Antimicrobial function of short amidated peptide fragments from the tick-derived OsDef2 defensin.

Previously Os, a 22 amino acid sequence of a defensin from the soft tick Ornithodoros savignyi, was found to kill Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, we evaluated synthetic peptide analogues of Os for antibacterial activity with an aim to identify minimalized active peptide sequences and in so doing obtain a better understanding of the structural requirements for activity. Out of eight partially overlapping sequences of 10 to 12 residues, only Os(3-12) and Os(11-22) exhibit activity when screened against Gram-positive and Gram-negative bacteria. Carboxyamidation of both peptides increased membrane-mediated activity, although carboxyamidation of Os(11-22) negatively impacted on activity against Staphylococcus aureus. The amidated peptides, Os(3-12)NH2 and Os(11-22)NH2 , have minimum bactericidal concentrations of 3.3 µM against Escherichia coli. Killing was reached within 10 minutes for Os(3-12)NH2 and only during the second hour for Os(11-22)NH2 . In an E. coli membrane liposome system, both Os and Os(3-12)NH2 were identified as membrane disrupting while Os(11-22)NH2 was less active, indicating that in addition to membrane permeabilization, other targets may be involved in bacterial killing. In contrast to Os, the membrane disruptive effect of Os(3-12)NH2 did not diminish in the presence of salt. Neither Os nor its amidated derivatives caused human erythrocyte haemolysis. The contrasting killing kinetics and effects of amidation together with structural and liposome leakage data suggest that the 3-12 fragment relies on a membrane disruptive mechanism while the 11-22 fragment involves additional target mechanisms. The salt-resistant potency of Os(3-12)NH2 identifies it as a promising candidate for further development.

Tick Paralysis: Solving an Enigma.

In comparison to other arachnids, ticks are major vectors of disease, but less than 8% of the known species are capable of inducing paralysis, as compared to the ~99⁻100% arachnids that belong to venomous classes. When considering the potential monophyly of venomous Arachnida, this review reflects on the implications regarding the classification of ticks as venomous animals and the possible origin of toxins. The origin of tick toxins is compared with scorpion and spider toxins and venoms based on their significance, functionality, and structure in the search to find homologous venomous characters. Phenotypic evaluation of paralysis, as caused by different ticks, demonstrated the need for expansion on existing molecular data of pure isolated tick toxins because of differences and discrepancies in available data. The use of in-vivo, in-vitro, and in-silico assays for the purification and characterization of paralysis toxins were critically considered, in view of what may be considered to be a paralysis toxin. Purified toxins should exhibit physiologically relevant activity to distinguish them from other tick-derived proteins. A reductionist approach to identify defined tick proteins will remain as paramount in the search for defined anti-paralysis vaccines.

Structural properties of bioactive peptides with α-glucosidase inhibitory activity.

Bioactive peptides are emerging as promising class of drugs that could serve as α-glucosidase inhibitors for the treatment of type 2 diabetes. This article identifies structural and physicochemical requirements for the design of therapeutically relevant α-glucosidase inhibitory peptides. So far, a total of 43 fully sequenced α-glucosidase inhibitory peptides have been reported and 13 of them had IC50 values several folds lower than acarbose. Analysis of the peptides indicates that the most potent peptides are tri- to hexapeptides with amino acids containing a hydroxyl or basic side chain at the N-terminal. The presence of proline within the chain and alanine or methionine at the C-terminal appears to be relevant for high activity. Hydrophobicity and isoelectric points are less important variables for α-glucosidase inhibition whilst a net charge of 0 or +1 was predicted for the highly active peptides. In silico simulated gastrointestinal digestion revealed that the high and moderately active peptides, including the most potent peptide (STYV), were gastrointestinally unstable, except SQSPA. Molecular docking of SQSPA, STYV, and STY (digestion fragment of STYV) with α-glucosidase suggested that their hydrogen bonding interactions and binding energies were comparable with acarbose. The identified criteria will facilitate the design of new peptide-derived α-glucosidase inhibitors.

Anti-inflammatory and anti-endotoxin properties of peptides derived from the carboxy-terminal region of a defensin from the tick Ornithodoros savignyi.

