longipalpis were obtained from santarm and camar colonies , maintained 
 at the department of entomology , oswaldo cruz foundation , rio de janeiro ( rj ) , 
 brazil .
salivary glands of three - five days - old non blood - fed adult female 
 lu .
longipalpis were dissected , transferred to microtubes 
 containing phosphate buffer saline ( pbs ) and lysed by sonication .
the homogenates 
 were centrifuged at 10,000 rpm for 1 min to remove debris and the supernatants were 
 transferred to new tubes and stored to -80c until use . 

parasites - leishmania amazonensis 
 ( mhom / br/75/josefa ) was maintained on schneider insect 's medium supplemented with 
 10% foetal calf serum and 2% human urine at 26c .
briefly , stationary promastigotes 
 of five - six day cultures were collected , washed and resuspended at 1 x 10 
  /ml density in rpmi-1640 . parasites ( 4 ml ) were then overlaid on 4 
 ml of ficoll ( 10% ) in pbs and centrifuged at room temperature ( rt ) for 10 min at 
 1,300 g with the brake off .
metacyclic forms were collected in the 
 10% ficoll layer , washed twice in pbs and resuspended in pbs for assays . 

reagents - factor xa was purchased from calbiochem ( san diego , ca , 
 usa ) .
human factor va and factor x were purchased from haematologic technologies 
 ( essex junction , vt , usa ) .
human factor ixa was purchased from american diagnostica 
 ( greenwich , ct , usa ) .
human fviii ( advate ) was purchased from baxter healthcare 
 corporation ( westlake village , ca , usa ) and was activated with human thrombin as 
 previously described ( astermark et al .
chromogenic substrates for factor xa 
 ( s-2765 , n--benzyloxycarbonyl - d - arg - gly - arg - p - nitroanilide ) and thrombin ( s-2238 , 
 h - d - phenylalanyl - l - pipecolyl - l - arginine - p - nitroanilinedihydrochloride ) were 
 purchased from diapharma ( westchester , oh , usa ) . activated partial thromboplastin 
 time ( aptt ) ( cephalin plus kaolin ) and prothrombin time ( pt ) ( thromboplastin with 
 calcium ) reagents were from biomrieux ( rj , brazil ) .
l--phosphatidylcholine ( pc ) 
 and ps were purchased from sigma chemical co ( st louis , mo , usa ) .
phospholipid 
 vesicles ( pc / ps ) composed of 75% pc , 25% ps ( w / w ) were prepared by sonication . 
 briefly , phospholipids in chloroform were dried with a stream of n2 , resuspended in 
 50 mm tris - hcl , 150 mm nacl , ph 7.5 and sonicated for 2 min . 
 evaluation of plasma coagulation by recalcification time - plasma 
 coagulation assays by recalcification time were performed on an amelung kc4a 
 coagulometer ( labcon , heppenheim , germany ) using plastic tubes .
human blood samples 
 were collected from healthy donors in 3.8% trisodium citrate ( 9:1 , v / v ) and platelet 
 poor plasma was obtained by further centrifugation at 2,000 g for 
 10 min .
the procoagulant activity of l. amazonensis was evaluated 
 by incubation of plasma ( 50 l ) with 50 l of tris buffered saline containing 
 different concentrations of metacyclic promastigotes .
after 1 min incubation at 
 37c , plasma clotting was initiated by addition of 100 l of 12.5 mm cacl 
 2 and coagulation time was recorded .
longipalpis sgl was evaluated by incubating 50 l of pbs 
 containing 1 sgl with plasma ( 50 l ) .
after 1 min at 37c , plasma clotting was 
 initiated by addition of 100 l of 12.5 mm cacl 2 and coagulation time 
 was recorded . for control ,
assays were also 
 performed upon incubation of sgl ( various dilutions ) with different concentrations 
 of metacyclic forms of l. amazonensis .
activated partial aptt and pt tests - clotting times of human 
 plasma were recorded on a coagulometer after activation of either the intrinsic or 
 the extrinsic coagulation pathways .
platelet - poor plasma was incubated with 50 l of 
 pbs containing or not 1 sgl and 50 l of aptt reagent for 2 min at 37c .
plasma 
 clotting was started by addition of 100 l of 12.5 mm cacl 2 and 
 coagulation time was recorded . for pt assays , 50 l of platelet - poor plasma were 
 incubated with 50 l of pbs containing or not 1 sgl for 1 min at 37c .
reaction was 
 started by addition of 100 l of pt reagent and the plasma clotting was 
 recorded . 

