Update Trends in Parasitology Vol.25 No.7

Research Focus

Platelet power: sticky problems for sticky parasites?

Richard J. Pleass

Institute of Genetics, School of Biology, University of Nottingham, NG7 2UH, UK

Platelets might have a crucial role in the pathogenesis ofboth human and rodent malarias by assisting in thesequestration of infected erythrocytes within thecerebral vasculature. However, recent elegant work byMcMorran et al. suggests that they are also involved ininnate protection during the early stages of infection.Here, we discuss the implications of their importantfindings in the context of immunity to malaria.

Platelet: friend or foe?The platelet, traditionally known for its role in blood clot-ting, is also known for its putative involvement in malariapathology [1,2].However, a recent studyusingC57BL6micegenetically deficient in the megakaryocyte growth anddifferentiation factor C-mpl (encoded by the Mpl gene),and resulting inmice with 90% fewer platelets, showed thatthese animals were significantly more susceptible to deathwhen infected with Plasmodium chabaudi [3]. Further-more, the authors went on to show that purified humanplatelets killed Plasmodium falciparum parasites withinred blood cells when added to in vitro cultures and thatvarious platelet antagonists, including aspirin, reversedthis antiparasitic activity both in vitro and in vivo. Thisresult raises concerns over the use of aspirin as an anti-pyretic in patients with malaria. Using specific receptorantagonists, the authors demonstrated that killing andcontrol of parasite growth in P. falciparum cultures wasdependent on platelet activation via P2Y1, an ADP-depend-ent metabotrophic puronergic receptor (Figure 1).

These results in animals seem counterintuitive, in thatplatelets are believed to be involved in pathological diseasestates that hasten death, such as cerebral malaria [1,2]. Forexample,micewith significantly compromisedplatelet func-tion (CXCL4 or CXCR3 deficient) have been shown to sur-vive longer than their wild-type counterparts [4]. Bycontrast, platelet depletion by anti-CD41 monoclonal anti-body injection early, but not late, in the course of disease isknown to protect C57BL6 mice from Plasmodium bergheiANKA-induced severe experimental cerebral malaria(ECM) by altering levels of pathogenic cytokines [5]. Unfor-tunately, the study by McMorran et al. used P. chabaudi, arodent malaria that – although capable of sequestering to anumber of organs – is not known to develop ECM. It shouldalso be noted that non-sequestering Plasmodium speciesalso give rise to ECM in some inbred mouse strains, Plas-modium yoelii 17XL in BALB/c mice being a good example[6].

It is also important to consider issues of mouse geneticbackground. All knockout studies to date, including those

Corresponding author: Pleass, R.J. (richard.pleass@nottingham.ac.uk).

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reportedbyMcMorran etal. for theMplgene,havebeendonein the C57BL6 mouse (susceptible to ECM). These exper-iments nowneed to be repeated in animals backcrossed ontodifferent genetic backgrounds, such as BALB/c and DBA/2mice (resistant to ECM), to determine whether other con-tributorygenetic factors areatplay.Agreat deal of caution isalso required inextrapolating thesemousemodelsofECMtothe involvement of platelets in human disease.

Although these findings are clearly important, theauthors did not address three other, equally sticky issues.First, how do platelets bind to infected erythrocytes? Sec-ond, what is or are the mechanism(s) by which plateletsinduce apoptosis and death for parasites hidden within theconfines of the parasitophorus vacuole? And third, giventhe known importance of the common g-chain in plateletactivation and function, what part might Fc receptors(FcRs) and antibodies play in this process (Figure 1)?

