malaria remains one of world s major infectious diseases and an impediment to economic development .
one third of the world s population are at risk of infection , around 250 million people develop clinical infections annually , and at least half a million die each year ; most are children aged under five years .
the disease is caused by infection of circulating red blood cells by one of five species of the protozoan parasite , plasmodium ; p. falciparum causes most morbidity and mortality .
the majority of antiplasmodial drugs are now severely compromised due to acquired resistance by the parasite , and the development of efficacious vaccines remains a challenge .
novel approaches to treatment and a greater understanding of disease pathogenesis and the host response to infection are desperately needed .
outcome to malaria infection is determined largely by the increasing parasite mass and the response of the host to the infection .
importantly , less than 1% of infections progress to life - threatening stages , underscoring the efficacy of host protective mechanisms .
the innate protective response limits parasite growth early in an infection in a non antigen - specific manner .
it also allows time for the subsequent development of an adaptive response , which is capable of clearing the infection and protecting against clinically symptomatic malaria .
it is also antigen - specific , and several years and many exposures are needed to build an effective immunity against the multitudinous array of parasite antigens in any given endemic region .
innate immune mechanisms are therefore crucial in all malarial infections to buffer against the early growth of blood - stage parasites .
we recently reported that platelets are an important component of the host innate immune response against malaria infection .
in addition to their well - defined role in hemostasis , platelets are increasingly implicated in immunological processes , including direct pathogen - killing functions ( reviewed by yeaman and colleagues , ref .
they express receptors that bind host immune response modulators ( e.g. , antibodies and cytokines ) and toll - like receptors that bind microbial products .
they also express the cd154 co - stimulatory molecule and influence the development of adaptive immune responses .
importantly , their location in the circulation makes them ideal  sentinels  against any nascent infection .
platelets respond to a variety of microbial cells by releasing immunomodulatory molecules and by directly killing microbial pathogens .
malaria infections are commonly accompanied by a thrombocytopenia or loss of platelets , the severity of which closely mirrors the increasing parasite mass .
it is now clear from our study and others that platelets protect the host during erythrocytic infection .
mice with pre - existing platelet deficiencies are more susceptible to infection and exhibit higher loads of viable parasites .
treatment of normal mice with aspirin , a platelet activation and aggregation inhibitor , also reduces survival to infection .
pre - treatment of these platelets with inhibitors ( including aspirin ) blocks the parasite killing effect .
platelets bind preferentially to p. falciparum ie , mainly through interactions between the platelet - expressed scavenger receptor protein , cd36 and the p. falciparum erythrocyte membrane protein ( pfemp1 ) , produced by the parasite and trafficked to the erythrocyte surface .
we believe that platelets are active early in infection to slow the initial growth of malaria parasites in the bloodstream , providing greater opportunity for other defense mechanisms to control the infection and ensure survival .
more recent studies by our group and others have provided additional mechanistic and molecular insight to how platelets kill the intraerythrocytic parasite .
central to these findings is a platelet derived cxc - type chemokine called platelet factor 4 ( pf4/cxcl4 ) , which is released by activated platelets and kills the parasite .
approximately 25% of the protein released by platelets comprises of pf4 , and concentrations surrounding activated platelets reach high micromolar ranges .
the parasite - killing activity of platelets appears to be entirely due to pf4 . neutralizing anti - pf4 antibodies
completely block the activity of human platelet lysate and pf4-deficient platelets from mice fail to kill parasites .
love and colleagues elegantly demonstrated that upon entering the cell , the protein relocates to the parasite digestive vacuole ( dv , site of hemoglobin digestion ) , resulting in specific lysis of the organelle and death of the parasite .
therefore pf4 is a unique example of a host - derived molecule with direct plasmocidal activity .
pf4 belongs to growing list of chemokine molecules called kinocidins , which have a remarkable capacity to function as both chemotactic and antimicrobial molecules .
the different functions are localized to structurally distinct domains , and the latter at least is separable .
chemotactic activity resides in the n - terminal portion of the protein where the classical cxc chemokine motif exists .
