Uncomplicated Plasmodium vivax malaria: mapping the proteome from circulating platelets.

BACKGROUND: Thrombocytopenia is frequent in Plasmodium vivax malaria but the role of platelets in pathogenesis is unknown. Our study explores the platelet (PLT) proteome from uncomplicated P. vivax patients, to fingerprint molecular pathways related to platelet function. Plasma levels of Platelet factor 4 (PF4/CXCL4) and Von Willebrand factor (VWf), as well as in vitro PLTs-P. vivax infected erythrocytes (Pv-IEs) interactions were also evaluated to explore the PLT response and effect on parasite development. METHODS: A cohort of 48 patients and 25 healthy controls were enrolled. PLTs were purified from 5 patients and 5 healthy controls for Liquid Chromatography-Mass spectrometry (LC-MS/MS) analysis. Plasma levels of PF4/CXCL4 and VWf were measured in all participants. Additionally, P. vivax isolates (n = 10) were co-cultured with PLTs to measure PLT activation by PF4/CXCL4 and Pv-IE schizonts formation by light microscopy. RESULTS: The proteome from uncomplicated P. vivax patients showed 26 out of 215 proteins significantly decreased. PF4/CXCL4 was significantly decreased followed by other proteins involved in platelet activation, cytoskeletal remodeling, and endothelial adhesion, including glycoprotein V that was significantly decreased in thrombocytopenic patients. In contrast, acute phase proteins, including SERPINs and Amyloid Serum A1 were increased. High levels of VWf in plasma from patients suggested endothelial activation while PF4/CXCL4 plasma levels were similar between patients and controls. Interestingly, high levels of PF4/CXCL4 were released from PLTs-Pv-IEs co-cultures while Pv-IEs schizont formation was inhibited. CONCLUSIONS: The PLT proteome analyzed in this study suggests that PLTs actively respond to P. vivax infection. Altogether, our findings suggest important roles of PF4/CXCL4 during uncomplicated P. vivax infection through a possible intracellular localization. Our study shows that platelets are active responders to P. vivax infection, inhibiting intraerythrocytic parasite development. Future studies are needed to further investigate the molecular pathways of interaction between platelet proteins found in this study and host response, which could affect parasite control as well as disease progression.

Practical Considerations of Dissolved Oxygen Levels for Platelet Function under Hypoxia.

Investigating human platelet function in low-oxygen environments is important in multiple settings, including hypobaric hypoxia (e.g., high altitude), sea level hypoxia-related disease, and thrombus stability. These studies often involve drawing blood from which platelets are isolated and analysed at atmospheric conditions or re-exposed to low oxygen levels in hypoxia chambers before testing. However, it remains unknown how the in vitro handling of the samples itself changes their dissolved oxygen concentration, which might affect platelet function and experimental results. Here, we prepared healthy donor platelet-rich plasma and washed platelet (WP) suspensions and exposed them to 2% oxygen. We found that the use of hypoxia pre-equilibrated tubes, higher platelet concentrations (>2 × 108/mL versus 2 × 107/mL), smaller volumes (600 µL versus 3 mL), and presence of plasma reduced the time for samples to reach 2% oxygen. Notably, oxygen levels decreased below 2% in most suspensions, but also in WP maintained at atmospheric 21% oxygen. Additionally, platelet spreading on fibrinogen was decreased when using hypoxic fibrinogen-coated culture plates regardless of the oxygen percentage (2% or 21%) in which platelet incubation took place. Thus, sample handling and experimental conditions should be carefully monitored in platelet-hypoxia studies as they might compromise results interpretation and comparison across studies.

Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbß3, and platelet factor 4.

Bacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbß3 engagement. Moreover, feedback agonists adenosine 5'-diphosphate and thromboxane A2 are mandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndrome α-granule-deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway.

Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbß3.

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet-bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbß3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbß3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5'-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.

Activation of glycoprotein VI (GPVI) and C-type lectin-like receptor-2 (CLEC-2) underlies platelet activation by diesel exhaust particles and other charged/hydrophobic ligands.