flow cytometric analysis - ps exposure by metacyclic forms of 
 l. amazonensis was evaluated by flow cytometric analysis . 

parasites were resuspended in annexin v binding buffer ( 10 mm hepes , 150 mm nacl , 
 2.5 mm cacl 2 , ph 7.2 ) and further incubated for 15 min at rt with 
 annexin v - alexa-488 ( molecular probes , eugene , or , usa ) according to the 
 instructions of the manufacturer .
propidium iodide ( pi ) was added to the samples at 
 the moment of acquisition ( 0.4 g / ml , final concentration ) .
the samples ( 10,000 
 events ) were acquired on facscalibur and analysed on cell quest software . 

prothrombin activation assay - activation of prothrombin by the 
 prothrombinase complex ( factor xa / factor va / calcium ) was performed in 50 mm hepes , 
 100 mm nacl , 5 mm cacl 2 , 1 mg / ml bovine serum albumin ( bsa ) , ph 7.5 
 ( hepes - bsa buffer ) , using a previously described discontinuous assay ( fernandes et al . 2006 ) .
factor xa ( 10 pm , final 
 concentration ) was incubated for 2 min at 37c with factor va ( 1 nm , final 
 concentration ) and pc / ps ( 20 m , final concentration ) in the presence of serially 
 diluted sgl .
reaction was initiated by addition of prothrombin ( 0.5 m , final 
 concentration ) and aliquots of 10 l were transferred into microplate wells 
 containing 40 l of tris - ethylenediamine tetraacetic acid ( edta ) buffer ( 50 mm 
 tris - hcl , 150 mm nacl , 20 mm edta , 1 mg / ml polyethyleneglycol 6,000 , ph 7.5 ) .
after 
 addition of 50 l of 100 m s-2238 prepared in tris - edta buffer , absorbance at 405 
 nm was recorded at 37c for 20 min at 6 s intervals using a versamax microplate 
 reader ( molecular devices , menlo park , ca , usa ) equipped with a microplate mixer and 
 heating system .
the ability of 
 metacyclic promastigotes in assembling the prothrombinase complex was evaluated by 
 the incubation of factor xa ( 10 pm , final concentration ) and factor va ( 1 nm , final 
 concentration ) for 2 min at 37c with l. amazonensis in hepes - bsa 
 buffer .
reaction was initiated by addition of prothrombin ( 0.5 m , final 
 concentration ) and thrombin formation was evaluated as described .
the inhibitory 
 effect of sgl on prothrombinase complex assembled in either pc / ps or l. 
 amazonensis was also evaluated . in this case , sgl ( at various 
 concentrations ) was incubated with factor xa , factor va and either pc / ps or 
 l. amazo - nensis , before addition of prothrombin . 

factor x activation assay - activation of factor x to factor xa by 
 the intrinsic tenase complex ( factor ixa / factor viiia / calcium ) was performed in 
 hepes - bsa buffer , using a previously described discontinuous assay ( fernandes et al .
factor ixa ( 0.2 nm , 
 final concentration ) was incubated for 2 min at 37c with factor viiia ( 10 iu / ml , 
 final concentration ) in the presence of serially diluted sgl .
reaction was initiated 
 by addition of factor x ( 50 nm , final concentration ) and after 5 min at rt , aliquots 
 of 25 l were transferred into microplate wells containing 25 l of tris - edta .
after 
 addition of 50 l of 200 m s-2765 prepared in tris - edta buffer , absorbance at 405 
 nm was recorded , at 37c , for 20 min at 6 s intervals using a versamax microplate 
 reader . 

