Juvenile hormone mediates lipid storage in the oocytes of Dipetalogaster maxima.

Triatomines are vectors of Chagas disease and important model organisms in insect physiology. "Kissing bugs" are obligatory hematophagous insects. A blood meal is required to successfully complete oogenesis, a process primarily controlled by juvenile hormone (JH). We used Dipetalogaster maxima as an experimental model to further understand the roles of JH in the regulation of vitellogenesis and oogenesis. A particular focus was set on the role of JH controlling lipid and protein recruitment by the oocytes. The hemolymph titer of JH III skipped bisepoxide increased after a blood meal. Following a blood meal there were increased levels of mRNAs in the fat body for the yolk protein precursors, vitellogenin (Vg) and lipophorin (Lp), as well as of their protein products in the hemolymph; mRNAs of the Vg and Lp receptors (VgR and LpR) were concomitantly up-regulated in the ovaries. Topical administration of JH induced the expression of Lp/LpR and Vg/VgR genes, and prompted the uptake of Lp and Vg in pre-vitellogenic females. Knockdown of the expression of LpR by RNA interference in fed females did not impair the Lp-mediated lipid transfer to oocytes, suggesting that the bulk of lipid acquisition by oocytes occurred by other pathways rather than by the endocytic Lp/LpR pathway. In conclusion, our results strongly suggest that JH signaling is critical for lipid storage in oocytes, by regulating Vg and Lp gene expression in the fat body as well as by modulating the expression of LpR and VgR genes in ovaries.

Trypanosoma cruzi could affect wild triatomine approaching behaviour to humans by altering vector nutritional status: A field test.

Hematophagous insects exhibit complex behaviour when searching for blood-meals, responding to several host stimuli. The hematophagous insect Mepraia spinolai is a wild vector of Trypanosoma cruzi, causative agent of Chagas disease in humans, in the semiarid-Mediterranean ecosystem of Chile. In this study, we evaluated the association between the approaching behaviour to a human host, with T. cruzi infection status and nutritional condition of M. spinolai. To this end, we captured 501 individuals in six consecutive 10 min-timespan, using a human as bait. Captured vectors were weighed, photographed and measured to calculate their nutritional status by means of a Standardized Body Mass Index. Trypanosoma cruzi infection was assessed in the intestinal content by using a real-time PCR assay. Ordinal logistic regressions were performed separately for infected and uninfected groups to evaluate if the nutritional status was associated with the approaching behaviour to a human host, recorded as the time-span of capture. Nutritional status of uninfected triatomines was higher than that from infected ones (p < 0.005). Among the infected, those with higher nutritional status approached first (p < 0.01); there was no effect of nutritional status in the uninfected group. Trypanosoma cruzi infection might affect the foraging behaviour of M. spinolai under natural conditions, probably deteriorating nutritional status and/or altering vector detection abilities.

Insights into a hotspot in the Brasiliensis subcomplex (Hemiptera, Triatominae) by analysis of D2 domain of the nuclear gene 28S.

The Brasiliensis subcomplex is a monophyletic group formed by the species Triatoma brasiliensis brasiliensis, T. b. macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki. However, using cytogenetic data and experimental hybrid crosses, T. lenti and T. petrochiae were also grouped into this subcomplex. This study aims to analyze the properties of hotspot in the D2 domain of the nuclear gene 28S in all species of the Brasiliensis subcomplex as well as T. lenti and T. petrochiae. These species show two transversions at position 385 (G↔C and T↔G). We suggest that this mutation in haplotype 4 may be an initial molecular tool that supports the relationship of these species with the subcomplex. In addition to the transversion at haplotype 4, these species, aside from T. melanica, also possess a transversion at position 385 (G↔T) in haplotype 1. Thus, we describe the hotspot mutations of the D2 domain of the nuclear gene 28S for species in Brasiliensis subcomplex as follows: three transversions are present at position 385 of haplotypes 1 and 4, which are shared by members of the subcomplex as well as T. lenti and T. petrochiae. These transversions may be considered a synapomorphy between these species. However, we emphasize that new phylogenetic studies should be conducted to evaluate whether T. lenti and T. petrochiae are truly members of the Brasiliensis subcomplex.

Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs) Formation and Arterial Thrombosis.

BACKGROUND: The saliva of blood-feeding arthropods contains a notable diversity of molecules that target the hemostatic and immune systems of the host. Dipetalodipin and triplatin are triatomine salivary proteins that exhibit high affinity binding to prostanoids, such as TXA2, thus resulting in potent inhibitory effect on platelet aggregation in vitro. It was recently demonstrated that platelet-derived TXA2 mediates the formation of neutrophil extracellular traps (NETs), a newly recognized link between inflammation and thrombosis that promote thrombus growth and stability. METHODOLOGY/PRINCIPAL FINDINGS: This study evaluated the ability of dipetalodipin and triplatin to block NETs formation in vitro. We also investigated the in vivo antithrombotic activity of TXA2 binding proteins by employing two murine models of experimental thrombosis. Remarkably, we observed that both inhibitors abolished the platelet-mediated formation of NETs in vitro. Dipetalodipin and triplatin significantly increased carotid artery occlusion time in a FeCl3-induced injury model. Treatment with TXA2-binding proteins also protected mice from lethal pulmonary thromboembolism evoked by the intravenous injection of collagen and epinephrine. Effective antithrombotic doses of dipetalodipin and triplatin did not increase blood loss, which was estimated using the tail transection method. CONCLUSIONS/SIGNIFICANCE: Salivary TXA2-binding proteins, dipetalodipin and triplatin, are capable to prevent platelet-mediated NETs formation in vitro. This ability may contribute to the antithrombotic effects in vivo. Notably, both molecules inhibit arterial thrombosis without promoting excessive bleeding. Our results provide new insight into the antihemostatic effects of TXA2-binding proteins and may have important significance in elucidating the mechanisms of saliva to avoid host's hemostatic responses and innate immune system.

Distribution of constitutive heterochromatin in Triatoma melanocephala (Hemiptera, Triatominae).

In principle, Triatoma melanocephala was included in the Brasiliensis subcomplex on the basis of morphological parameters and geographical layout, since there were no other relevant data available in the literature. On the basis of karyotype, it has been proposed to exclude T. melanocephala, as well as of T. vitticeps and T. tibiamaculata, from the subcomplex, which shows fragmentation of the X sex chromosomes, thereby approaching the species of North America. Therefore, the present study aimed to determine the pattern of constitutive heterochromatin of T. melanocephala to provide new data on the cytotaxonomy of this vector of Chagas disease and especially to try to relate this species to some group, complex or subcomplex of triatomine species, aiding in their classification. This species showed no constitutive heterochromatin in the autosomes or X sex chromosome, but only the Y sex chromosome. The number of chromosomes and heterochromatin pattern of T. melanocephala proved to be identical to that described for Panstrongylus lutzi. Thus, the present study demonstrated a tentative relationship between T. melanocephala and P. lutzi. However, we emphasize that other comparative studies should be conducted between these species, such as experimental crosses and molecular, enzymatic, morphological, and morphometric analyses to determine whether these species are actually evolutionarily related or if the number of chromosomes and the heterochromatin pattern emerged as homoplasies in T. melanocephala and P. lutzi.

Biochemical changes in the transition from vitellogenesis to follicular atresia in the hematophagous Dipetalogaster maxima (Hemiptera: Reduviidae).