Antimicrobial peptides are small cationic peptides that possess a large spectrum of bioactivities, including antimicrobial, anti-inflammatory and antioxidant activities. Several antimicrobial peptides are known to inhibit lipopolysaccharide (LPS)-induced inflammation in vitro and to protect animals from sepsis. In this study, the cellular anti-inflammatory and anti-endotoxin activities of Os and Os-C, peptides derived from the carboxy-terminal of a tick defensin, were investigated. Both Os and Os-C were found to bind LPS in vitro, albeit to a lesser extent than polymyxin B and melittin, known endotoxin-binding peptides. Binding to LPS was found to reduce the bactericidal activity of Os and Os-C against Escherichia coli confirming the affinity of both peptides for LPS. At a concentration of 25 µM, the nitric oxide (NO) scavenging activity of Os was higher than glutathione, a known NO scavenger. In contrast, Os-C showed no scavenging activity. Os and Os-C inhibited LPS/IFN-γ induced NO and TNF-α production in RAW 264.7 cells in a concentration-dependent manner, with no cellular toxicity even at a concentration of 100 µM. Although inhibition of NO and TNF-α secretion was more pronounced for melittin and polymyxin B, significant cytotoxicity was observed at concentrations of 1.56 µM and 25 µM for melittin and polymyxin B, respectively. In addition, Os, Os-C and glutathione protected RAW 264.7 cells from oxidative damage at concentrations as low as 25 µM. This study identified that besides previously reported antibacterial activity of Os and Os-C, both peptides have in addition anti-inflammatory and anti-endotoxin properties.

Investigation into the mechanism of action of the antimicrobial peptides Os and Os-C derived from a tick defensin.

Os and Os-C are two novel antimicrobial peptides, derived from a tick defensin, which have been shown to have a larger range of antimicrobial activity than the parent peptide, OsDef2. The aim of this study was to determine whether the peptides Os and Os-C are mainly membrane acting, or if these peptides have possible additional intracellular targets in Escherichia coli and Bacillus subtilis. Transmission electron microscopy revealed that both peptides adversely affected intracellular structure of both bacteria causing different degrees of granulation of the intracellular contents. At the minimum bactericidal concentrations, permeabilization as determined with the SYTOX green assay seemed not to be the principle mode of killing when compared to melittin. However, fluorescent triple staining indicated that the peptides caused permeabilization of stationary phase bacteria and TEM indicated membrane effects. Studies using fluorescently labeled peptides revealed that the membrane penetrating activity of Os and Os-C was similar to buforin II. Os-C was found to associate with the septa of B. subtilis. Plasmid binding studies showed that Os and Os-C binds E. coli plasmid DNA at a similar charge ratio as melittin. These studies suggest membrane activity for Os and Os-C with possible intracellular targets such as DNA. The differences in permeabilization at lower concentrations and binding to DNA between Os and Os-C, suggest that the two peptides have dissimilar modes of action.

Evolution of the tissue factor pathway inhibitor-like Kunitz domain-containing protein family in Rhipicephalus microplus.

One of the principle mechanisms utilised by ticks to obtain a blood meal is the subversion of the host's haemostatic response. This is achieved through the secretion of saliva containing anti-haemostatic proteins into the feeding lesion. Lineage-specific expansion of predicted secretory protein families have been observed in all previously studied ticks and occurred in response to adaptation to a blood-feeding environment. Of these, the predominant families are common between both hard and soft ticks. One of these families, namely the Kunitz domain-containing protein family, includes proven tissue factor pathway inhibitor-like (TFPI-like) anti-haemostatics such as ixolaris and penthalaris that play a crucial role during tick feeding. Although Kunitz-type proteins have been found in Rhipicephalus microplus, the TFPI-like Kunitz protein family has not yet been studied. We report a comprehensive search for TFPI-like Kunitz domain-containing proteins in R. microplus expressed sequence tag libraries, resulting in the identification of 42 homologues. The homologues were bioinformatically and phylogenetically studied, including the application of an intensive Bayesian Markov Chain Monte Carlo (MCMC) analysis of the individual Kunitz domain nucleotide sequences. We show that the R. microplus TFPI-like Kunitz protein family groups into two main clades that presumably underwent ancient duplication, which indicates that a whole genome duplication event occurred at least 150 million years ago. Evidence for recent and ancient gene and domain duplication events was also found. Furthermore, the divergence times of the various tick lineages estimated in this paper correspond with those presented in previous studies. The elucidation of this large protein family's evolution within R. microplus adds to current knowledge of this economically important tick.

