Characterization of the salivary apyrase activity of three rodent flea species

1. Salivary gland lysates of the adult female fleas Oropsylla bacchi, Orchopea howardi and Xenopsylla cheopis hydrolyse ATP and ADP, but not AMP, thus characterizing the existence of a salivary apyrase activity. 2. In all species Mg++ or Ca++ function as activators, and a pH optimum between 7 and 8 is observed. 3. Salivary gland lysates of male fleas contain significantly smaller amounts of the enzyme activity than do those of female fleas. 4. Immediately following a blood meal, apyrase activity and protein content of female X. cheopis salivary glands are 2-3-fold less than that of unfed fleas, indicating that salivary apyrase activity is secreted during feeding. 5. It is suggested that, as in other arthropods, salivary apyrase may facilitate blood location and blood feeding by preventing ADP-induced platelet aggregation at the site of the bite.     

Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation

Apyrase activity is present in the saliva of haematophagous arthropods. It is related to blood-feeding because of the apyrase ability to hydrolyse ADP, a key component of platelet aggregation. Five apyrases with apparent molecular masses of 88, 82, 79, 68 and 67 kDa were identified in the saliva of the vector of Chagas disease, Triatoma infestans. The large size observed during purification of these enzymes suggested oligomerization. In the present study, we confirmed, using gel-filtration and analytical ultracentrifugation, the presence of apyrase oligomers with molecular masses of 200 kDa in the saliva. Electrophoretic analyses showed that disulphide bonds were involved in homo-oligomerization. In addition, heterogeneity in disulphide bonds and in pI was detected, with the pI ranging from 4.9 to 5.4. The present study gives the first insights into the quaternary structure of soluble apyrases.     

Purification and biochemical characterization of a novel ecto-apyrase, MP67, from Mimosa pudica

We have previously reported the presence of an apyrase in Mimosa pudica. However, only limited information is available for this enzyme. Thus, in this study, the apyrase was purified to homogeneity. The purified enzyme had a molecular mass of around 67 kD and was able to hydrolyze both nucleotide triphosphate and nucleotide diphosphate as substrates. The ratio of ATP to ADP hydrolysis velocity of the purified protein was 0.01 in the presence of calcium ion, showing extremely high substrate specificity toward ADP. Thus, we designated this novel apyrase as MP67. A cDNA clone of MP67 was obtained using primers designed from the amino acid sequence of trypsin-digested fragments of the protein. In addition, rapid amplification of cDNA ends-polymerase chain reaction was performed to clone a conventional apyrase (MpAPY2). Comparison of the deduced amino acid sequences showed that MP67 is similar to ecto-apyrases; however, it was distinct from conventional apyrase based on phylogenetic classification. MP67 and MpAPY2 were expressed in Escherichia coli, and the recombinant proteins were purified. The recombinant MP67 showed high substrate specificity toward ADP rather than ATP. A polyclonal antibody raised against the recombinant MP67 was used to examine the tissue distribution and localization of native MP67 in the plant. The results showed that MP67 was ubiquitously distributed in various tissues, most abundantly in leaves, and was localized to plasma membranes. Thus, MP67 is a novel ecto-apyrase with extremely high substrate specificity for ADP.     

Triatoma infestans apyrases belong to the 5'-nucleotidase family

Apyrases are nucleoside triphosphate-diphosphohydrolases (EC 3.6.1.5) present in a variety of organisms. The apyrase activity found in the saliva of hematophagous insects is correlated with the prevention of ADP-induced platelet aggregation of the host during blood sucking. Purification of apyrase activity from the saliva of the triatomine bug Triatoma infestans was achieved by affinity chromatography on oligo(dT)-cellulose and gel filtration chromatography. The isolated fraction includes five N-glycosylated polypeptides of 88, 82, 79, 68 and 67 kDa apparent molecular masses. The isolated apyrase mixture completely inhibited aggregation of human blood platelets. Labeling with the ATP substrate analogue 5'-p-fluorosulfonylbenzoyladenosine showed that the five species have ATP-binding characteristic of functional apyrases. Furthermore, tandem mass spectroscopy peptide sequencing showed that the five species share sequence similarities with the apyrase from Aedes aegypti and with 5'-nucleotidases from other species. The complete cDNA of the 79-kDa enzyme was cloned, and its sequence confirmed that it encodes for an apyrase belonging to the 5'-nucleotidase family. The gene multiplication leading to the unusual salivary apyrase diversity in T. infestans could represent an important mechanism amplifying the enzyme expression during the insect evolution to hematophagy, in addition to an escape from the host immune response, thus enhancing acquisition of a meal by this triatomine vector of Chagas' disease.     