In this work, we have explored the biochemical changes characterizing the transition from vitellogenesis to follicular atresia, employing the hematophagous insect vector Dipetalogaster maxima as a model. Standardized insect rearing conditions were established to induce a gradual follicular degeneration stage by depriving females of blood meal during post-vitellogenesis. For the studies, hemolymph and ovaries were sampled at representative days of pre-vitellogenesis, vitellogenesis and early and late follicular atresia. When examined by scanning electron microscopy, ovarioles at the initial stage of atresia were small but still showed some degree of asynchronism, a feature that was lost in an advanced degeneration state. At late follicular atresia, in vivo uptake assays of fluorescently labeled vitellogenin (Vg-FITC) showed loss of competitiveness of oocytes to uptake vitellogenin. Circulating vitellogenin levels in atresia were significantly higher than those registered at pre-vitellogenesis, most likely to maintain appropriate conditions for another gonotrophic cycle if a second blood meal is available. Follicular atresia was also characterized by partial proteolysis of vitellin, which was evidenced in ovarian homogenates by western blot. When the activity of ovarian peptidases upon hemoglobin (a non-specific substrate) was tested, higher activities were detected at early and late atresia whereas the lowest activity was found at vitellogenesis. The activity upon hemoglobin was significantly inhibited by pepstatin A (an aspartic peptidase inhibitor), and was not affected by E64 (a cysteine peptidase inhibitor) at any tested conditions. The use of specific fluorogenic substrates demonstrated that ovarian homogenates at early follicular atresia displayed high cathepsin D-like activity, whereas no activity of either, cathepsin B or L was detected. Mass spectrometry analysis of the digestion products of the substrate Abz-AIAFFSRQ-EDDnp further confirmed the presence of a cathepsin D-like peptidase in ovarian tissue. In the context of our findings, the early activation of cathepsin D-like peptidase could be relevant in promoting yolk protein recycling and/or enhancing follicle removal.

On the mechanisms involved in biological heme crystallization.

Blood-feeding organisms digest hemoglobin, releasing large quantities of heme inside their digestive tracts. Free heme is very toxic, and these organisms have evolved several mechanisms to protect against its deleterious effects. One of these adaptations is the crystallization of heme into the dark-brown pigment hemozoin (Hz). Here we review the process of Hz formation, focusing on organisms other than Plasmodium that have contributed to a better understanding of heme crystallization. Hemozoin has been found in several distinct classes of organisms including protozoa, helminths and insects and Hz formation is the predominant form of heme detoxification. The available evidence indicates that amphiphilic structures such as phospholipid membranes and lipid droplets accompanied by specific proteins play a major role in heme crystallization. Because this process is specific to a number of blood-feeding organisms and absent in their hosts, Hz formation is an attractive target for the development of novel drugs to control illnesses associated with these hematophagous organisms.

Insight into the salivary transcriptome and proteome of Dipetalogaster maxima.

Dipetalogaster maxima is a blood-sucking Hemiptera that inhabits sylvatic areas in Mexico. It usually takes its blood meal from lizards, but following human population growth, it invaded suburban areas, feeding also on humans and domestic animals. Hematophagous insect salivary glands produce potent pharmacologic compounds that counteract host hemostasis, including anticlotting, antiplatelet, and vasodilatory molecules. To obtain further insight into the salivary biochemical and pharmacologic complexity of this insect, a cDNA library from its salivary glands was randomly sequenced. Salivary proteins were also submitted to one- and two-dimensional gel electrophoresis (1DE and 2DE) followed by mass spectrometry analysis. We present the analysis of a set of 2728 cDNA sequences, 1375 of which coded for proteins of a putative secretory nature. The saliva 2DE proteome displayed approximately 150 spots. The mass spectrometry analysis revealed mainly lipocalins, pallidipins, antigen 5-like proteins, and apyrases. The redundancy of sequence identification of saliva-secreted proteins suggests that proteins are present in multiple isoforms or derive from gene duplications.

Dipetalodipin, a novel multifunctional salivary lipocalin that inhibits platelet aggregation, vasoconstriction, and angiogenesis through unique binding specificity for TXA2, PGF2alpha, and 15(S)-HETE.