Expression profiling, gene silencing and transcriptional networking of metzincin metalloproteases in the cattle tick, Rhipicephalus (Boophilus) microplus.

Tick proteins functioning in vital physiological processes such as blood meal uptake, digestion and reproduction are potential targets for anti-tick vaccines, since vaccination could inhibit these essential functions and ultimately affect tick survival. In this study we identified metzincin metalloproteases from Rhipicephalus microplus as potential vaccine candidates since they are implicated as essential to blood-cavity formation, bloodmeal digestion and reproduction in ixodid ticks. Eight transcripts encoding proteins that contain the characteristic metzincin zinc-binding motif HEXXHXXG/NXXH/D and a unique methionine containing "methionine-turn" were identified from native and in-house assembled R. microplus expressed sequence tag (EST) databases. These were representative of five reprolysin-like and three astacin-like metzincin metalloproteases. Reverse transcription-PCR analysis indicated that the reprolysins were most abundantly expressed in the salivary glands, whereas the astacins were most abundant in the midgut and ovaries. In vivo gene silencing was performed to assess a possible phenotype of these metalloproteases during adult female R. microplus blood feeding and reproduction. RNA interference (RNAi) against two of the reprolysins and one of the astacins significantly affected the average egg weight and oviposition rate. Evidently, this reverse genetic approach enabled the evaluation of the overall vital impact of tick proteins. Finally, integrated real time-PCR studies also revealed an extensive cross organ network between the R. microplus metzincin transcripts, supporting the use of a combinatorial metzincin-based anti- R. microplus vaccine.

Savicalin, a lipocalin from hemocytes of the soft tick, Ornithodoros savignyi.

Savicalin, is a lipocalin found in the hemocytes of the soft tick, Ornithodoros savignyi. It could be assigned to the tick lipocalin family based on BLAST analysis. Savicalin is the first non-salivary gland lipocalin described in ticks. The mature sequence is composed of 188 amino acids with a molecular mass of 21481.9 Da. A homolog for savicalin was found in a whole body EST-library from a related soft tick O. porcinus, while other tick salivary gland derived lipocalins retrieved from the non-redundant sequence database are more distantly related. Homology modeling supports the inclusion of savicalin into the lipocalin family. The model as well as multiple alignments suggests the presence of five disulphide bonds. Two conserved disulphide bonds are found in hard and soft tick lipocalins. A third disulphide bond is shared with the TSGP4-clade of leukotriene C4 binding soft tick lipocalins and a fourth is shared with a lipocalin from the hard tick Ixodes scapularis. The fifth disulphide bond is unique and links strands D-E. Phylogenetic analysis showed that savicalin is a distant relative of salivary gland derived lipocalins, but groups within a clade that is possibly non-salivary gland derived. It lacks the biogenic amine-binding motif associated with tick histamine and serotonin binding proteins. Expression profiles indicate that savicalin is found in hemocytes, midgut and ovaries, but not in the salivary glands. Up-regulation occurs in hemocytes after bacterial challenge and in midguts and ovaries after feeding. Given its tissue distribution and up-regulation of expression, it is possible that this lipocalin functions in tick development after feeding or in an anti-microbial capacity.

A proteomics approach for the analysis of hemolymph proteins involved in the immediate defense response of the soft tick, Ornithodoros savignyi, when challenged with Candida albicans.

A proteomics approach was employed to identify proteins secreted into the hemolymph of Ornithodorus savignyi ticks 2 h after immune-challenge with the yeast, Candida albicans. Profiling of the proteins present in hemolymph of unchallenged ticks versus ticks challenged with heat-killed yeast revealed five proteins to be differentially expressed. The modulated protein spots were subjected to tandem mass spectrometry (MS/MS) analysis, but could not be positively identified. These proteins can be assigned to the immune response as they were not induced after aseptic injury. In an attempt to identify hemolymph proteins that recognize and bind to yeast cells, hemolymph obtained from both unchallenged and challenged ticks was incubated with C. albicans. Elution of the bound proteins followed by SDS-PAGE analysis indicated that three proteins (97, 88 and 26 kDa) present in both unchallenged and challenged hemolymph samples bind to yeast cells. The constant presence of these three proteins in tick hemolymph leads us to believe that they may be involved in non-self recognition and participate in yeast clearance from tick plasma. The analyzed yeast-binding proteins could also not be positively identified, suggesting that all the tick immune proteins investigated in this study are novel.