Structure and protein design of a human platelet function inhibitor

Hematophagous arthropods secrete a salivary apyrase that inhibits platelet activation by catabolizing ADP released from damaged tissues and blood cells. We report the X-ray crystal structures of a human enzyme of the soluble apyrase family in its apo state and bound to a substrate analog. The structures reveal a nucleotide binding domain comprising a five-blade beta propeller, binding determinants of the substrate and the active site, and an unusual calcium binding site with a potential regulatory function. Using a comparative structural biology approach, we were able to redesign the human apyrase so as to enhance its ADPase activity by more than 100-fold. The engineered enzyme is a potent inhibitor of platelet aggregation and may serve as the basis for the development of a new class of antithrombotic agents.     

Anti-platelet activity of a three-finger toxin (3FTx) from Indian monocled cobra (Naja kaouthia) venom

A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor.     

Saliva of the soft tick, Ornithodoros moubata, contains anti-platelet and apyrase activities.

1. Pilocarpine-induced saliva of the soft tick Ornithodoros moubata inhibits platelet aggregation induced by ADP or collagen, even when diluted 2000 times into platelet rich plasma. 2. Saliva contains apyrase (ATP-diphosphohydrolase) activity, which has an optimal pH of 7.0 for ADP and of 8.0 for ATP hydrolysis, respectively. Both Ca2+ and Mg2+ activate the reactions. 3. The mean specific activities for ATP and ADP hydrolysis at pH 7.5 were 0.97 and 0.74 mumoles orthophosphate/min/mg protein. 4. These results, which demonstrate for the first time such activities in the saliva of soft ticks, support the hypothesis that the saliva of blood sucking arthropods serves an anti-hemostatic role during feeding and that large amounts of salivary apyrase activity have evolved independently in hematophagous arthropods.     

Disaggregation of aggregated platelets by apyrase from the tick, Ornithodoros savignyi

Apyrase, secreted by ticks during feeding, is a platelet aggregation inhibitor that functions as a regulator of the host's hemostatic system. This present study concerns the disaggregation effect of salivary gland apyrase from the tick Ornithodoros savignyi. Secondarily aggregated platelets, disaggregated by apyrase, exhibited a reversal of shape from a spherical (aggregated) form to a discoid form, reminiscent of reversible aggregation at low ADP concentrations in citrated platelet-rich plasma. However, they showed a dilatory open canaliculary system and an absence of granules indicating disaggregation after degranulation had taken place. In contrast, disaggregation by the fibrin(ogen)olytic enzyme, plasmin, showed that platelets degranulated, but retained a spherical form with numerous extended pseudopods. While thrombin had no effect on aggregation or clotting of platelets disaggregated with plasmin, it did activate those platelets disaggregated with apyrase and clotted the plasma. This is the first study to describe the disaggregating effects of tick derived apyrase on aggregated platelets. It also shows that apyrase can disaggregate platelets even after secondary aggregation and degranulation of platelets has taken place. Platelet aggregation is one of the main barriers encountered by ticks during feeding and counteraction of this process by ticks is an important factor for successful feeding.     

Stimulus-response coupling in human platelets activated by monoclonal antibodies to the CD9 antigen, a 24 kDa surface-membrane glycoprotein.