Platelets are activated by a range of stimuli that share little or no resemblance in structure to each other or to recognized ligands, including diesel exhaust particles (DEP), small peptides [4N1-1, Champs (computed helical anti-membrane proteins), LSARLAF (Leu-Ser-Ala-Arg-Leu-Ala-Phe)], proteins (histones) and large polysaccharides (fucoidan, dextran sulfate). This miscellaneous group stimulate aggregation of human and mouse platelets through the glycoprotein VI (GPVI)-FcR γ-chain complex and/or C-type lectin-like receptor-2 (CLEC-2) as shown using platelets from mice deficient in either or both of these receptors. In addition, all of these ligands stimulate tyrosine phosphorylation in GPVI/CLEC-2-double-deficient platelets, indicating that they bind to additional surface receptors, although only in the case of dextran sulfate does this lead to activation. DEP, fucoidan and dextran sulfate, but not the other agonists, activate GPVI and CLEC-2 in transfected cell lines as shown using a sensitive reporter assay confirming a direct interaction with the two receptors. We conclude that this miscellaneous group of ligands bind to multiple proteins on the cell surface including GPVI and/or CLEC-2, inducing activation. These results have pathophysiological significance in a variety of conditions that involve exposure to activating charged/hydrophobic agents.

A subset of group A-like var genes encodes the malaria parasite ligands for binding to human brain endothelial cells.

Cerebral malaria is the most deadly manifestation of infection with Plasmodium falciparum. The pathology of cerebral malaria is characterized by the accumulation of infected erythrocytes (IEs) in the microvasculature of the brain caused by parasite adhesins on the surface of IEs binding to human receptors on microvascular endothelial cells. The parasite and host molecules involved in this interaction are unknown. We selected three P. falciparum strains (HB3, 3D7, and IT/FCR3) for binding to a human brain endothelial cell line (HBEC-5i). The whole transcriptome of isogenic pairs of selected and unselected parasites was analyzed using a variant surface antigen-supplemented microarray chip. After selection, the most highly and consistently up-regulated genes were a subset of group A-like var genes (HB3var3, 3D7_PFD0020c, ITvar7, and ITvar19) that showed 11- to >100-fold increased transcription levels. These var genes encode P. falciparum erythrocyte membrane protein (PfEMP)1 variants with distinct N-terminal domain types (domain cassette 8 or domain cassette 13). Antibodies to HB3var3 and PFD0020c recognized the surface of live IEs and blocked binding to HBEC-5i, thereby confirming the adhesive function of these variants. The clinical in vivo relevance of the HBEC-selected parasites was supported by significantly higher surface recognition of HBEC-selected parasites compared with unselected parasites by antibodies from young African children suffering cerebral malaria (Mann-Whitney test, P = 0.029) but not by antibodies from controls with uncomplicated malaria (Mann-Whitney test, P = 0.58). This work describes a binding phenotype for virulence-associated group A P. falciparum erythrocyte membrane protein 1 variants and identifies targets for interventions to treat or prevent cerebral malaria.

Induction of strain-transcending antibodies against Group A PfEMP1 surface antigens from virulent malaria parasites.

Sequence diversity in pathogen antigens is an obstacle to the development of interventions against many infectious diseases. In malaria caused by Plasmodium falciparum, the PfEMP1 family of variant surface antigens encoded by var genes are adhesion molecules that play a pivotal role in malaria pathogenesis and clinical disease. PfEMP1 is a major target of protective immunity, however, development of drugs or vaccines based on PfEMP1 is problematic due to extensive sequence diversity within the PfEMP1 family. Here we identified the PfEMP1 variants transcribed by P. falciparum strains selected for a virulence-associated adhesion phenotype (IgM-positive rosetting). The parasites transcribed a subset of Group A PfEMP1 variants characterised by an unusual PfEMP1 architecture and a distinct N-terminal domain (either DBLα1.5 or DBLα1.8 type). Antibodies raised in rabbits against the N-terminal domains showed functional activity (surface reactivity with live infected erythrocytes (IEs), rosette inhibition and induction of phagocytosis of IEs) down to low concentrations (<10 µg/ml of total IgG) against homologous parasites. Furthermore, the antibodies showed broad cross-reactivity against heterologous parasite strains with the same rosetting phenotype, including clinical isolates from four sub-Saharan African countries that showed surface reactivity with either DBLα1.5 antibodies (variant HB3var6) or DBLα1.8 antibodies (variant TM284var1). These data show that parasites with a virulence-associated adhesion phenotype share IE surface epitopes that can be targeted by strain-transcending antibodies to PfEMP1. The existence of shared surface epitopes amongst functionally similar disease-associated P. falciparum parasite isolates suggests that development of therapeutic interventions to prevent severe malaria is a realistic goal.