chromogenic assay for measuring factor xa activity - the ability of 
 sgl in interfering with factor xa catalytic activity was evaluated using a 
 chromogenic assay .
fxa ( 1 nm , final concentration ) was incubated for 10 min at rt in 
 the presence of sgl serially diluted in hepes - bsa buffer .
after addition of 50 l of 
 200 m s-2765 , absorbance at 405 nm was recorded at 37c for 20 min at 6 s intervals 
 in a versamax microplate reader .
the analysis was performed using 
 prism 3.0 ( graphpad software ) . in all cases ,

 l. amazonesis metacyclic promastigotes activate blood coagulation in a 
 ps - dependent manner - a number of studies have reported presence of ps 
 on leishmania parasites ( wassef et 
 al .
however this phenomenon has been refuted by other 
 groups that were unable to detect this phospholipid on viable parasites ( zufferey et al .
we demonstrate that metacyclic promastigote forms of l. 
 amazonensis expose ps in viable parasites .
1a shows a parasite population that stains for annexin v , 
 but is negative for labelling with pi , a known marker of cell membrane rupture .
ps 
 exposure by leishmania amastigote forms accelerate blood 
 coagulation in vitro , as previously demonstrated in parasites isolated from balb / c 
 mice lesions ( balanco et al .
1b shows that incubation of human 
 plasma with 10   and 10   parasites reduced the human plasma 
 coagulation time by 32% and 28% , respectively , as compared to control , thus 
 evidencing a parasite - number dependent procoagulant effect . 

parasites were analysed by flow cytometry in a dot plot 
 of alexa 488 conjugated - annexin v vs. propidium iodide ( pi ) double 
 staining .
data are representative of two independent experiments ; b : 
 recalcification time of human plasma was measured in the absence ( ct ) or 
 in the presence of different numbers of metacyclic promastigotes . 

results from 10 independent experiments are expressed as mean  standard 
 error of the means ( sem ) .
asterisks mean p < 0.01 relative to ct ; c : 
 l. amazonensis metacyclic promastigotes support the prothrombinase 
 complex assembly .
activation of prothrombin into thrombin by factor 
 xa / factor va was performed in the absence or in the presence of 
 logarithmic or metacyclic promastigotes .
the amount of thrombin formed 
 was quantified as described in the materials and methods section .
data 
 from three independent experiments are expressed as mean  sem ; d : 
 annexin v blocks the assembly of the prothrombinase complex on l. 
 amazonensis .
metacyclic promastigotes were pre - incubated with different 
 concentrations of annexin v and further assayed for thrombin generation 
 in the presence of factor xa / factor va as described in the materials and 
 methods section .
it is generated by the 
 cleavage of prothrombin by prothrombinase complex , which consists in a 
 calcium - dependent , membrane - assembled complex between the enzyme factor xa and its 
 cofactor factor va .
assembly of the prothrombinase complex on ps - containing 
 membranes is essential to achieve a high catalytic efficiency ( mann 1999 ) . in order to confirm ps exposure in l. 
 amazonensis
, we further evaluated the ability of parasites in promoting 
 thrombin formation by the prothrombinase complex .
1c shows that metacyclic promastigotes , but not logarithmic 
 promastigotes , support the coagulation complex assembly resulting in prothrombin 
 activation into thrombin .
this process was highly dependent on ps exposure , since 
 pre - incubation of parasites with annexin v completely blocked thrombin formation 
 ( fig .
longipalpis saliva counteracts l. amazonensis procoagulant 
 activity - a number of anticoagulant proteins have been identified in 
 lu .
the presence of one sgl caused a 2.5-fold increase in 
 plasma coagulation time , as compared to control in the absence of sgl ( fig .
next , we investigated the putative 
 coagulation pathways inhibited by sgl by means of the activated partial aptt and pt 
 tests .
our data showed that the coagulation time was not altered in the presence or 
 in the absence of one sgl as evaluated by the pt test ( fig .
2a ) , which contains tissue factor and is used to screen 
 the extrinsic pathway . on the other hand , the aptt test , which is used to evaluate 
 the intrinsic pathway , showed an increase of 25% in the coagulation time when one 
 sgl was added to the reaction medium ( fig . 