Dipetalodipin (DPTL) is an 18 kDa protein cloned from salivary glands of the triatomine Dipetalogaster maxima. DPTL belongs to the lipocalin superfamily and has strong sequence similarity to pallidipin, a salivary inhibitor of collagen-induced platelet aggregation. DPTL expressed in Escherichia coli was found to inhibit platelet aggregation by collagen, U-46619, or arachidonic acid without affecting aggregation induced by ADP, convulxin, PMA, and ristocetin. An assay based on incubation of DPTL with small molecules (e.g. prostanoids, leukotrienes, lipids, biogenic amines) followed by chromatography, mass spectrometry, and isothermal titration calorimetry showed that DPTL binds with high affinity to carbocyclic TXA(2), TXA(2) mimetic (U-46619), TXB(2), PGH(2) mimetic (U-51605), PGD(2,) PGJ(2), and PGF(2α). It also interacts with 15(S)-HETE, being the first lipocalin described to date to bind to a derivative of 15-lipoxygenase. Binding was not observed to other prostaglandins (e.g. PGE(1), PGE(2), 8-iso-PGF(2α), prostacyclin), leukotrienes (e.g. LTB(4), LTC(4), LTD(4), LTE(4)), HETEs (e.g. 5(S)-HETE, 12(S)-HETE, 20-HETE), lipids (e.g. arachidonic acid, PAF), and biogenic amines (e.g. ADP, serotonin, epinephrine, norepinephrine, histamine). Consistent with its binding specificity, DPTL prevents contraction of rat uterus stimulated by PGF(2α) and induces relaxation of aorta previously contracted with U-46619. Moreover, it inhibits angiogenesis mediated by 15(S)-HETE and did not enhance inhibition of collagen-induced platelet aggregation by SQ29548 (TXA(2) antagonist) and indomethacin. A 3-D model for DPTL and pallidipin is presented that indicates the presence of a conserved Arg(39) and Gln(135) in the binding pocket of both lipocalins. Results suggest that DPTL blocks platelet aggregation, vasoconstriction, and angiogenesis through binding to distinct eicosanoids involved in inflammation.

Study of the nucleolar cycle and ribosomal RNA distribution during meiosis in triatomines (Triatominae, Heteroptera).

Aspects of nucleolar activity during spermatogenesis were assessed in three triatomine species (Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans) using cytochemical and fluorescent staining techniques. Toluidine blue and a variant of critical electrolytic concentration (CEC) allowed the discrimination of rRNA providing structural details of the nucleolus and RNA distribution during meiotic cell division. Acridine orange fluorochrome staining permitted the differentiation of nucleic acids, and silver-ion impregnation made possible the observation of pre-nucleolar bodies (PNBs). Our results support the phenomenon known as "persistence of the nucleolar material", and the hypothesis of post-meiotic reactivation of rRNA genes. Nucleolar organizer regions (NORs) were observed in some metaphasic spermatogonial chromosomes in P. megistus and T. infestans. In P. megistus at diplotene-diakinesis, NORs were also detected in one of the sex chromosomes and in an autosome. Therefore, it may be inferred that, in triatomines, the nucleolus does not completely disappear, but persists in the form of small bodies that get together to form the next nucleolar cycle which, in the case of meiosis, will be completed if fertilization occurs and a new zygote is formed.

An alternative, less invasive blood sample collection technique for serologic studies utilizing triatomine bugs (Heteroptera; Insecta).

The collection of blood samples for serological studies is often stressful for the focus animal. Recently, the use of bloodsucking bugs, such as Dipetalogaster maximus or Triatoma infestans (Reduviidae; Triatominae; Heteroptera), has been suggested as a new and less invasive method for blood collection. To evaluate this technique, we collected paired blood samples from 20 domestic rabbits (Oryctolagus cuniculus) during a study of rabbit hemorrhagic disease virus (RHDV). For each rabbit, blood samples were collected by the conventional method (needle and syringe from the vena auricularis) and through feeding by D. maximus. Samples were tested for RHDV antibodies using standard test kits at three different dilutions. Antibody titers were identical for 56 paired samples and differed in only four cases. The simple matching indices were 1 for the 1:10 dilution and 0.9 for the 1:100 and 1:1000 dilutions. The major advantages of the new technique are 1) the possibility to obtain blood from animals where veins are inaccessible and 2) the fact that anesthesia of focus animals may not be necessary.

Oogenesis and egg development in triatomines: a biochemical approach

Em triatomíneos, assim como em outros insetos, o acúmulo de vitelo é um processo no qual um tecido extraovariano, o corpo gorduroso, produz proteínas que são empacotadas no interior de um ovo. A principal proteína, sintetizada pelo corpo gorduroso, que é acumulada no interior de um ovócito, é a vitelogenina. Este processo é também conhecido por vitelogênese. Existem crescentes evidências em triatomíneos, que além do corpo gorduroso, o ovário também produz proteínas de vitelo. A forma como estas proteínas de vitelo entram nos ovócitos será aqui comentada. O vitelo é um material complexo composto por proteínas, lipídeos, carboidratos e outros compostos minoritários que são empacotados de uma maneira organizada no interior dos ovócitos. A fertilização dispara a embriogênese, um processo que culmina com o desenvolvimento do embrião. Durante a embriogênese o vitelo será utilizado para a construção de um novo indivíduo, a ninfa de primeiro estádio. O desafio para a próxima década é entender onde e como estas proteínas de vitelo são utilizadas junto com os seus componentes não protéicos, em compasso com o programa genético do embrião, que comanda a diferenciação celular (fase inicial da embriogênese) e diferenciação do embrião (fase final da embriogênese) no interior do ovo.