this domain interacts with chemokine receptors on various target cells , leading to receptor - mediated signaling events .
the c - terminal domain , rich in positive charged amino acids , mediates the antimicrobial effects of pf4 and other kinocidins .
peptide scanning analyses have mapped the bactericidal and fungicidal activity of the rabbit pf4 ortholog , platelet microbiocidal protein 1 to the carboxyl terminal 15 residues , 5 of which are lysines .
notably , love and colleagues showed that a peptide encompassing the carboxyl terminal 12 residues of human pf4 ( c12 ) had plasmocidal activity .
this function was abrogated when three out of the four lysine residues were substituted to alanines .
the charged , amphipathic nature of kinocidin c - terminal domains gives them propensity to interact with cell membranes . in bacteria ,
this has been variously shown to result in disruption of membrane potential , cell permeabilization and inhibition of protein and dna synthesis .
consistent with these former effects , pf4 and c12 treatment of p. falciparum results in lysis of the dv .
however , exactly how and why the dv is specifically targeted by pf4 is unclear , especially given that the molecule must cross three separate membranes in order to reach the dv ( red cell plasma membrane ( pm ) , parasitophorous vacuole membrane and parasite pm ) .
pf4 binds to duffy , a red cell antigen receptor for chemokines ( darc / fy ) and our results show that platelet and pf4-mediated killing of intraerythrocytic parasites requires darc .
the likely mechanism involves the binding of pf4 to darc on the infected cell surface , following by internalization of the pf4-darc complex .
binding occurs via a domain on darc known to bind a number of other chemokines .
also , other chemokines with a high affinity for darc out - compete the pf4 parasite binding and killing of parasites .
the molecular details of how the complex may translocate into the parasite ( as well as the existence of the complex within the parasite ) remain to be demonstrated .
but interestingly the uptake of host membrane proteins by intraerythrocytic plasmodium , including darc , has been documented .
uptake of darc appears to be an active process and requires the tubovesicular membrane ( tvm ) network that derives from the parasitophorous vacuole membrane and connects the parasite with the erythrocyte pm .
the tvm develops within the first 24 h following merozoite invasion and host proteins accumulate in conjunction with parasite maturation .
uptake is also selective for certain host proteins , especially those that reside in cholesterol - rich detergent - resistant host membranes ; more abundant cytoskeletal molecules such as glycophorin - a , band-3 and glut1 are excluded .
interestingly , establishment of the tvm and maximal accumulation of darc coincide with the parasite stage most sensitive to pf4 ( trophozoites ) .
the intracellular fate ( and function ) of these imported host proteins remains to be determined . in the case of darc , details of parasite intracellular localization are not known . as well as establishing
if pf4-directed lysis of the dv requires darc , it will be of interest to determine if darc translocates to the parasite dv membrane , and thereby provide a plausible mechanism for the delivery of pf4 from the activated platelet to this organelle .
the protective role played by platelets in human malarial infections has yet to be directly answered , but there are a number of lines of evidence that support the affirmative .
depletion of platelets ( or inhibition of platelet function ) in murine models of malaria where outcome is determined by bloodstream parasitemia ( as opposed to the inflammatory response - based cerebral malaria syndrome ) results in reduced survival .
although a similar platelet depletion study is difficult to perform in humans , we do know that thrombocytopenia , which is a common clinical accompaniment of all malarial infections , has been correlated with a poor outcome in falciparum malaria .
platelets have also been implicated as susceptibility factors in the development of cerebral malaria ( cm ) , a complex collection of syndromes specific to p. falciparum infections and a major cause of death .
central to the pathophysiology of cm is the accumulation or sequestration of ie in the cerebral microvasculature , causing the obstruction of blood flow , leukocyte accumulation , localized intravascular inflammation , activation and damage of the endothelium and disruption of the blood - brain barrier .
platelets are often found at sites of ie sequestration in both human cm and mouse cm models where they are believed to mediate ie binding in the microvasculature , and release molecules that affect endothelial cell viability and promote leukocyte adhesion .