2:anticoagulant effect of lutzomyia longipalpis saliva counteracts 
 procoagulant activity of leishmania amazonensis metacyclic 
 promastigotes . a : anticoagulant activity of sandfly saliva . 

recalcification time of human plasma was measured in the absence ( ct ) or 
 in the presence of salivary gland lysate ( sgl ) .
prothrombin time and 
 thromboplastin time assays were also performed in the absence ( ct ) or in 
 the presence of sgl . in all cases ,
data from 
 three independent experiments are expressed as mean  standard error of 
 the means ( sem ) .
asterisks mean p < 0.001 or p < 0.01 relative to 
 ct ; b : recalcification time of human plasma was measured in the absence 
 ( plasma alone ) or in the presence of different numbers of promastigotes , 
 co - incubated or not with 0.5 sgl ; c : recalcification time of human 
 plasma was measured in the absence ( plasma alone ) or in the presence of 
 10 6 promastigotes , co - incubated or not with different numbers of sgl . 
 in b and c ,
data were analysed by student t test . * : p < 0.05 ; * * : p 
 < 0.01 ( relative to control ) . 
 
considering that the procoagulant activity of l. amazonensis 
 metacyclic forms might be deleterious to vector blood feeding ( kimblin et al .
2b shows that 0.5 sgl 
 efficiently counteracted the procoagulant effect of either 10   or 10 
  parasites .
alternatively , assays performed at a fixed concentration 
 of 10   parasites and varying sgl amounts showed a dose - dependent 
 inhibitory effect of sandfly saliva ( fig . 
 2c ) . 

sandfly saliva inhibits prothrombinase complex activity - plasma 
 coagulation results from the enzymatic cleavage of soluble fibrinogen into an 
 insoluble fibrin polymer .
this reaction is specifically catalysed by thrombin in a 
 reaction that is subsequent to massive prothrombin activation by membrane - assembled 
 prothrombinase complex ( mann 1999 ) . in this 
 context , we further evaluated the ability of lu .
this was assessed by incubating saliva 
 with factor xa , factor va and prothrombin in the presence of calcium ions and 
 artificial pc / ps membranes . as shown in fig . 

3a , the presence of increasing sgl concentrations progressively inhibited 
 thrombin formation by pc / ps - assembled prothrombinase complex .
we next examined the 
 ability of sandfly saliva to inhibit l. amazonensis -assembled 
 prothrombinase complex .
3b shows that the 
 presence of one sgl efficiently counteracted thrombin formation in all parasite 
 concentrations tested . 