Oogenesis and egg development in triatomines: a biochemical approach.

In triatomines, as well as in other insects, accumulation of yolk is a process in which an extra-ovarian tissue, the fat body, produces yolk proteins that are packed in the egg. The main protein, synthesized by the fat body, which is accumulated inside the oocyte, is vitellogenin. This process is also known as vitellogenesis. There are growing evidences in triatomines that besides fat body the ovary also produces yolk proteins. The way these yolk proteins enter the oocyte will be discussed. Yolk is a complex material composed of proteins, lipids, carbohydrates and other minor components which are packed inside the oocyte in an organized manner. Fertilization triggers embryogenesis, a process where an embryo will develop. During embryogenesis the yolk will be used for the construction of a new individual, the first instar nymph. The challenge for the next decade is to understand how and where these egg proteins are used up together with their non-protein components, in pace with the genetic program of the embryo, which enables cell differentiation (early phase of embryogenesis) and embryo differentiation (late phase) inside the egg.

Validation of a minimally invasive blood-sampling technique for the analysis of hormones in domestic rabbits, Oryctolagus cuniculus (Lagomorpha).

Previous studies in small mammals showed that blood-sucking bugs (Reduviidae, Heteroptera) can be used to obtain blood from veins difficult to access by human experimenters. In the present study, we validated the use of reduviid bugs for endocrinological studies in endotherms using domestic rabbits as a model organism. Two processes could alter the hormone concentrations in the blood ingested by the bug: (1) Mixing of ingested blood with saliva, gut fluid, or hemolymph and (2) digestive processes. We compared concentrations of progesterone, testosterone, and hydrocortisone in blood samples that were acquired from domestic rabbits (Oryctolagus cuniculus) by bugs (Dipetalogaster maxima) with hormone concentrations in blood obtained from the same individual rabbits with a conventional method, i.e., syringe. We found no significant differences in hormone concentrations between the two methods. Thus, the mixing effect is negligible immediately after the blood meal. In addition, we also could not find significant changes in concentrations of progesterone and hydrocortisone for up to 8h after the blood meal. Whereas levels of hydrocortisone remained unchanged for even 24h, progesterone levels significantly increased between eight and 24h. Thus, the bugs' excretory apparatus did not fractionate between water and hormones. Thirdly, we hypothesized that reduviid bugs impose less stress on the rabbits than the conventional method. We showed that deviations in hydrocortisone concentrations between the two blood sampling routines were lower when the bug method was used first and higher when the conventional method was used first. Thus, bugs imposed less stress on the study animals than the conventional method. Overall, we conclude that reduviid bugs present a minimally invasive method for obtaining blood from endotherm animals for endocrinological studies.

Effect of fasting on the composition of the fat body lipid of Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus (Hemiptera: Reduviidae).

Modifications in content and lipid composition induced by fasting were examined in fat bodies from adults of Triatominae, Dipetalogaster maximus, Triatoma infestans and Panstrongylus megistus. With fasting, total lipid stores dropped approximately 50% for T. infestans and more than 70% for P. megistus. Total lipids analyzed by thin layer chromatography and fractionated by column chromatography on Unisil showed triacylglycerols as the main component in the three species, although P. megistus showed high levels of diacylglycerols (31-46%). Cholesterol amounted to 8-15%. In diacylglycerol fractions, C16:0, C18:1 and C18:0 fatty acids were detected; their ratio varied with species but it was not dependent on nutritional status. In triacylglycerol fractions C18:1 fatty acid was the major component at different times (48-68%); the ratio of monounsaturated to saturated in this fraction was 1.3, 2.6 and 1.2 for D. maximus, T. infestans and P. megistus respectively. The remarkable drop in lipid stores without noticeable changes in their relative composition would suggest that all types of lipid are used at similar rates. The higher content of diacylglycerols in P. megistus may be associated with the better flight performance of this species.