the parasite - killing activity of platelets may also contribute to the pathophysiology of cm .
platelet - directed killing may moderate the local inflammatory responses to live parasites , or dead parasite toxins and exudate could contribute to the inflammatory and cell - damaging milieu .
the effect of non - steroidal anti - inflammatory drugs , especially aspirin , may add an additional level of complexity in assigning the importance of platelets in human malaria .
we showed previously that aspirin prevented platelet - mediated killing of cultured p. falciparum and reduced survival in p. chabaudi - infected mice .
aspirin is cheap and readily available , and its usage as an antipyretic remains widespread ( although contraindications such as dengue fever and reyes syndrome have led to revisions on usage guidelines in many westernized countries ) .
a recent cochrane review found that the available data are insufficient to conclude if aspirin ( as well as other antipyretics ) are beneficial or detrimental in malaria . therefore studies to access the impact of aspirin on malaria are highly warranted .
the polymorphism responsible for the duffy - negative blood group , a t to c substitution in a gata1 promoter binding site , prevents erythrocytic expression of the duffy gene .
the c allele is very common in african populations ; frequencies are close to 100% in most of central and western africa . our findings on the requirement for darc in killing of parasites by platelets have led us to speculate that platelet - mediated protection may be inadequate in duffy - negative ( darc deficient ) individuals .
duffy - negativity provides protection against p. vivax infection by virtue of the requirement of the receptor for merozoite invasion ( although the simplicity of this assumption has been recently challenged ) , and p. vivax is rarely seen in africa .
however since erythrocytic expression of duffy is also necessary for the platelet - mediated killing of p. falciparum parasites , we might expect to see increased severity or a poorer outcome to falciparum malaria in duffy - negative populations . at a broad level , this appears to be the case .
using the best available global estimates , rates of infection and death due to p. falciparum are much higher in africa compared with other parts of world ( asia and south america ) , where duffy - negativity is rare .
but many other factors also contribute to this difference , such as the type of parasite and its level of virulence , vector control measures , rates of host immunity , access to healthcare and other socioeconomic factors .
in africa as much as half of malaria - associated deaths are attributable to cm .
the failure of platelets to kill sequestered parasites growing in duffy - negative red cells may counter intuitively exacerbate this complication by allowing viable parasites to remain within the cerebral vasculature .
importantly , studies are needed to test the relationship between duffy - antigen status and platelet protection where , ideally , p. falciparum disease severity , incidence and outcome would be compared within duffy - negative and positive individuals living in the same area and affected by the same parasite strains and other environmental factors . if a lack of platelet protection is indeed detrimental in duffy - negative individuals , it is challenging to reconcile evolutionarily why the duffy - negative polymorphism should remain common in africa .
however the events that led to , and maintained , the duffy - negative allele in africans are likely to be complicated .
it is widely held that the polymorphism arose through positive selection at a distant time in human evolutionary history to protect against , what was presumably then , a lethal form of p. vivax . compared with p. vivax , the advent of p. falciparum infection in humans appears to have been a more recent event .
therefore fixation of the duffy - negative allele may have preceded p. falciparum in humans .
there may also be other positive selective pressures for the polymorphism that we do not know about .
the protective power of the platelet in malaria infection is only beginning to be appreciated . while convincing evidence of their importance in clinical infection is still needed , advances in understanding the molecular detail of parasite killing process have led to some very interesting findings .
useful novel antimalarial molecules based on the pf4 platelet effector molecule are an exciting prospect .
the findings may also help to explain why p. falciparum is a particular problem in africa .
we must continue to direct our efforts to control this scourge of humankind , especially in this part of the world .
 platelets , as well as regulating blood hemostasis , are an important component of the body s defense against invading microbial pathogens . 
 we previously reported that platelets protect during malaria infection by binding plasmodium - infected erythrocytes ( ie ) and killing the parasite within . 
 more recent studies have now revealed the platelet plasmocidal factor , platelet factor 4 ( pf4 ) and the red cell - expressed duffy - antigen molecule as the central players in the parasite killing activity of platelets . 