Bruton's Tyrosine Kinase Inhibitors Impair FcγRIIA-Driven Platelet Responses to Bacteria in Chronic Lymphocytic Leukemia.

Bacterial infections are a major cause of morbidity and mortality in chronic lymphocytic leukemia (CLL), and infection risk increases in patients treated with the Bruton's tyrosine kinase (Btk) inhibitor, ibrutinib. Btk and related kinases (like Tec) are expressed in non-leukemic hematopoietic cells and can be targeted by ibrutinib. In platelets, ibrutinib therapy is associated with bleeding complications mostly due to off-target effects. But the ability of platelets to respond to bacteria in CLL, and the potential impact of ibrutinib on platelet innate immune functions remain unknown. FcγRIIA is a tyrosine kinase-dependent receptor critical for platelet activation in response to IgG-coated pathogens. Crosslinking of this receptor with monoclonal antibodies causes downstream activation of Btk and Tec in platelets, however, this has not been investigated in response to bacteria. We asked whether ibrutinib impacts on FcγRIIA-mediated activation of platelets derived from CLL patients and healthy donors after exposure to Staphylococcus aureus Newman and Escherichia coli RS218. Platelet aggregation, α-granule secretion and integrin αIIbß3-dependent scavenging of bacteria were detected in CLL platelets but impaired in platelets from ibrutinib-treated patients and in healthy donor-derived platelets exposed to ibrutinib in vitro. While levels of surface FcγRIIA remained unaffected, CLL platelets had reduced expression of integrin αIIbß3 and GPVI compared to controls regardless of therapy. In respect of intracellular signaling, bacteria induced Btk and Tec phosphorylation in both CLL and control platelets that was inhibited by ibrutinib. To address if Btk is essential for platelet activation in response to bacteria, platelets derived from X-linked agammaglobulinemia patients (lacking functional Btk) were exposed to S. aureus Newman and E. coli RS218, and FcγRIIA-dependent aggregation was observed. Our data suggest that ibrutinib impairment of FcγRIIA-mediated platelet activation by bacteria results from a combination of Btk and Tec inhibition, although off-target effects on additional kinases cannot be discarded. This is potentially relevant to control infection-risk in CLL patients and, thus, future studies should carefully evaluate the effects of CLL therapies, including Btk inhibitors with higher specificity for Btk, on platelet-mediated immune functions.

Adhesion of Plasmodium falciparum-infected erythrocytes to human cells: molecular mechanisms and therapeutic implications.

Severe malaria has a high mortality rate (15-20%) despite treatment with effective antimalarial drugs. Adjunctive therapies for severe malaria that target the underlying disease process are therefore urgently required. Adhesion of erythrocytes infected with Plasmodium falciparum to human cells has a key role in the pathogenesis of life-threatening malaria and could be targeted with antiadhesion therapy. Parasite adhesion interactions include binding to endothelial cells (cytoadherence), rosetting with uninfected erythrocytes and platelet-mediated clumping of infected erythrocytes. Recent research has started to define the molecular mechanisms of parasite adhesion, and antiadhesion therapies are being explored. However, many fundamental questions regarding the role of parasite adhesion in severe malaria remain unanswered. There is strong evidence that rosetting contributes to severe malaria in sub-Saharan Africa; however, the identity of other parasite adhesion phenotypes that are implicated in disease pathogenesis remains unclear. In addition, the possibility of geographic variation in adhesion phenotypes causing severe malaria, linked to differences in malaria transmission levels and host immunity, has been neglected. Further research is needed to realise the untapped potential of antiadhesion adjunctive therapies, which could revolutionize the treatment of severe malaria and reduce the high mortality rate of the disease.

Experimental conditions affect the outcome of Plasmodium falciparum platelet-mediated clumping assays.