Metabolic post-feeding changes in fat body and hemolymph of Dipetalogaster maximus (Hemiptera: Reduviidae).

Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult ecdysis (time 0) and during a 30-day period after ingestion of blood meal, total lipids, high density lipophorin (HDLp), carbohydrates, total proteins and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day only 42% of the maximum weight. Lipids stored in the fat body increased up to day 15 reach 24% of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3% of the fat body weight. HDLp represented at all times between 17-24% of the total proteins, whose levels ranged between 35 and 47 mg/ml uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the one shown at days 10 and 15. Hemolymphatic lipids fluctuated during starvation between 3-4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves.

Role of salivary antihemostatic components in blood feeding by triatomine bugs (Heteroptera).

Salivary gland homogenates from 4 genera of triatomine bugs were assayed for anticlotting, apyrase, and vasodilatory activities, and these activities were correlated with the efficiency of each bug species to initiate a blood meal. Antihemostatic activities spanned a large range of values. Apyrase activity in members of the genus Rhodnius was markedly different from that in other genera with respect to their sensitivity to divalent cation activators. Apyrase and vasodilatory activities, but not anticlotting activity, correlated with feeding efficiency of bugs taking a blood meal on a rat. Results are discussed within the context of the evolution of blood-feeding by insects.

Metabolic post-feeding changes in flat body and hemolymph of Dipetalogaster maximus (Hemiptera: Reduviidae)

Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult acdysis (time 0) and during a 30-day period after ingestion of blood meal. Total lipids, high density lipophorin (HDLp), carbohydrates, total protein and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day 30 only 42 per cent of the maximum weight. Lipids stored in the fat body increased up to day 15 reaching 24 per cent of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3 per cent of the fat body weight. HDLp represented at all times between 17-24 per cent of the total proteins, whose levels ranged between 35 and 47 mg/ml. Uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the ones shown at days 10 an 15. Hemolymphatic lipids fluctuated during starvation between 3-4.4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves.

Preservaçäo do Trypanosoma cruzi em tritomíneos alimentos em galinha e em camundongo / Trypanosoma cruzi preservation in triatomines fed in chicken and mouse

Estudou-se a preservaçäo do Trypanosoma cruzi em diferentes espécies de triatomíneos, alimentados em galinha e em camundongo: Rhodnius prolixus (Stal, 1859), R. robustus (Larrouse, 1927), Triatoma brasiliensis (Neiva, 1911) e T. rubrovaria (Blanchard, 1843). Os triatomíneos infectaram-se com o T. cruzi, em camundongos com infecçäo aguda. Para estimar a permanência do T. cruzi nos triatomíneos examinaram-se, individualmente, 20 insetos de cada espécie. Os triatomíneos permaneceram infectados pelo T. cruzi, tanto em camundongo quanto em galinha, ao longo de 5 meses de observaçäo, sem definir uma alteraçäo significativa da influência da fonte sangüinea sobre a replicaçäo do tripanosoma

An inhibitor of collagen-induced platelet aggregation from the saliva of Triatoma pallidipennis.

The saliva of Triatoma pallidipennis, a blood-sucking triatomine bug (Hemiptera, family Reduviidae, subfamily Triatominae) was found to contain a factor that specifically inhibits collagen-induced platelet aggregation. The 19-kDa protein was purified to homogeneity and named pallidipin. Collagen-mediated aggregation of platelets in plasma and of washed platelets was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors (ADP, thrombin, thromboxane A2 mimetic U46619, phorbol ester) was detected. Pallidipin had no effect on platelet adhesion to collagen but inhibited ATP release from platelets. It interacted reversibly with platelets and may share with collagen a common target on them. The protein exhibits a unique primary structure (predicted from cDNA clones) with no significant similarity to other previously described sequences. The protein produced in recombinant baby hamster kidney cells had antiaggregatory effects similar to those of native pallidipin. Availability of recombinant pallidipin will allow further investigation of the precise mechanism of action.