecule expression leads to iRBC sequestration, vascular inflammation, and potentially disruption of the brain microvasculature. With sustained presence of iRBC, there is continued platelet activation, brain vascular inflammation, iRBC sequestration, and thrombus formation, leading to multifocal brain vascular injury and potentially death. Since first proposing this hypothesis, much has been proven true and been further built upon. Platelets are activated very early postinfection by iRBC [30]. Within 24 h of infection, there is in-vivo evidence of platelet activation in both humans and mouse ECM models. Platelet activation is, in part, driven by direct interactions between iRBC PfEMP1 and platelet CD36 [4]. Activated platelets then release multiple inflammatory mediators, form microthrombi, and drive more endothelial cell inflammation. Platelets may also be activated postinfection via less understood and multifactorial mechanisms that may include activation of the coagulation cascade, interactions with activated endothelial cells, or secreted inflammatory factors. Platelet activation then leads to more vascular inflammation through direct interactions between platelets and endothelial cell. We have found that platelets amplify T-cell responses to infection by interactions between platelet costimulatory molecules and T-cells [31]. Platelets also have all the machinery necessary to directly present antigen to T-cells and may do so in the experimental malaria model system [31].

It has recently been described that platelets may be protective in models of uncomplicated malaria infection. Platelet interactions with iRBC can lead to direct parasite killing and genetically platelet deficient mice had increased parasitemia in a P. chabaudi model of infection [8]. This is perhaps similar to the pathogen killing functions for platelets noted in bacteria infections. These differences in platelet protection vs. adverse platelet-mediated cerebral vascular inflammation are most easily ascribed to the type of infection response that leads to severe or uncomplicated malaria. The platelet-dependent outcomes are, therefore, due to an interplay between platelets and immune responses to infection. However, it may also be dependent on the timing of platelet activation and its effect on immune responses in the immediate stages postinfection.

Using a mouse ECM model, platelet depletion postinfection results in ECM protection and improved ECM survival [30]. In contrast, platelet depletion 24 h preinfection results in no ECM protection, and perhaps a worsened disease outcome. These results are in large part due to platelet induction of a protective acute phase response to infection. Platelet activation within the first 24 h of infection induces the production of acute phase proteins, in part via an IL-1β-dependent mechanism. Acute phase proteins, such as serum amyloid P and C-reactive protein, bind to iRBC leading to a reduction in the parasite burden [30]. These results highlight the concept that even in ECM platelet activation very early postinfection helps induce a protective acute phase response. Continued, ongoing platelet activation postinfection, however, leads to adverse inflammatory and thrombotic consequences that result in cerebral vascular compromise (Fig. 1).

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PF4 represents the type of molecule that makes understanding platelet involvement in malaria challenging. PF4 was the first discovered CXC class of chemokine and PF4 makes up approximately 25% of platelet α-granule content. Plasma PF4 concentration is approximately 200 ng/ml in humans, and in thrombotic and inflammatory conditions can increase to greater than 1 μg/ml. PF4 has been most studied in the context of heparin-induced thrombocytopenia, but it has diverse physiologic roles. PF4–/– mice have only a mild thrombotic defect and PF4 has been suggested to be an inhibitor of angiogenesis, an inhibitor of platelet production and a mediator of vascular inflammation and atherosclerosis [32,33]. We have found that PF4–/– mice have significant protection from ECM [4]. PF4–/– mice infected with P. berghei had reduced T-cell CXCR3 expression and fewer T-cells trafficking to the brain. Platelet-derived PF4, therefore, appears to have a major role in T-cell trafficking that drives the ECM-associated lesion. We have recently found that PF4 also has a central role in T-helper cell differentiation by reducing T helper 17 cell development both basally and in immune-activated states using a model of cardiac transplant rejection [2]. This may indicate that PF4 effects in ECM may also include a reduced T helper 1 response and an increased T helper 17 response. PF4 also activates monocytes and induces monocyte IL-6 and tumor necrosis factor-α secretion that can contribute to the cerebral vascular injury response in ECM [34]. Others have found that PF4 has direct parasite killing effects in uncomplicated malaria infection [9,35]. PF4 structurally has a chemokine domain and an antimicrobial peptide domain (AMP). The AMP domain when separated from the chemokine domain kills Plasmodium both in vitro and in vivo[9]. Together these data demonstrate that PF4 has a chemokine-mediated immune-activating function that in ECM contributes to T-cell responses and cerebral injury. PF4 AMP activities have direct parasite killing effects that in the absence of a robust immune response lead to decreased parasitemia and malaria protective effects. PF4 adaptive and innate (including direct parasite killing) immune functions, therefore, warrant much additional study to better understand how it can be used therapeutically.

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CONCLUSION

Platelets have diverse roles in infection and immunity. The combined thrombotic, immune, and antimicrobial properties of platelets make it difficult to place generic roles for platelets in infections, particularly malaria infection. An understanding of these many platelet properties, the pathogenesis of disease manifestation, and the model systems used helps unravel the outcomes of platelet interactions in malaria infection. These are lessons that can be applied to numerous other vascular and infectious disease pathologies in considering platelet functions.

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Acknowledgements

None.

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Conflicts of interest

None declared.

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Keywords

inflammation; malaria; platelet; platelet factor 4

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