Ornithodoros savignyi: soft tick apyrase belongs to the 5'-nucleotidase family.

Salivary apyrases are nucleotide-metabolising enzymes that blood-feeding parasites utilise for modulation of extracellular nucleotides to prevent platelet activation and aggregation. In this study a 5'-nucleotidase specific degenerate primer was used to identify homologous transcripts from Ornithodoros savignyi salivary gland cDNA. Two 5'-nucleotidase isoforms that share significant sequence identity to putative apyrases from Rhipicephalus appendiculatus and Ixodes scapularis were identified. Structure prediction showed a tertiary structure similar to periplasmic ecto-5'-nucleotidase from Escherichia coli, with high conservation of functional residues. The O. savignyi 5'-nucleotidase isoform I was recombinantly expressed in Pichia pastoris. Cross-reactivity was demonstrated with polyclonal anti-apyrase antisera produced against O. savignyi apyrase. Subsequent Edman sequencing and MS/MS analysis of purified O. savignyi apyrase identified peptide sequence fragments that shared sequence identity with both newly identified 5'-nucleotidase isoforms. It was concluded that wild-type apyrase is a mixture of the isoforms identified from the salivary glands of O. savignyi. These results represent the first confirmation of a soft (argasid) tick apyrase that belongs to the 5'-nucleotidase family of enzymes.

Tick anti-hemostatics: targets for future vaccines and therapeutics.

For ticks, a significant obstacle in obtaining a blood meal is counteracting the hemostatic system of the host. To this end, ticks have developed a broad array of anti-hemostatics, which is reflected in the presence of structurally related tick proteins with different functions. Disruption of blood flow which blocks successful tick feeding makes anti-hemostatics attractive targets for anti-tick vaccines. Moreover, the limited number of drugs currently available for a range of important cardio-vascular diseases makes ticks a potential source of novel therapeutics. This review aims to summarize the key features of tick anti-hemostatics, their structures, mode of action and possible future application as vaccines and novel therapeutic agents.

A reassessment of argasid tick salivary gland ultrastructure from an immuno-cytochemical perspective.

Previous morphological and histochemical studies of argasid tick salivary glands indicated that they were less complex than ixodid salivary glands, with only three granular cell types. The present study shows that there exist at least four different granular cell types in the salivary glands of the argasid tick Ornithodoros savignyi, based on immuno-localization of the anti-hemostatic factors, apyrase and savignygrin. Both anti-hemostatic factors were localized to dense core granule type 'a' and to granule type 'b', that shares a similar homogenous morphology with non-labeled granule type 'd'. Furthermore, the major tick salivary gland proteins (TSGPs), previously implicated in granule biogenesis, were localized to all the granular cell types. This indicates that granular cell types with different morphologies can express the same proteins, while cell types that show similar morphologies may not express the same proteins. Argasid tick salivary glands seem to be more complex than previously thought and might not be amenable to morphological classification alone. Alternative classification methodologies that rely on physical expression patterns of the salivary gland proteome might be more reliable as markers for a specific granular cell type.

Exon-intron structure of outlier tick lipocalins indicate a monophyletic origin within the larger lipocalin family.