BACKGROUND: Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes (IE) is a parasite adhesion phenotype that has been associated with severe malaria in some, but not all, field isolate studies. A variety of experimental conditions have been used to study clumping in vitro, with substantial differences in parasitaemia (Pt), haematocrit (Ht), and time of reaction between studies. It is unknown whether these experimental variables affect the outcome of parasite clumping assays. METHODS: The effects of Pt (1, 4 and 12%), Ht (2, 5 and 10%) and time (15 min, 30 min, 1 h, 2 h) on the clumping of P. falciparum clone HB3 were examined. The effects of platelet freshness and parasite maturity were also studied. RESULTS: At low Ht (2%), the Pt of the culture has a large effect on clumping, with significantly higher clumping occurring at 12% Pt (mean 47% of IE in clumps) compared to 4% Pt (mean 26% IE in clumps) or 1% Pt (mean 7% IE in clumps) (ANOVA, p=0.0004). Similarly, at low Pt (1%), the Ht of the culture has a large effect on clumping, with significantly higher clumping occurring at 10% Ht (mean 62% IE in clumps) compared to 5% Ht (mean 25% IE in clumps) or 2% Ht (mean 10% IE in clumps) (ANOVA, p=0.0004). Combinations of high Ht and high Pt were impractical because of the difficulty assessing clumping in densely packed IE and the rapid formation of enormous clumps that could not be counted accurately. There was no significant difference in clumping when fresh platelets were used compared to platelets stored at 4 degrees C for 10 days. Clumping was a property of mature pigmented-trophozoites and schizonts but not ring stage parasites. CONCLUSION: The Pt and Ht at which in vitro clumping assays are set up have a profound effect on the outcome. All previous field isolate studies on clumping and malaria severity suffer from potential problems in experimental design and methodology. Future studies of clumping should use standardized conditions and control for Pt, and should take into account the limitations and variability inherent in the assay.

A method for positive and negative selection of Plasmodium falciparum platelet-mediated clumping parasites and investigation of the role of CD36.

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes (IEs) is a frequently observed parasite adhesion phenotype. The importance of clumping in severe malaria and the molecular mechanisms behind this phenomenon are incompletely understood. Three platelet surface molecules have previously been identified as clumping receptors: CD36, globular C1q receptor (gC1qR/HABP1/p32), and P-selectin (CD62P), but the parasite ligands mediating this phenotype are unknown. The aim of this work was to develop a selection method to facilitate investigations of the molecular mechanisms of clumping in selected P. falciparum lines. Magnetic beads coated with anti-platelet antibodies were used to positively and negatively select clumping IEs from P. falciparum strains IT, HB3, 3D7 and Dd2. Clumping in all four positively selected parasite lines was abolished by antibodies to CD36, but was not affected by antibodies to gC1qR or P-selectin. Clumping positive lines showed significantly higher binding to CD36 than clumping negative lines in flow adhesion assays (strains IT, HB3 and 3D7, p<0.05 for all strains, paired t test) and static assays (strain Dd2, p<0.0001 paired t test). However, clumping negative lines IT, HB3 and 3D7 did show some binding to CD36 under flow conditions, indicating that CD36-binding is not sufficient for clumping. These data show that CD36-dependent clumping positive and negative lines can easily be selected from P. falciparum laboratory strains. CD36-binding is necessary but not sufficient for clumping, and the molecular differences between clumping positive and negative parasite lines responsible for the phenotype require further investigation.

The human CD6 gene is transcriptionally regulated by RUNX and Ets transcription factors in T cells.

CD6 is a lymphocyte surface receptor involved in lymphocyte activation and differentiation processes that is constitutively expressed on developing and mature T cells and on the B1a cells. To define the molecular basis for the tissue-specific expression of CD6 we have identified the transcription factors that control the activity of the proximal regulatory region of the human CD6 gene. The TATA-less CD6 promoter contains multiple transcriptional start sites, and its preferential activity in human T lymphocytes is dependent on RUNX- and Ets-binding sites located within a highly conserved region. RUNX and Ets-1 factors transactivated the CD6 promoter through recognition of the -215 and -230 binding sites, respectively. Chromatin immunoprecipitation assays revealed that RUNX1 constitutively occupies the CD6 promoter in vivo, and knockdown experiments demonstrated that the steady-state level of CD6 mRNA is dependent on the expression of RUNX1, RUNX3 and Ets-1 transcription factors. Therefore, RUNX1/3 and Ets1 control the expression of CD6 in human T lymphocytes, thus expanding the range of T-cell specific and developmentally regulated lymphocyte gene targets involved in T-cell activation and differentiation.

In vitro inhibition of Plasmodium falciparum rosette formation by Curdlan sulfate.