a : activation of prothrombin into thrombin was carried out in the 
 presence of factor xa , factor va and l - -phosphatidylcholine 
 ( pc)/phosphatidylserine ( ps ) vesicles as described in the materials and 
 methods section .
assays were performed in the presence of serially 
 diluted salivary gland lysate ( sgl ) or hepes - bovine serum albumin buffer 
 alone ( pc / ps ) .
data from five independent experiments are expressed as 
 mean  standard error of the means ( sem ) [ * * * : p < 0.001 ; * : p < 
 0.05 ( relative to control ) ( pc / ps ) ] ; b : inhibition of prothrombinase 
 complex by sgl ( 0.5 equivalent per assay ) in the presence of different 
 numbers of leishmania amazonensis metacyclic promastigotes . a negative 
 control of thrombin generation in the presence of factor xa / factor va 
 alone and in the absence of parasites and sgl
sandfly saliva inhibits tenase complex and fxa activity - 
 activation of zymogen factor x into factor xa is efficiently catalysed by the 
 intrinsic tenase complex , which consists in a calcium - dependent , membrane - assembled 
 complex between the enzyme factor ixa and its cofactor factor viiia ( mann 1999 ) . in order to evaluate whether the 
 intrinsic tenase complex is inhibited by sandfly saliva , we incubated factor ixa , 
 factor viiia and pc / ps vesicles with serially diluted sandfly saliva .
4:inhibition of intrinsic tenase complex and factor xa catalytic 
 activity by lutzomyia longipalpis saliva .
a : activation of factor x was 
 carried out in the presence of factor viiia , factor ixa and 
 l - -phosphatidylcholine / phosphatidylserine vesicles as described in the 
 materials and methods section .
assays were performed in the presence of 
 serially diluted salivary gland lysate ( sgl ) or hepes - bovine serum 
 albumin ( bsa ) buffer alone ( ct ) .
data from three independent experiments 
 are expressed as mean  standard error of the means ( sem ) ( asterisk 
 means p < 0.05 relative to ct ) ; b : factor xa catalytic activity was 
 assayed by measuring the cleavage of s-2765 chromogenic substrate in the 
 absence ( ct ) or in the presence of different numbers of sgl serially 
 diluted in hepes - bsa buffer .
data were analysed by student t test . * * : p 
 < 0.01 ; * : p < 0.05 ( relative to ct ) . 
  in general , many arthropods anticoagulants target thrombin or factor x and/or factor 
 xa , this last a nexus of the intrinsic and extrinsic pathways of blood coagulation 
 ( champagne 2005 ) . in order to evaluate 
 whether the sandfly saliva anticoagulants have a direct effect on factor xa and can 
 thus contribute to extend the observed coagulation time , chromogenic assays were 
 performed with factor xa and sandfly saliva .
4b , the presence of 0.5 sgl caused a 50% inhibition in 
 factor xa catalytic activity , thus indicating that the anticoagulant effect of 
 sandfly saliva is , at least in part , related to direct inhibition of factor xa 
 activity .
the loss of membrane asymmetry during programmed cell death , i.e. apoptosis , is 
 typically associated with ps exposure .
the presence of ps in 
 leishmania parasites has been reported by a number of groups 
 ( wassef et al .
1985 , henriques et al . 2003 , wanderley et al . 2006 , 2009 , yoneyama et al . 2006 , frana - costa et 
 al .
2013 ) and 
 the phenomenon of ps exposure by viable parasites has been named " apoptotic 
 mimicry " . this process is often referred to as a strategy of the parasites to evade 
 host inflammatory and immune responses ( wanderley et 
 al .
2006 , mercer & heleniu 
 2008 , laliberte & moss 2009 , 
 santos et al .
this process , which 
 resembles ps exposure during apoptosis , consists in the externalisation of ps on 
 living parasite 's surface . in the case of leishmania
it is believed 
 that most of the amastigote forms expose ps without evidences of apoptotic death 
 ( balanco et al . 2001 , wanderley et al . 2006 ) .
interestingly , it has 
 been reported a differential ps exposure between amastigotes isolated from either 
 susceptible or resistant mice ( wanderley et al . 

the recognition of ps - exposing metacyclic 
 promastigotes by phagocytic cells modulates host immune response by eliciting the 
 production of transforming growth factor - beta and interleukin-10 , which facilitate 
 the establishment and maintenance of the infection by the non - ps exposing metacyclic 
 forms ( van zandbergen et al .
we 
 demonstrate that metacyclic promastigote forms of l. amazonensis 
 expose ps , while not presenting evidences of cell membrane rupture .
ps exposure by activated platelets is an essential phenomenon during physiological 
 haemostasis ( heemskerk et al .
in fact , ps - containing membranes 
 allow the assembly of multimolecular enzymatic complexes that ultimately lead to 
 thrombin generation and fibrin deposition ( mann 
 1999 ) . as a result of ps exposure
, we herein demonstrate that l. 
 amazonensis accelerates plasma clotting and supports the assembly of 
 the prothrombin activating complex , prothrombinase .
this process was fully dependent 
 of ps exposure since annexin v completely reverted thrombin formation in the 
 presence of l. amazonensis .
the ability of l. 
 amazo - nensis in recruiting and activating coagulation proteins might be 
 correlated with non - haemostatic properties of these proteins . in this context
it has 
 been demonstrated that activation of protease - activated receptor-1 , known as the 
 thrombin receptor , contribute to l. amazonensis infection in 
 macrophages ( rana et al . 2011 ) .
saliva of haematophagous arthropods contains a diverse mixture of compounds that 
 counteracts host haemostasis ( ribeiro & 
 francischetti 2003 ) .
these compounds are essential for achieving a 
 successful blood feeding . in this context ,
an anti - clotting 
 molecule containing a carbohydrate - recognition domain - like structure , with a c - type 
 ( ca   -dependent ) lectin domain , similar to the snake venom active 
 proteins has been identified ( kini 2006 ) .
six 
 other members of a family of putative anticoagulants were identified on secreted 
 proteins from lu .
however , the 
 effect of saliva towards extrinsic pathway can be eventually underestimated , due to 
 the large amount of thromboplastin reagent that is used in the pt test .
this 
 observation agrees with the estimated concentration of the factor xa inhibitor , 
 lufaxin , which has been recently characterised in the salivary gland of lu . 