The CD9 molecule is a 24 kDa surface-membrane glycoprotein present on platelets and a variety of haematopoetic and non-haematopoetic tissues. In the present study we utilized specific inhibitors of thromboxane A2 (TxA2) formation (aspirin), protein kinase C [H-7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine]] and autocrine stimulation by secreted ADP (apyrase) to modify platelet activation by a monoclonal antibody ALB-6 to the CD9 antigen. This activation is only partially inhibited by aspirin alone but, in combination with either H-7 or apyrase, more than 50% inhibition of platelet aggregation and secretion was observed. This combination of inhibitors was also required to inhibit effectively the phosphorylation of myosin light chain and the 47 kDa substrate of protein kinase C. Intracellular Ca2+ flux monitored by the fluorescent dye fura-2 showed that this was almost completely mediated by the aspirin-sensitive TxA2 pathway. We suggest that the aspirin-insensitive pathway is primarily mediated by phospholipase C formation of diacylglycerol to activate protein kinase C. The inhibition by apyrase suggests a strong dependency on autocrine stimulation by secreted ADP to fully activate both phospholipase C and express fibrinogen-binding sites mediating platelet aggregation. This alternate pathway of phospholipase C activation by ALB-6 may be mediated by cytoplasmic alkalinization [monitored by SNARF-1 (5'(6')-carboxy-10-bismethylamino-3-hydroxy-spiro-[7H- benzo[c]xanthine-1',7(3H)-isobenzofuran]-3'-one) fluorescence of the dye]. Both activation pathways are dependent on intact antibodies, since F(ab')2 fragments of SYB-1, a monoclonal antibody against the CD9 antigen with activation characteristics identical with those of ALB-6, do not elicit activation. Besides thrombin, collagen is another physiological agonist shown to induce aspirin-insensitive activation. Similarities to ALB-6 in collagen sensitivity to apyrase in combination with aspirin inhibitors were noted with respect to aggregation and secretion, as well as a complete block of Ca2+ flux by aspirin. However, it is unlikely that collagen activation is mediated by the CD9 antigen, since SYB-1 F(ab')2 fragments had no effect on collagen activation and aspirin also completely blocked the alkalinization response to collagen, in contrast with ALB-6.     

Salivary apyrase activity of some old world phlebotomine sand flies

Salivary gland homogenates of three Old World phlebotomine sand flies (Phlebotomus papatasi, P. argentipes and P. perniciosus) contained abundant ATPase and ADPase activities, indicating the presence of an apyrase activity. These activities had an optimum pH around 8.0 and were activated by Ca²⁺ but not Mg²⁺. Both hydrolytic activities and salivary protein content were significantly reduced after the female sand fly took a blood meal indicating a secretory fate for the enzymic activities and salivary gland contents during the feeding process. In contrast to the above mentioned species, the salivary apyrase activity of P. colabaensis is much less abundant. Salivary gland homogenates of P. papatasi, P. argentipes and P. perniciosus inhibited ADP-induced platelet aggregation of citrated rabbit platelet rich plasma. It is suggested that salivary apyrase activity, as in some other blood-sucking arthropods, helps the blood-feeding process by preventing host platelet aggregation.     

Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.     

Demonstration of a novel ecto-enzyme on human erythrocytes, capable of degrading ADP and of inhibiting ADP-induced platelet aggregation

The role of ADP as an important inducer of platelet aggregation is generally accepted. Therefore it has been postulated by many authors that the enzymatic removal of extracellular ADP from the circulation is essential to avoid platelet aggregation and thrombus formation. Here we show that erythrocytes essentially contribute to the clearance of ADP. The removal of ADP from suspensions of washed human erythrocytes was due to at least two different activities. One activity, which had already been observed by earlier workers, was identified as adenylate kinase, on the basis of the reaction products and the inhibition by adenosine(5')pentaphospho(5')adenosine (Ap5A). This enzyme was not associated with the cells and was always detectable in cell-free supernatants, indicating that the enzyme had leaked from the cells into the extracellular medium. In contrast, the second activity, which is described here for the first time, was tightly bound to the cells. The activity was not inhibited by Ap5A. The main product of the reaction was AMP, and enzyme activity depended on the presence of divalent cations. The Michaelis constant was about 28 mumol/l. This activity seemed to be an ecto-ADPase. Studies with various inhibitors revealed that degradation of ADP was not due to a non-specific phosphatase. Besides the well known ADPase on the endothelium, the ecto-activity on erythrocytes may play an important part in destroying pro-aggregatory ADP.     