A cornucopia of receptorsThe first of these questions is easier to explain for P.falciparum than for Plasmodium vivax or the murinemalarias. Platelet-mediated clumping is common in P.falciparum field isolates, is distinctive from other adhesivephenotypes and involves the host receptors CD36 [7] andgC1qR/HABP1/p32 [8]. Whether these are the only plateletreceptors involved is debatable and worth exploring.Although GPIIb/IIIa (CD41/CD61) and GPIb/IX (CD42a/CD42b)-deficient platelets still clump to infected erythro-cytes [7], the role of other key platelet adhesion or aggre-gation receptors – including GPVI, a2b1, a11b3, a5b1 anda6b1, PSGL-1, and platelet-endothelial cell adhesion mol-ecule-1 (PECAM-1) – have not been explored (Figure 1).Furthermore, although not primary receptors involved inbinding, the recruitment of other receptors after initialtethering could nonetheless be important for stabilizationof the platelet-infected erythrocyte complex or for trigger-ing functions from them, as is known for other immuno-logical synapses. PECAM-1 is particularly interesting inthis respect because it is known to be a ligand for the P.falciparum erythrocyte membrane protein 1 (PfEMP1)family of variant surface antigens [9], binds glycosamino-glycans [10] and has been shown to inhibit plateletresponses [11,12], suggesting that PECAM-1 triggeringmight be advantageous to the parasite.

These issues certainly need to be explored, and theavailability of increasing numbers of mice deficient invarious platelet-adhesion receptors and ligands mightprovide novel insights into the role of platelets in protec-tion from malaria, especially under hydrodynamic shearflow in the bloodstream [13]. In addition, many of thesereceptors (including CD36 and gC1qR/HABP1/p32) areexpressed by other important immune cells, including

Figure 1. Hypotheses on the interaction of platelets with infected erythrocytes (IEs). (1) Platelets are activated by unknown molecules released from IEs through the

metabotrophic puronergic receptor P2Y1. It is unclear whether activation requires prior binding and tethering of IEs via platelet-expressed CD36 and gC1qR (also known as

HABP1/p32) [7,8]. The potential roles of other platelet receptors in tethering and triggering are unclear, as are the identities of the parasite ligands interacting with them. (2)

Activation of platelets results in the release of both a-granules and dense granules, loaded with numerous potent pharmacological and immunological mediators.

Serotonin results in increased vascular permeability and smooth muscle contraction and has been shown to activate dendritic cells (DCs). It might also influence the IE

directly; serotonin receptor agonists and tryptophan catabolites are known to modulate the parasite life cycle and inhibit parasite growth in culture [17,18]. (3) Recent

analysis of the secreted platelet proteome have detected numerous chemokines including CXCL4, CXCL7 and regulated upon activation normal T-cell expressed and

secreted (RANTES, or CCL5) that have important roles in the phased arrival of leukocytes and natural killer (NK) cells and granulocytes (eosinophils, or Eos),

polymorphonuclear neutrophils (PMNs) and mast cells (Mast) [15]. CXCL4 and its cognate receptor CXCR3 expressed on T helper (Th) cells have been shown to impact

directly on the severity of experimental cerebral malaria in rodents [4]. CXCL4 stimulates monocyte release of tumour necrosis factor (TNF-a) and reactive oxygen

intermediates (ROIs) and has been shown to induce apoptosis of endothelial cells (ECs) that, together, might compromise the integrity of the blood–brain barrier. Soluble

factors released by IEs are known to induce apoptosis in human brain ECs. CXCL7 recruits PMNs in particular that release large quantities of platelet-activating factor (PAF).

RANTES is a potent pro-inflammatory chemokine and inhibitor of HIV replication in vitro and is known to bind the Duffy antigen receptor for chemokines (DARC),

coincidentally required for invasion of erythrocytes by P. vivax [16]. Whether RANTES can inhibit growth of malaria parasites when administered to growing cultures is

unknown. (4) How all these molecules eventually lead to apoptosis in the parasite and the pathways leading to death have also yet to be worked out. Although parasites are

known to possess two metacaspase proteins, whether their expression is increased after culture in the presence of platelets now needs to be determined. (5) What role

antibodies and/or immune complexes (ICs) have in thrombocytopenia is still unclear. Given that platelets express numerous Fc receptors for antibody, what part these play

in the function of platelets should be investigated. What role platelets might have in subsequent adaptive immune responses to malaria is unclear. Whether Mpl-deficient

mice can be immunized successfully or whether passive transfer experiments with antibody can be done in the absence of platelets could be usefully explored.