Spontaneous binding of infected erythrocytes to uninfected erythrocytes to form rosettes is a property of some strains of Plasmodium falciparum that is linked to severe complications of malaria. Curdlan sulfate (CRDS) is a sulfated glycoconjugate compound that is chemically similar to known rosette-inhibiting drugs such as heparin. CRDS has previously been shown to have antimalarial activity in vitro and is safe for clinical use. Here we show that CRDS at therapeutic levels (10 to 100 microg/ml) significantly reduces rosette formation in vitro in seven P. falciparum laboratory strains and in a group of 18 African clinical isolates. The strong ability to inhibit rosetting suggests that CRDS has the potential to reduce the severe complications and mortality rates from P. falciparum malaria among African children. Our data support further clinical trials of CRDS.

Platelet-mediated clumping of Plasmodium falciparum infected erythrocytes is associated with high parasitemia but not severe clinical manifestations of malaria in African children.

Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is an adhesive phenotype commonly found in field isolates that has previously been associated with severe malaria. Here, clumping was assessed in 131 isolates from Malian children. The clumping phenotype was seen in 6% (N = 51) of uncomplicated malaria, 24% (N = 51) of severe malaria, and 45% (N = 29) of high parasitemia non-severe malaria isolates. Multivariate analysis indicated that clumping was strongly positively associated with parasitemia (F(1,122) = 24.1, P < 0.001) but not with disease category (F(2,122) = 1.8, P = 0.17). Therefore platelet-mediated clumping in Malian P. falciparum isolates is primarily associated with high parasitemia and not with severe clinical manifestations of malaria.

Transcriptional regulation of human CD5: important role of Ets transcription factors in CD5 expression in T cells.

CD5 is a surface receptor constitutively expressed on thymocytes and mature T and B-1a cells. CD5 expression is tightly regulated during T and B cell development and activation processes. In this study we shown that the constitutive expression of CD5 on human T cells correlates with the presence of a DNase I-hypersensitive (DH) site at the 5'-flanking region of CD5. Human CD5 is a TATA-less gene for which 5'-RACE analysis shows multiple transcriptional start sites, the most frequent of which locates within an initiator sequence. Luciferase reporter assays indicate that a 282-bp region upstream of the initiation ATG displays full promoter activity in human T cells. Two conserved Ets-binding sites (at positions -239 and -185) were identified as functionally relevant to CD5 expression by site-directed mutagenesis, EMSAs, and cotransfection experiments. A possible contribution of Sp1 (-115 and -95), c-Myb (-177), and AP-1-like (-151) motifs was also detected. Further DH site analyses revealed an inducible DH site 10 kb upstream of the human CD5 gene in both T and B CD5(+) cells. Interestingly, a 140-bp sequence showing high homology with a murine inducible enhancer is found within that site. The data presented indicate that the 5'-flanking region of human CD5 is transcriptionally active in T cells, and that Ets transcription factors in conjunction with other regulatory elements are responsible for constitutive and tissue-specific CD5 expression.

Cloning of S4D-SRCRB, a new soluble member of the group B scavenger receptor cysteine-rich family (SRCR-SF) mapping to human chromosome 7q11.23.

The scavenger receptor cysteine-rich superfamily (SRCR-SF) is a highly conserved group of membrane and/or secreted proteins related to the innate and adaptive immune system. Here, we report the cloning of the gene encoding human S4D-SRCRB, a novel soluble member of the SRCR-SF, which is composed of four group B SRCR domains separated by Pro-, Ser- and Thr-rich polypeptides. The longest cDNA sequence found is 2,806 bp in length and encodes a mature protein of 528 aa, with a predicted molecular mass of M(r) 55,600. The S4D-SRCRB gene is located at Chromosome 7q11.23, telomeric to the Williams-Beuren syndrome deletion. It extends over 20 kb and consists of 11 exons, with each SRCR domain being encoded by a single exon. Northern blot analysis indicated that S4D-SRCRB has a broad tissue distribution and is expressed as two major mRNA species: one of 2.8 kb, with a restricted tissue expression pattern (mainly kidney and placenta), and another of 1.5 kb, with a broader distribution. A similar mRNA expression pattern was observed during the analysis of several tumor cell lines. The highest degree of similarity found between S4D-SRCRB and other group B SRCR-SF members was with human DMBT1 (a mosaic protein composed of fourteen SRCR domains, which is involved in innate defense and epithelia polarization) and chicken 18-B (a turpentine-induced soluble acute-phase protein composed of four SRCR domains). Our data indicate that S4D-SRCRB constitutes a novel SRCR-SF member, which could be involved in basic homeostatic functions such as innate host defense.