All tick proteins assigned to the lipocalin family lack the structural conserved regions (SCRs) that are characteristic of the kernel lipocalins and can thus be classified as outliers. These tick proteins have been assigned to the tick lipocalin family based on database searches that indicated homology between tick sequences and the fact that the histamine binding protein (HBP2) from the hard tick Rhipicephalus appendiculatus (Ixodidae) shows structural similarity to the lipocalin fold. Sequence identity between kernel and outlier lipocalins falls below 20% and the question raised is whether the outlier and kernel lipocalins are truly homologous. More specifically in the case of the tick lipocalins, whether their structural fold is derived from the lipocalin fold or whether convergent evolution resulted in the generation of the basic lipocalin-like fold which consists of an eight stranded continuous anti-parallel beta-barrel terminated by a C-terminal alpha-helix that lies parallel to the barrel. The current study determined the gene structure for HBP2 and TSGP1, TSGP2 and TSGP4, lipocalins identified from the soft tick Ornithodoros savignyi (Argasidae). All tick lipocalins have four introns (A-D) with conserved positions and phases within the tick lipocalin sequence alignment. The positions and phase information are also conserved with regard to the rest of the lipocalin family. Phylogenetic analysis using this information shows conclusively that tick lipocalins are evolutionary related to the rest of the lipocalin family. Tick lipocalins are grouped within a monophyletic clade that indicates a monophyletic origin within the tick lineage and also group with the other arthropod lipocalins in a larger clade. Phylogenetic analysis of sequence alignments based on conserved secondary structure of the lipocalin fold support the conclusions from the gene structure trees. These results indicate that exon-intron arrangement can be useful for the inclusion of outlier lipocalins within the larger lipocalin family.

Adaptation of ticks to a blood-feeding environment: evolution from a functional perspective.

Ticks had to adapt to an existing and complex vertebrate hemostatic system from being free-living scavengers. A large array of anti-hemostatic mechanisms evolved during this process and includes blood coagulation as well as platelet aggregation inhibitors. Several questions regarding tick evolution exist. What was the nature of the ancestral tick? When did ticks evolve blood-feeding capabilities? How did these capabilities evolve? Did host specificity influence the adaptation of ticks to a blood-feeding environment? What are the implications of tick evolution for future research into tick biology and vaccine development? We investigate these questions in the light of recent research into protein superfamilies from tick saliva. Our conclusions are that the main tick families adapted independently to a blood-feeding environment. This is supported by major differences observed in all processes involved with blood-feeding for hard and soft ticks. Gene duplication events played a major role in the evolution of novel protein functions involved in tick-host interactions. This occurred during the late Cretaceous and was stimulated by the radiation of birds and placental mammals, which provided numerous new niches for ticks to adapt to a new lifestyle. Independent adaptation of the main tick families to a blood-feeding environment has several implications for future tick research in terms of tick genome projects and vaccine development.

The major tick salivary gland proteins and toxins from the soft tick, Ornithodoros savignyi, are part of the tick Lipocalin family: implications for the origins of tick toxicoses.

The origins of tick toxicoses remain a subject of controversy because no molecular data are yet available to study the evolution of tick-derived toxins. In this study we describe the molecular structure of toxins from the soft tick, Ornithodoros savignyi. The tick salivary gland proteins (TSGPs) are four highly abundant proteins proposed to play a role in salivary gland granule biogenesis of the soft tick O. savignyi, of which the toxins TSGP2 and TSGP4 are a part. They were assigned to the lipocalin family based on sequence similarity to known tick lipocalins. Several other tick lipocalins were also identified using Smith-Waterman database searches, bringing the tick lipocalin family up to 20. Phylogenetic analysis showed that most tick lipocalins group within genus-specific clades, suggesting that gene duplication and divergence of tick lipocalin function occurred after tick speciation, most probably during the evolution of a hematophagous lifestyle. TSGP2 and TSGP3 show high sequence identity and group terminal to moubatin, an inhibitor of collagen-induced platelet aggregation from the tick, O. moubata. However, no platelet aggregation inhibitory activity is associated with the TSGPs using ADP or collagen as agonists, suggesting that TSGP2 and TSGP3 duplicated after divergence of O. savignyi and O. moubata. This timing is supported by the absence of TSGP2-4 in the salivary gland extracts of O. moubata. The absence of TSGP2 and TSGP4 in salivary gland extracts from O. moubata correlates with the nontoxicity of this tick species. The implications of this study are that the various forms of tick toxicoses do not have a common origin, but must have evolved independently in those tick species that cause pathogenesis.

Evolution of hematophagy in ticks: common origins for blood coagulation and platelet aggregation inhibitors from soft ticks of the genus Ornithodoros.