Update Trends in Parasitology Vol.25 No.7

dendritic cells, neutrophils and B cells. Are these cells alsofound within platelet clumps in vivo, and if not, why not?Equally weighty issues concern the affinity and avidity ofsuch interactions and whether binding occurs in the pre-sence or absence of the native ligands for these receptors.This is particularly interesting for gC1qR/HABP1/p32 thatbinds the globular head domains of C1q and hyaluronicacid [8,14]. Do these host ligands share overlapping bind-ing sites on gC1qR/HABP1/p32 with the parasite mol-

ecules expressed on the infected erythrocytes? And whatis the hierarchy of binding in terms of affinity? What rolecomplement and/or immune complexes (abundant inmalaria-infected individuals) containing C1q might playin the observations made by McMorran et al. clearly needto be investigated in future studies because these alsocorrelate with the severity of severe pathology. The in vitroplatelet assays set up by the authors will be particularlyuseful for addressing these thorny problems.

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The receptors on the infected erythrocyte responsible forthe binding to platelets also remain a mystery for bothhuman and rodent malarias. Although no direct evidenceexists to show that PfEMP1 is directly involved in plateletbinding via either CD36 or gC1qR/HABP1/p32, the plateletplasmamembrane does contact knob-like structures on theinfected erythrocyte, where PfEMP1 resides [7]. If PfEMP1does turn out to be responsible for platelet binding, whichDBL domains are involved? And what happens in vivowhen PfEMP1 is saturated with other known solubleligands that might influence this interaction?

To die upon a platelet’s kissThe second question concerning the mechanism of killing,especially for an intracellular parasite residing in the redblood cell, is less easy to explain. Perhaps the recruitedplatelets release toxic mediators or cytokines. Plateletssecrete dense granules (containing ADP, ATP, Ca2+ andserotonin) and alpha granules (containing CXCL4, PDGF,fibronectin, von Willebrand factor, fibrinogen and coagu-lation factors V and XIII), as well as microparticles(Figure 1). Platelet-derived microparticles account for90% of the plasma microparticles from healthy individualsand recent proteomic analysis has shown them to containchemokines CXCL4, CXCL7 and RANTES (CCL5) [15]. Itis not known whether these secreted factors directly affectintracellular developing parasites or whether theyindirectly recruit other mechanisms of killing, perhapsvia pro-inflammatory mediators including cytokines.

The Duffy antigen receptor for chemokines (DARC),which is needed for invasion of erythrocytes by P. vivax,is characterized by its ability to bind a wide array of pro-inflammatory chemokines (especially RANTES, which ishighly effective in suppressing HIV-1 replication) [16].Whether these platelet-derived chemokines have a similarrole in malaria, perhaps by blocking access of P. vivaxmerozoites to DARC, is unknown. Another possibility isthat platelets clumped around an infected erythrocytemight starve the parasite of key metabolites by blockingnutrient uptake through parasite or host transporterproteins located within the erythrocyte plasma membrane(Figure 1). Given that serotonin is found in platelet densegranules, the observation that serotonin receptor agonistsand other products of tryptophan catabolism inhibit P.falciparum by blocking membrane channels [17] and mod-ulating the parasite cell cycle [18] is very exciting. It willnow be important to throw some of these plateletmediatorsdirectly into parasite cultures to determine what effectthey have on inducing apoptosis in the authors’ TUNELassays. Unfortunately, these assays were only carried outover a 24-h period, so the effect of platelet clumping onmerozoite egress or invasion of fresh erythrocytes was notinvestigated.