our results demonstrate that the anticoagulant effect of 
 sandfly saliva is partially due to the direct inhibition of factor xa activity , 
 resulting in decreased thrombin formation by prothrombinase complex and decreased 
 factor xa activity towards a chromogenic substrate . in this context
, lufaxin 
 specifically inhibits factor xa in a tight , reversible , ca 
 -independent and non - competitive manner ( collin et 
 al . 2012 ) .
remarkably , sandfly saliva counteracts l. 
 amazonensis procoagulant activity as well as thrombin formation by 
 l. amazonensis -assembled prothrombinase complex .
it is important to emphasise that other anticoagulant proteins might exist in the 
 saliva of lu . longipalpis . while anophelines display 
 thrombin - directed anticoagulants , culicine mosquitoes display factor xa - directed 
 anticoagulants ( stark & james 1996 , prez de len et al
albimanus , anophelin , a 6.5 kda saliva - derived 
 peptide , functions as a specific and tight - binding thrombin inhibitor ( francischetti et al .
in addition , a 
 kazal - type thrombin inhibitor has been also characterised in aedes 
 aegypti ( watanabe et al .
aedes albopictus , the 
 protein alboserpin , a factor xa inhibitor , was characterised as an atypical serpin , 
 which binds tightly to factor xa and also contains high affinity for heparin and 
 interacts with pc and phosphatidyl ethanolamine ( calvo et al .
altogether we conclude that the previously described immunosuppressive properties 
 evoked by ps exposure on l. amazonensis parasites are concomitant 
 with procoagulant responses . on the other hand , the potent anticoagulant effect of 
 leishmania parasites ' vector 's saliva
most of the anticoagulant properties of haematophagous organisms are driven against 
 host procoagulant enzymes or multimolecular coagulation complexes .
therefore it is 
 conceivable that vector 's saliva impairs procoagulant , but not immunosuppressive 
 properties of ps exposing parasites in the first steps of infection . on the other 
 hand , the subsequent spread of the parasite on the vertebrate host
possibly unmasks 
 the procoagulant properties of ps exposing parasites which may evoke important 
 biological responses during leishmania infection .
 
 leishmania parasites expose phosphatidylserine ( ps ) on their 
 surface , a process that has been associated with regulation of host 's immune 
 responses . in this study 
 we demonstrate that ps exposure by metacyclic 
 promastigotes of leishmania amazonensis favours blood 
 coagulation . 
 l. amazonensis accelerates in vitro coagulation of 
 human plasma . 
 in addition , l. amazonensis supports the assembly 
 of the prothrombinase complex , thus promoting thrombin formation . 
 this process 
 was reversed by annexin v which blocks ps binding sites . during blood meal , 
 
 lutzomyia longipalpis sandfly inject saliva in the bite 
 site , which has a series of pharmacologically active compounds that inhibit 
 blood coagulation . 
 since saliva and parasites are co - injected in the host during 
 natural transmission , we evaluated the anticoagulant properties of sandfly 
 saliva in counteracting the procoagulant activity of l. 
 amazonensis . 
 lu . 
 longipalpis saliva reverses 
 plasma clotting promoted by promastigotes . 
 it also inhibits thrombin formation 
 by the prothrombinase complex assembled either in phosphatidylcholine ( pc)/ps 
 vesicles or in l. amazonensis . 
 sandfly saliva inhibits factor 
 x activation by the intrinsic tenase complex assembled on pc / ps vesicles and 
 blocks factor xa catalytic activity . 
 altogether our results show that metacyclic 
 promastigotes of l. amazonensis are procoagulant due to ps 
 exposure . 
 notably , this effect is efficiently counteracted by sandfly 
 saliva . 
MATERIALS AND METHODS
RESULTS
DISCUSSION