Identification and characterization of antihemostatic components from hematophagous organisms are useful for the elucidation of the evolutionary mechanisms involved in adaptation to a highly complex host hemostatic system. Although many bioactive components involved in the regulation of the host's hemostatic system have been described, the evolutionary mechanisms of how arthropods adapted to a blood-feeding environment have not been elucidated. This study describes common origins of both blood coagulation inhibitors and platelet aggregation inhibitors (PAIs) from soft ticks of the genus Ornithodoros. Neighbor-joining analysis indicates that fXa, thrombin, and PAIs share a common ancestor. Maximum parsimony analysis and a phylogeny based on root mean square deviation values of alpha-carbon backbone structures suggest a novel evolutionary pathway by which different antihemostatic functions have evolved through a series of paralogous gene duplication events. In this scenario, the thrombin inhibitors preceded the fXa and PAIs. This evolutionary model explains why the tick serine protease inhibitors have inhibition mechanisms that differ from that of the canonical bovine pancreatic trypsin inhibitor (BPTI)-like inhibitors. Higher nonsynonymous-to-synonymous substitution rates indicate positive Darwinian selection for the fXa and PAIs. Comparison with hemostatic inhibitors of hard ticks suggests that the two main tick families have independently evolved novel antihemostatic mechanisms. Independent evolution of these mechanisms in ticks points to a rapid divergence between tick families that could be dated between 120 and 92 MYA. This coincides with current molecular phylogeny views on the early divergence of modern birds and placental mammals in the Late Cretaceous, which suggests that this event might have been a driving force in the evolution of hematophagy in ticks.

Pathogenic mechanisms of sand tampan toxicoses induced by the tick, Ornithodoros savignyi.

The tick, Ornithodoros savignyi has been implicated in inducing paralysis and tampan toxicosis. In this study, a basic toxin (TSGP4) was identified and the presence of an acidic toxin (TSGP2) was confirmed. Both basic and acidic toxins were more lethal than previously described, with TSGP4 (34microg) and TSGP2 (24microg) causing mortality of adult mice within 30min. Pathological effects on the cardiac system, notably of salivary gland extract on an isolated rat heart perfusion system and of purified toxins on mouse electrocardiogram patterns could be observed. TSGP4 caused Mobitz type ventricular block, while TSGP2 induced ventricular tachycardia. Conversely, fractions from reversed phase high performance liquid chromatography preparations caused paralysis-like symptoms of the limbs after only 48h. The toxins also differ from previously described tick paralysis toxins in terms of molecular behavior and properties. These results indicate that tampan toxicoses and tick paralysis are unrelated pathogenic phenomena.

Savignygrin, a platelet aggregation inhibitor from the soft tick Ornithodoros savignyi, presents the RGD integrin recognition motif on the Kunitz-BPTI fold.

Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of alpha-CD41 (P2) to platelets, the binding of purified alpha(IIb)beta3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin alpha(v)beta3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward alpha(IIb)beta3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tick Ornithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold.

Disaggregation of aggregated platelets by savignygrin, a alphaIIbeta3 antagonist from Ornithodoros savignyi.

Ticks control their host's hemostatic system by secretion of bioactive components during feeding that inhibit blood coagulation and platelet aggregation. Dissolution of platelets that have already aggregated can enhance control over the hemostatic system. It has been shown that disaggregation of aggregated platelets by the enzyme apyrase was accompanied by a shape change from the aggregated spherical form back to the discoid form associated with un-activated platelets. The present study concerns the disaggregation effect of the alpha IIb/beta3 antagonist, savignygrin. Aggregated platelets that were disaggregated by savignygrin and platelets pre-incubated with savignygrin before activation with ADP, retained a spherical form similar to platelets disaggregated by the fibrinogenolytic enzyme plasmin. The number of pseudopods were however, markedly reduced suggesting a disruption of the focal adhesion points that act as a localization point of alpha IIb/beta3. These results are concurrent with targeting of alpha IIb/beta3 and dissociation of fibrinogen from its receptor, once aggregation has taken place. This is the second mediator of platelet disaggregation found in soft ticks and suggests that disaggregation of aggregated platelets might play an important part in the anti-hemostatic strategy of ticks.