A spoonful of antibodies makes the platelets go down?Close examination of the in vivo data presented by McMor-ran et al. suggests that the impact of thrombocytopenia onmouse survival occurs ten days after parasite injection(when platelet levels are low), a time point thatwill coincidewith theproduction of antibodies. Are antibodies involved inthe observed malaria-induced thrombocytopenia? Most

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malaria patients with thrombocytopenia possess platelet-associated antibodies that bind to platelet-bound malariaantigens [19], and the extent of thrombocytopenia correlateswith disease severity and immunoglobulin E (IgE) levels[20]. One hypothesis is that platelet autoantibodies andsubsequent phagocytosis by splenic macrophages areresponsible for the observed thrombocytopenia, althoughin experiments with P. berghei in mMT mice (which areconsidered devoid of functional B cells and IgG antibodies),the mice still develop thrombocytopenia [21]. However, thiswork needs reappraisal in the light of more recent workshowing mice deficient in m- or d-chain expression, onnumerous genetic backgrounds, generate robust and func-tional IgAand IgE responses (humanplatelets expressFcRsfor both these antibody classes), although these studies areyet to be extended to malaria [22,23].

Irrespective of their controversial role in inducing throm-bocytopenia, could antibodies recruit platelet killing duringa secondary adaptive immune response? Human plateletsexpressFcRs for IgG, IgAand IgE (Figure1) [24]. As the onlyIgGFcRexpressedbyhumanplatelets,FcgRIIA contributesto the pathophysiology of diseases such as heparin-inducedand antibody-mediated thrombocytopenias and to anti-phospholipid antibody syndrome-mediated arterial throm-bosis. In addition, FcgRIIA has been shown to stimulateplatelets through interaction with other molecules andreceptors, including a2bb3, GP1b-IX-V and vonWillebrandfactor [25–27]. Furthermore, FcgRIIA and PECAM-1 arephysically and functionally associated on the surface ofhuman platelets [28]. Human platelets have been shownto internalize IgG-containing immune complexes, abundantduring malaria infection, and polymorphisms in FcgRIIAhave been implicated in susceptibility to severe malaria[24,29]. Whether these polymorphic variants of FcgRIIAhave functional consequences for killing of malaria para-sites by human platelets, in either the presence or theabsence of specific antibody, is unknown. Mice transgenicfor human FcgRIIA and whose platelets express this re-ceptor are available and might provide interesting modelswith which to explore the role of both mouse and humanantibodies in platelet killing of infected erythrocytes duringthe adaptive immune response [24].

Concluding remarksExciting times lie ahead for the humble platelet.

AcknowledgementsI am particularly grateful to the Wellcome Trust, the Medical ResearchCouncil, European Union and the Sir Halley Stewart trust for fundingwork in my laboratory. I apologize to those authors whose work I wasunable to cite directly owing to space constraints imposed by the journal.

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Letters

Treatment of clinical schistosomiasis at the prepatentphase: an option?

Paulo M.Z. Coelho, Martin J. Enk and Naftale Katz

Laboratory of Schistosomiasis, Rene Rachou Research Center/The Oswaldo Cruz Foundation, Av. Augusto de Lima 1715, 30190-002

Belo Horizonte, Brazil

The excellent result of treatment during the early stageof human infection with Schistosoma mansoni (i.e. up toone week after exposure, during the skin and lungstages), using 50 mg kg�1 oxamniquine (oral dose), pro-vides strong evidence for its application during the pre-patent phase [1]. This therapeutic approach blocked thedevelopment of larval forms into adult worms, avoidingthe pathology and symptoms caused by the presence ofadult schistosomes and subsequent eggs deposited intissues.

The decision to treat without prior laboratory confir-mation of infection is based on a combination of clinical andepidemiological evidence, such as cercarial dermatitis(swimmer’s itch), with water contact in which occurs theinfected Biomphalaria shedding cercariae of S. mansoni.

The detection of schistosomiasis during the prepatentphase is difficult, mainly because proving the existenceof infected snails requires skilled technicians and an earlydiagnosticmethod is not available. The routinely used stoolexaminations are not appropriate because they depend onthe identification of parasite eggs, which appear in thefaeces �40 days after S. mansoni infection. Diagnostictechniques, such as PCR and the detection of circulatingantigens, could be valuable in early detection of schistoso-miasis; studies of animal models using PCR indicate thatinfection can be detected two weeks after exposure [2].More studies are needed to improve the sensitivity ofcirculating antigen detection. It is still unclear what typeof antigen is most suitable and how much time is neededafter infection until it can be detected with certainty [3].More research is needed to elucidate the early diagnosticpotential of these techniques.

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