Kinetics of expression of the salivary apyrases in Triatoma infestans

Apyrases are nucleoside triphosphate-diphosphohydrolases that remove Pi from ATP and ADP. The blood feeding reduviid Triatoma infestans, which transmits the Trypanosoma cruzi agent of Chagas disease to animals and man, presents in its salivary glands five apyrases with molecular masses of 88, 82, 79, 68 and 67 kDa. These triatomine apyrases have been associated with the prevention of ADP induced platelet aggregation in the host. Here we provide biochemical data showing that these apyrases are stored in the lumen of the salivary gland D1 pairs, and that about one half of the pool of the enzyme is consumed during feeding. After the feeding recovery of apyrases to maximal activity level takes days, thus suggesting de novo protein synthesis. This hypothesis is supported by quantitative RT-PCR analysis which shows an upregulation of the 79 kDa apyrase mRNA level after feeding.     

Characterization of ATP-diphosphohydrolase activities in the intima and media of the bovine aorta: evidence for a regulatory role in platelet activation in vitro.

The inner layer of the aorta contains the enzyme ATP diphosphohydrolase (ATPDase: EC 3.6.1.5) which catalyzes the sequential phosphorolysis of ATP----ADP----AMP. Two zones of the inner layer, the intima and media, were separated and both were shown to contain ATPDase activity of similar specific activity (0.08 and 0.10 U/mg protein, respectively). However, the media exhibited about 100-times more enzyme activity than the intima. Both preparations were virtually identical with respect to pH optima (7.5), migration patterns after electrophoresis under non-denaturing conditions, relative rates of ATP and ADP hydrolysis and potency to inhibit ADP-induced platelet aggregation in both human platelet-rich plasma and whole blood. The IC50 values for ADP (2 microM)-induced aggregation were 6.8 and 12.9 mU/ml in platelet-rich plasma and whole blood, respectively. Addition of ATPDase to platelets pre-aggregated with ADP resulted in a dose-dependent disaggregation in platelet-rich plasma (IC50 4.9 mU/ml), but not in whole blood. When both ATPDase (5.6-58.7 mU/ml) and ATP (0.5-10 microM) were added to platelet-rich plasma, there was an immediate dose-dependent aggregation of platelets followed by a slowly developing disaggregation. These data show that ATPDase is present in both the intima and media layers of bovine aorta and suggest a dual role for this enzyme in platelet activation. By converting ATP released from damaged cells into ADP, the enzyme could facilitate platelet aggregation at the site of vascular injury, whereas the subsequent conversion of ADP to AMP could inhibit or reverse platelet aggregation. The consequence of these activities would be to control the growth of a platelet thrombus.     

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status

Abstract. Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum , a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 pM 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 pM 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 pM 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S.(Ps.)ochraceum (24.8 pM 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum , a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S.(N.) exiguum (10.9 pM 0.6mU) than in Simulium (Notolepria) gonzalezi (5.9pM1.9mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O. volvulus.     

Apyrase Activity and Platelet Aggregation Inhibitors in the Tick Ornithodoros Savignyi (Acari: Argasidae)

Ticks are ectoparasites that cause considerable damage to their hosts while feeding. The feeding process is facilitated by anti-haemostatic factors present in the tick saliva. Apyrase (ATP diphosphohydrolase, EC 3.6.1.5) is a platelet aggregation inhibitor found in most haematophagous organisms studied. The present study describes the identification and characterization of such an activity in the tick Ornithodoros savignyi. The enzyme conformed to many properties common to apyrases. These included a low substrate specificity, dependence on bivalent metal ions for activity and insensitivity to the classical ATPase inhibitors. Heat denaturation studies, pH optima and similar effects of inhibitors on the enzyme's ATP and ADP hydrolysing activities supported its classification as an apyrase. Salivary gland extracts inhibited the platelet aggregation induced by ADP, collagen and thrombin and disaggregated aggregated platelets. The results suggest the presence of two or more anti-platelet factors present in the salivary glands of this tick species.